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Sample records for pt32 sperm protein

  1. Moonlighting proteins in sperm-egg interactions.

    Science.gov (United States)

    Petit, François M; Serres, Catherine; Auer, Jana

    2014-12-01

    Sperm-egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI-MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm-ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins.

  2. Detecting coevolution in mammalian sperm-egg fusion proteins.

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    Claw, Katrina G; George, Renee D; Swanson, Willie J

    2014-06-01

    Interactions between sperm and egg proteins can occur physically between gamete surface-binding proteins, and genetically between gamete proteins that work in complementary pathways in which they may not physically interact. Physically interacting sperm-egg proteins have been functionally identified in only a few species, and none have been verified within mammals. Candidate genes on both the sperm and egg surfaces exist, but gene deletion studies do not support functional interactions between these sperm-egg proteins; interacting sperm-egg proteins thus remain elusive. Cooperative gamete proteins undergo rapid evolution, and it is predicted that these sperm-egg proteins will also have correlated evolutionary rates due to compensatory changes on both the sperm and egg. To explore potential physical and genetic interactions in sperm-egg proteins, we sequenced four candidate genes from diverse primate species, and used regression and likelihood methods to test for signatures of coevolution between sperm-egg gene pairs. With both methods, we found that the egg protein CD9 coevolves with the sperm protein IZUMO1, suggesting a physical or genetic interaction occurs between them. With regression analysis, we found that CD9 and CRISP2 have correlated rates of evolution, and with likelihood analysis, that CD9 and CRISP1 have correlated rates. This suggests that the different tests may reflect different levels of interaction, be it physical or genetic. Coevolution tests thus provide an exploratory method for detecting potentially interacting sperm-egg protein pairs.

  3. Protein restriction affects sperm number but not sperm viability in male ants.

    Science.gov (United States)

    Dávila, Francisco; Aron, Serge

    2017-07-01

    Sperm cells are costly to produce; diet should therefore affect sperm number and/or viability. In non-social insects and vertebrates, there is compelling evidence that diet influences sperm production. Less is known about this relationship in eusocial hymenopterans (all ants and some bees and wasps), whose mating systems impose unique selective pressures on sperm production. Males face physiological constraints: they acquire all of the resources they will use in future reproductive efforts as larvae and emerge from the pupal stage with a fixed, non-renewable amount of sperm. Furthermore, males die shortly after copulation, but their genetic material persists for years since their spermatozoa are stored in their mates' spermathecae. We examined the effects of protein restriction during larval development on sperm number and viability in the Argentine ant Linepithema humile. We also looked at its impact on male development, adult mass, and adult fluctuating asymmetry. We found that protein restriction during larval development significantly reduced sperm production, but not sperm viability. It did not affect the number of males reared, male mass, or male asymmetry. However, males from protein-restricted nests developed much more slowly than males from protein-supplemented nests. These results suggest investing in sperm quality and in somatic growth, which enhances a male's ability to disperse and find a mate, are critical to successful male reproduction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Egg jelly proteins stimulate directed motility in Xenopus laevis sperm.

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    Burnett, Lindsey A; Sugiyama, Hitoshi; Bieber, Allan L; Chandler, Douglas E

    2011-06-01

    Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose-dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488-conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0-1 µg/ml range and correlated well with previously published dose-dependent sperm attraction data. Binding was rapid with a half-time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm-orienting behavior.

  5. Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm-zona pellucida interaction.

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    Luna, C; Colás, C; Casao, A; Serrano, E; Domingo, J; Pérez-Pé, R; Cebrián-Pérez, J A; Muiño-Blanco, T

    2015-03-01

    Incubation of ram spermatozoa in capacitating conditions with cAMP-elevating agents promotes a progressive time-dependent increase in the capacitated sperm subpopulation. In this study, the fertilizing capacity of ram spermatozoa (ability to bind to the zona pellucida, ZBA rate) capacitated in these conditions was determined. The results showed an increase (P ram spermatozoa. Likewise, the presence of two seminal plasma (SP) proteins able to protect sperm against cold shock (RSVP14 and RSVP20) was evidenced in both SP and the ram sperm surface, and their influence in the fertilizing ability of spermatozoa capacitated in basal medium or with cAMP-elevating agents was determined. The results verified that RSVP14 and RSVP20 act as decapacitating factors given that their addition to SP-free sperm samples previously to capacitation maintained high proportions of the noncapacitated sperm pattern with no increase in protein tyrosine phosphorylation. However, the obtained ZBA rate in the high-cAMP-containing samples was increased in the presence of RSVP20 (P < 0.05). These findings would indicate that the stimulating effect exerted by this protein on the sperm-oocyte binding occurs downstream from the cAMP generation and that the mechanisms by which RSVP20 promotes the zona pellucida binding might be independent of protein tyrosine phosphorylation.

  6. Egg Activation at Fertilization by a Soluble Sperm Protein.

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    Swann, Karl; Lai, F Anthony

    2016-01-01

    The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca(2+) increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca(2+) increase by initiating Ca(2+) release from intracellular Ca(2+) stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca(2+) oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca(2+) oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species.

  7. Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm.

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    Ledesma, Alba; Fernández-Alegre, Estela; Cano, Adriana; Hozbor, Federico; Martínez-Pastor, Felipe; Cesari, Andreína

    2016-10-01

    This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13±2.99 and 72.27±2.99 for AV and EE, respectively) and progressive motility (64.97±2.64 and 63.73±2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p>0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60±2.87) and sperm with stable plasma membrane (44.56±2.49) comparing with the addition of SP collected by EE (35.80±2.47 and 36.67±1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters.

  8. Hepatitis B virus s protein enhances sperm apoptosis and reduces sperm fertilizing capacity in vitro.

    Directory of Open Access Journals (Sweden)

    Jihua Huang

    Full Text Available OBJECTIVE: Studying the impact of Hepatitis B virus S protein (HBs on early apoptotic events in human spermatozoa and sperm fertilizing capacity. METHODOLOGY/PRINCIPAL FINDINGS: Spermatozoa were exposed to HBs (0, 25, 50, 100 µg/ml for 3 h, and then fluo-4 AM calcium assay, Calcein/Co(2+ assay, protein extraction and ELISA, ADP/ATP ratio assay, sperm motility and hyperactivation and sperm-zona pellucida (ZP binding and ZP-induced acrosome reaction (ZPIAR tests were performed. The results showed that in the spermatozoa, with increasing concentration of HBs, (1 average cytosolic free Ca(2+ concentration ([Ca(2+]i rose; (2 fluorescence intensity of Cal-AM declined; (3 average levels of cytochrome c decreased in mitochondrial fraction and increased in cytosolic fraction; (4 ADP/ATP ratios rose; (5 average rates of total motility and mean hyperactivation declined; (6 average rate of ZPIAR declined. In the above groups the effects of HBs exhibited dose dependency. However, there was no significant difference in the number of sperms bound to ZP between the control and all test groups. CONCLUSION: HBs could induce early events in the apoptotic cascade in human spermatozoa, such as elevation of [Ca(2+]i, opening of mitochondrial permeability transition pore (MPTP, release of cytochrome c (cyt c and increase of ADP/ATP ratio, but exerted a negative impact on sperm fertilizing capacity.

  9. Development of antifertility vaccine using sperm specific proteins

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    A H Bandivdekar

    2014-01-01

    Full Text Available Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4, protein hyaluronidase (PH-20, and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80kDa HSA is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80kDa HSA (Peptide NT and its peptides (Peptides 1, 2, 3 and 4 obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80kDA HAS suggest that the synthetic Peptide-1 of 80kDa HSA is the promising candidate for development of male contraceptive vaccine.

  10. Changes in exposed membrane proteins during in vitro capacitation of boar sperm

    Energy Technology Data Exchange (ETDEWEB)

    Berger, T. (Univ. of California, Davis (USA))

    1990-11-01

    Exposed plasma membrane proteins were labeled with {sup 125}I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.

  11. Valosin-containing protein/p97 interacts with sperm-activating and sperm-attracting factor (SAAF) in the ascidian egg and modulates sperm-attracting activity.

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    Kondoh, Eri; Konno, Aru; Inaba, Kazuo; Oishi, Tohru; Murata, Michio; Yoshida, Manabu

    2008-10-01

    Sperm chemotaxis toward an egg is observed in many animals, and the control of sperm-attracting activity is thought to play an important role in ensuring fertilization. However, the mechanism underlying the release of a sperm attractant from an egg is still obscure. In this study, we examined the systems involved in the release of sperm-activating and sperm-attracting factor (SAAF), which is the sperm attractant of the ascidian Ciona intestinalis. Here, we show that the egg acquires sperm-attracting activity after germinal vesicle breakdown. Further, since the cytoplasmic extracts of immature oocytes exhibit no sperm-attracting activity, the SAAF in oocytes may be activated after germinal vesicle breakdown. We found 13 SAAF-binding proteins in an egg plasma membrane extract and identified five proteins by proteomic analysis: valosin-containing protein (VCP)/p97, proteasome alpha 2 subunit, MGC97756 protein, proteasome subunit Y, and beta-tubulin. In particular, the interaction between VCP/p97 and SAAF was confirmed by a pull-down assay. VCP/p97 is initially localized in the germinal vesicle, and during oocyte maturation, it shifts to the endoplasmic reticulum in the cortical regions. Thus, VCP/p97 is a potential modulator of SAAF release from the egg.

  12. Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation

    NARCIS (Netherlands)

    Saadi, H.A.S.; Riemsdijk, van E.L.C.; Dance, A.L.; Rajamanickam, G.D.; Kastelic, J.P.; Thundathil, J.C.

    2013-01-01

    The objective was to investigate expression patterns of proteins in pyriform sperm, a common morphological abnormality in bull sperm. Ejaculates were collected from sexually mature Holstein bulls (n = 3) twice weekly for 10 weeks (pre-thermal insult samples). Testicular temperature was elevated in a

  13. Protein phosphatase PP1γ2 in sperm morphogenesis and epididymal initiation of sperm motility

    Institute of Scientific and Technical Information of China (English)

    Rumela Chakrabarti; Lina Cheng; Pawan Puri; David Soler; Srinivasan Viiayaraghavan

    2007-01-01

    The serine/threonine phosphatase (PP1) isoform PP1γ2, predominantly expressed in the testis, is a key enzyme in spermatozoa. High PP1γ2 catalytic activity holds motility in check in immature spermatozoa. Inhibition of PP1γ2 causes motility initiation in immature spermatozoa and motility stimulation and changes in flagellar beat parameters in mature spermatozoa. The PP1γ2 isoform is present in all mammalian spermatozoa studied: mouse, rat, hamster,bovine, non-human primate and man. We have now identified at least four of its regulatory proteins that regulate distinct pools of PP1γ2 within spermatozoa. Our studies provide new insights into biochemical mechanisms underlying development and regulation of sperm motility. We hypothesize that changes in sperm PP1γ2 activity as a result of phosphorylation and reversible binding of the regulatory proteins to the catalytic subunit are critical in the development and regulation of motility and the ability of sperm to fertilize eggs. Targeted disruption of the Ppp1cc gene,which encodes the PP1γ1 or PP1γ2 isoforms, causes male infertility in mice as a result of impaired spermiogenesis.Our observations suggest that, in addition to motility, the protein phosphatase PP1γ2 might play an isoform-specific function in the development of specialized flagellar structures of mammalian spermatozoa.

  14. Heat shock protein 90 has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-responsive sperm function in human sperm.

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    Kun Li

    Full Text Available Heat shock protein 90 plays critical roles in client protein maturation, signal transduction, protein folding and degradation, and morphological evolution; however, its function in human sperm is not fully understood. Therefore, our objective in this study was to elucidate the mechanism by which heat shock protein 90 exerts its effects on human sperm function. By performing indirect immunofluorescence staining, we found that heat shock protein 90 was localized primarily in the neck, midpiece, and tail regions of human sperm, and that its expression increased with increasing incubation time under capacitation conditions. Geldanamycin, a specific inhibitor of heat shock protein 90, was shown to inhibit this increase in heat shock protein 90 expression in western blotting analyses. Using a multifunctional microplate reader to examine Fluo-3 AM-loaded sperm, we observed for the first time that inhibition of heat shock protein 90 by using geldanamycin significantly decreased intracellular calcium concentrations during capacitation. Moreover, western blot analysis showed that geldanamycin enhanced tyrosine phosphorylation of several proteins, including heat shock protein 90, in a dose-dependent manner. The effects of geldanamycin on human sperm function in the absence or presence of progesterone was evaluated by performing chlortetracycline staining and by using a computer-assisted sperm analyzer. We found that geldanamycin alone did not affect sperm capacitation, hyperactivation, and motility, but did so in the presence of progesterone. Taken together, these data suggest that heat shock protein 90, which increases in expression in human sperm during capacitation, has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-stimulated sperm function. In this study, we provide new insights into the roles of heat shock protein 90 in sperm function.

  15. Functional features and protein network of human sperm-egg interaction.

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    Sabetian, Soudabeh; Shamsir, Mohd Shahir; Abu Naser, Mohammed

    2014-12-01

    Elucidation of the sperm-egg interaction at the molecular level is one of the unresolved problems in sexual reproduction, and understanding the molecular mechanism is crucial in solving problems in infertility and failed in vitro fertilization (IVF). Many molecular interactions in the form of protein-protein interactions (PPIs) mediate the sperm-egg membrane interaction. Due to the complexity of the problem such as difficulties in analyzing in vivo membrane PPIs, many efforts have failed to comprehensively elucidate the fusion mechanism and the molecular interactions that mediate sperm-egg membrane fusion. The main purpose of this study was to reveal possible protein interactions and associated molecular function during sperm-egg interaction using a protein interaction network approach. Different databases have been used to construct the human sperm-egg interaction network. The constructed network revealed new interactions. These included CD151 and CD9 in human oocyte that interact with CD49 in sperm, and CD49 and ITGA4 in sperm that interact with CD63 and CD81, respectively, in the oocyte. These results showed that the different integrins in sperm may be involved in human sperm-egg interaction. It was also suggested that sperm ADAM2 plays a role as a protein candidate involved in sperm-egg membrane interaction by interacting with CD9 in the oocyte. Interleukin-4 receptor activity, receptor signaling protein tyrosine kinase activity, and manganese ion transmembrane transport activity are the major molecular functions in sperm-egg interaction protein network. The disease association analysis indicated that sperm-egg interaction defects are also reflected in other disease networks such as cardiovascular, hematological, and breast cancer diseases. By analyzing the network, we identified the major molecular functions and disease association genes in sperm-egg interaction protein. Further experimental studies will be required to confirm the significance of these new

  16. Sperm and spermatids contain different proteins and bind distinct egg factors.

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    Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J; Gurdon, John B; Jullien, Jerome

    2014-09-19

    Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.

  17. Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse spermatozoa.

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    Vicens, Alberto; Lüke, Lena; Roldan, Eduardo R S

    2014-01-01

    Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm's life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

  18. Identification of calcium-binding proteins associated with the human sperm plasma membrane

    National Research Council Canada - National Science Library

    Naaby-Hansen, Soren; Diekman, Alan; Shetty, Jagathpala; Flickinger, Charles J; Westbrook, Anne; Herr, John C

    2010-01-01

    The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation...

  19. A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization.

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    Xu, X-Z Shawn; Sternberg, Paul W

    2003-08-08

    Fertilization, a critical step in animal reproduction, is triggered by a series of specialized sperm-egg interactions. However, the molecular mechanisms underlying fertilization are not well understood. Here, we identify a sperm-enriched C. elegans TRPC homolog, TRP-3. Mutations in trp-3 lead to sterility in both hermaphrodites and males due to a defect in their sperm. trp-3 mutant sperm are motile, but fail to fertilize oocytes after gamete contact. TRP-3 is initially localized in intracellular vesicles, and then translocates to the plasma membrane during sperm activation. This translocation coincides with a marked increase in store-operated calcium entry, providing an in vivo mechanism for the regulation of TRP-3 activity. As C. elegans oocytes lack egg coats, our data suggest that some TRPC family channels might function to mediate calcium influx during sperm-egg plasma membrane interactions leading to fertilization.

  20. Molecular kinetics of proteins at the surface of porcine sperm before and during fertilization.

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    Tsai, P S; Gadella, B M

    2009-01-01

    Fertilization is a decisive moment in life and enables the combination of the DNA from two gametes to ultimately form a new organism. The sperm surface, especially the head area, has distinguishable subdomains that are involved in distinct fertilization processes. It is known that the sperm head surface undergoes constant remodelling during epididymal maturation and migration in the male and female genital tract. But intriguingly, the identity, origin and spatial ordering of proteins at the sperm surface that are involved in mammalian fertilization are essentially unknown. This review deals with sperm surface protein modifications that are under somatic cell control. As soon as the sperm is released from the seminiferous tubules it is subjected to these modifications. These surface reorganisations continue until the sperm reside in the fallopian tube where they meet the oocyte and may fertilize it. Most likely, a selective process allows only functionally mature and intact sperm to optimally interact and fertilize the oocyte. Recent data suggest that even the perivitelline fluid is involved in sperm surface remodelling as it contains factors which could facilitate the first penetrating sperm to fertilize the oocyte. In this contribution, the kinetics of proteins at the sperm surface will be overviewed. Better understanding of this would help to design strategies to improve male fertility or to devise novel contraceptives.

  1. Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane

    Directory of Open Access Journals (Sweden)

    C.E.A. Souza

    2011-06-01

    Full Text Available The present study was designed to investigate the topographical distribution of seminal plasma (SP proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.

  2. Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

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    Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

    2005-08-01

    This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

  3. Evolutionary conservation of mammalian sperm proteins associates with overall, not tyrosine, phosphorylation in human spermatozoa.

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    Schumacher, Julia; Ramljak, Sanja; Asif, Abdul R; Schaffrath, Michael; Zischler, Hans; Herlyn, Holger

    2013-12-06

    We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.

  4. Boar seminal plasma exosomes: effect on sperm function and protein identification by sequencing.

    Science.gov (United States)

    Piehl, Lidia L; Fischman, M Laura; Hellman, Ulf; Cisale, Humberto; Miranda, Patricia V

    2013-04-15

    Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a

  5. Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

    Science.gov (United States)

    Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

    2015-01-01

    The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

  6. Sperm protein 17 is expressed in human nervous system tumours

    Directory of Open Access Journals (Sweden)

    Frezza Eldo E

    2006-01-01

    Full Text Available Abstract Background Human sperm protein 17 (Sp17 is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. Methods The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma, 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma,. Results A number of neuroectodermal (21% and meningeal tumours (4% were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. Conclusion The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells.

  7. Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm-zona pellucida receptors.

    Science.gov (United States)

    Zigo, Michal; Dorosh, Andriy; Pohlová, Alžběta; Jonáková, Věra; Šulc, Miroslav; Maňásková-Postlerová, Pavla

    2015-03-01

    Primary binding of the sperm to the zona pellucida (ZP) is one of the many steps necessary for successful fertilization. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-defined sequential process. Primary binding receptors, many of which have been disclosed in various mammals, are localized throughout the acrosomal region of the sperm surface. A panel of monoclonal antibodies against proteins from the sperm surface was prepared. Antibodies were screened by immunofluorescence for protein localization and Western blotting. Proteins localized on the sperm head and simultaneously detected by Western blotting were further studied in terms of immunolocalization in reproductive tissues and fluids, binding to ZP, immunoprecipitation and sequencing. Of 17 prepared antibodies, 8 recognized proteins localized on the sperm head and also detected proteins of interest by Western blotting. Only three other antibodies recognized proteins that also coincided in binding to ZP. These three antibodies were used for immunoprecipitation, and further protein sequencing of immunoprecipitates revealed that these antibodies distinguished acrosin precursor, RAB-2A protein, and lactadherin P47. This is not the first time we have detected acrosin on the surface of ejaculated and capacitated sperm. However, to our knowledge, this is the first time RAB-2A has been detected on the sperm surface. Lactadherin P47 has already been characterized and its physiological function in reproduction has been proposed.

  8. Detection of boar sperm plasma membrane protein using Rhodamine 640; implications for cryobiology and physiology

    Science.gov (United States)

    Rhodamine 640 (R640) was used to detect changes in boar sperm plasma membrane protein (PMP) during cryopreservation; a poorly understood phenomenon. The protocol was adapted for boar sperm so that semen samples (n = 17) could be analyzed for PMP (R640 positive) and plasma membrane integrity (PMI; Y...

  9. Sperm proteome of Mytilus galloprovincialis: Insights into the evolution of fertilization proteins in marine mussels.

    Science.gov (United States)

    Zhang, Yanjie; Mu, Huawei; Lau, Stanley C K; Zhang, Zhifeng; Qiu, Jian-Wen

    2015-12-01

    Cataloging the sperm proteome of an animal can improve our understanding of its sperm-egg interaction and speciation, but such data are available for only a few free-spawning invertebrates. This study aimed to identify the sperm proteome of Mytilus galloprovincialis, a free-spawning marine mussel. We integrated public transcriptome datasets by de novo assembly, and applied SDS-PAGE coupled LC-MS/MS analysis to profile the sperm proteome, resulting in the identification of 550 proteins. Comparing the homologous sperm protein coding genes between M. galloprovincialis and its closely related species M. edulis revealed that fertilization proteins have the highest mean nonsynonymous substitution rate (Ka/Ks = 0.62) among 11 functional groups, consistent with previous reports of positive selection of several fertilization proteins in Mytilus. Moreover, 78 sperm proteins in different functional groups have Ka/Ks values > 0.5, indicating the presence of many candidate sperm proteins for further analysis of rapid interspecific divergence. The MS data are available in ProteomeXchange with the identifier PXD001665.

  10. D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.

    Science.gov (United States)

    Leahy, T; Rickard, J P; Aitken, R J; de Graaf, S P

    2016-05-01

    Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

  11. Equatorial segment protein (ESP) is a human alloantigen involved in sperm-egg binding and fusion.

    Science.gov (United States)

    Wolkowicz, M J; Digilio, L; Klotz, K; Shetty, J; Flickinger, C J; Herr, J C

    2008-01-01

    The equatorial segment of the sperm head is known to play a role in fertilization; however, the specific sperm molecules contributing to the integrity of the equatorial segment and in binding and fusion at the oolemma remain incomplete. Moreover, identification of molecular mediators of fertilization that are also immunogenic in humans is predicted to advance both the diagnosis and treatment of immune infertility. We previously reported the cloning of Equatorial Segment Protein (ESP), a protein localized to the equatorial segment of ejaculated human sperm. ESP is a biomarker for a subcompartment of the acrosomal matrix that can be traced through all stages of acrosome biogenesis (Wolkowicz et al, 2003). In the present study, ESP immunoreacted on Western blots with 4 (27%) of 15 antisperm antibody (ASA)-positive serum samples from infertile male patients and 2 (40%) of 5 ASA-positive female sera. Immunofluorescent studies revealed ESP in the equatorial segment of 89% of acrosome-reacted sperm. ESP persisted as a defined equatorial segment band on 100% of sperm tightly bound to the oolemma of hamster eggs. Antisera to recombinant human ESP inhibited both oolemmal binding and fusion of human sperm in the hamster egg penetration assay. The results indicate that ESP is a human alloantigen involved in sperm-egg binding and fusion. Defined recombinant sperm immunogens, such as ESP, may offer opportunities for differential diagnosis of immune infertility.

  12. Location of the Protein of RSG6,a Predominantly Expressed Gene in Rice Sperm Cells

    Institute of Scientific and Technical Information of China (English)

    Lan Li-qiong; Miao chen; Zeng yu; Wang Sheng-hua; Xu ying; Tang Lin; BAI Yu; Chen Fang

    2004-01-01

    Using Western blot and immunohistochemistry analysis, here the localization of RSG6 protein was determined in various tissues of rice. Western blot showed only a weak signal in mature pollen. Nevertheless, according to the result of immunohistochemistry with DAB and fluorescent staining, the expression of RSG6 protein appeared to begin at the bicellular microspore stage, and then keep activity in the sperm cells of mature pollens. The fluorescence pattern showed RSG6 polypeptide was present close to or attached to the surface of the isolated sperm cells. And this suggested that RSG6 might take an important part in the process of recognition of sperm cell and ovum.

  13. Membrane proteins associated with sperm-oocyte interaction: A proteomic comparison between Kedah Kelantan (Bos indicus) and Mafriwal (Bos taurus × Bos indicus) sperm

    Science.gov (United States)

    Ashrafzadeh, Ali; Nathan, Sheila; Othman, Iekhsan; Yee, Tee Ting; Karsani, Saiful Anuar

    2013-11-01

    Production performance of European cattle breeds has significantly improved through various breeding programs. However, European breeds are more susceptible to heat stress compared to zebu cattle (Bos indicus) as their conception rate can range between 20 to 30% in hot seasons compared to winter. To identify cattle sperm proteins associated with zebu cattle higher fertility and heat tolerance in tropical environments, we utilised a proteomics-based approach to compare sperm from the highly fertile Malaysian indigenous breed, Kedah Kelantan (Bos indicus), with sperm from the sub-fertile crossbreed, Mafriwal (Bos taurus × Bos indicus). Frozen semen of three high performance bulls from each breed was processed to obtain live and pure sperm. Proteins were separated and gel bands were processed by in-gel tryptic digestion. For each breed, mass spectrometry data was acquired over 11 replicates. The analyzed data identified peptides with different expression levels (99% confidence level) and protein identification was determined by targeted MS/MS. Among the identified proteins associated with sperm-oocyte interaction, two proteins were up-regulated in Kedah Kelantan sperm and 7 proteins were up-regulated in or specific to Mafriwal. Our results suggest that the higher fertility of zebu cattle in tropical areas may not be related to more efficient sperm-oocyte interaction. Further analysis of the other regulated proteins in these two breeds may contribute further knowledge on the physiological reason/s for higher fertility and heat tolerance of Zebu cattle in tropical areas.

  14. Effect of Pseudomonas aeruginosa on sperm capacitation and protein phosphorylation of boar spermatozoa.

    Science.gov (United States)

    Sepúlveda, Lilian; Bussalleu, Eva; Yeste, Marc; Bonet, Sergi

    2016-05-01

    Several studies have reported the detrimental effects that bacteriospermia causes on boar sperm quality, but little is known about its effects on IVC. Considering that, the present study sought to evaluate the effects of different concentrations of Pseudomonas aeruginosa on different indicators of capacitation status (sperm viability, membrane lipid disorder, sperm motility kinematics, and protein phosphorylation of boar spermatozoa) after IVC. Flow cytometry and computer assisted sperm analysis (CASA) revealed that the presence of P aeruginosa in boar sperm samples, mostly at concentrations greater than 10(6) CFU/mL, is associated with a significant (P sperm membrane integrity and sperm with low membrane lipid disorder, and also with a reduction in sperm motility kinetic parameters when compared with results obtained from the control sample, which presented the typical motility pattern of capacitated-like boar spermatozoa. Moreover, Western blot results also showed significant (P boar sperm, being the most relevant. Indeed, after 3 hours of IVC, phosphotyrosine levels of p32 in the control sample were 3.13 ± 0.81, whereas in the tubes with 10(6) and 10(8) CFU/mL were 1.05 ± 0.20 and 0.36 ± 0.07, respectively. Therefore, the present study provides novel data regarding the effects of bacterial contamination on boar sperm, suggesting that the presence of P aeruginosa affects the fertilizing ability of boar sperm by altering its ability to accomplish IVC.

  15. Protein differences between normal and oligospermic human sperm demonstrated by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Morgentaler, A; Schopperle, W M; Crocker, R H; DeWolf, W C

    1990-11-01

    Protein expression by sperm obtained from men with normal semen analysis and men with oligospermia were evaluated by two-dimensional gel electrophoresis. Proteins were solubilized in a 9.5 M urea/2% Nonidet-P40 (LKB, Bromma, Sweden) lysis buffer and underwent second dimension separation on 10 to 16% polyacrylamide gradient gels. A set of 36 invariant proteins was identified in all normospermic samples, whereas 8 of 10 evaluable oligospermic samples lacked 1 or more of the invariant proteins. Proteins absent in oligospermic samples may be critical to normal sperm function and may serve as markers for infertility.

  16. Modeling how reproductive ecology can drive protein diversification and result in linkage disequilibrium between sperm and egg proteins.

    Science.gov (United States)

    Tomaiuolo, Maurizio; Levitan, Don R

    2010-07-01

    Gamete-recognition proteins determine whether sperm and eggs are compatible at fertilization, and they often evolve rapidly. The source of selection driving the evolution of these proteins is still debated. It has been suggested that sexual conflict can result in proliferation of genetic variation and possibly linkage disequilibrium between sperm and egg proteins. Empirical evidence suggests that both male and female reproductive success can be predicted by their sperm ligand genotype, but why female success can be predicted by a protein expressed only in males is unknown. Here we use mathematical modeling to investigate the interaction between reproductive behavior and sperm availability on the evolution of sperm ligands and egg receptors. We consider haploid and diploid expression in gametes in two possible ecological scenarios, monogamous spawning and competitive spawning. Reproductive behavior plays an important role in determining possible outcomes resulting from sexual conflict. Sperm limitation selects for common genotypes regardless of mating behavior. Under conditions of sperm abundance, competitive spawning provides conditions for the persistence of allelic variation and gametic disequilibrium. With monogamous spawning, such conditions are more restrictive.

  17. Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Jana Capkova

    2016-01-01

    Full Text Available Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs, which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A, evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

  18. Investigation of Function of Novel Sperm Binding Protein HBRP in Human

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate the biology function of novel protein related to bovie seminal plasma protein in human testis.Methods Recombination pcDNA3/HBRP was constructed and transfected to HEK293 cell and permanently expression cell line was established.The activity of protein kinase C (PKC) of the cell line was detected by autoradiography method.Results The stable expression cell line of HBRP was obtained.The HBRP inhibited the activity of PKC significantly.Conclusion One of the newfunctions of novel sperm binding protein in human is the inhibitor action on activity of PKC.It may be involved in the sperm capacitation,and acrosome reaction.

  19. Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

    Science.gov (United States)

    Ledesma, Alba; Zalazar, Lucía; Fernández-Alegre, Estela; Hozbor, Federico; Cesari, Andreina; Martínez-Pastor, Felipe

    2017-09-01

    Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as "hyperactivated" (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Antiidiotypic antibody related to the 84 kD human sperm membrane protein

    Institute of Scientific and Technical Information of China (English)

    YUJUN; WANGLINFANG; 等

    1990-01-01

    Wistar rats were inoculated with purified YWK-I antibody.The anti-idiotypic antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs.Specificity of anti-Id antibody was established by ELISA.The 84kD protein inhibited the binding of anti-Id to YWK-I mAb,but failed to repress antibody against normal mouse Ig binding to YWK-I mAb.In competitive inhibition assay,84kD protein had shown the ability to compete with anti-Id binding to YWK-I mAb in a dose-dependent manner.Crude sperm extract showed a lower competitive ability.No effect was found with the irrelevant 36kD sperm protein.The antisera from the Balb/C micr immunized with AId contained Ab3 that reacted with 84kD sperm protein.The binding of anti-Id to YWK-I mAb was inhibited by Ab3 in a dose-dependent fashion and Ab3 was shown to be able to induce human sperm agglutination.These results indicate that anti-Id which may mimic an epitope of the 84kD protein could be exploited as an antigen to raise antibodies against sperm protein.

  1. Hamster oviductin regulates tyrosine phosphorylation of sperm proteins during in vitro capacitation.

    Science.gov (United States)

    Saccary, Laurelle; She, Yi-Min; Oko, Richard; Kan, Frederick W K

    2013-08-01

    Oviductin or OVGP1, also known as oviduct-specific glycoprotein, has been shown to enhance sperm capacitation in addition to its other beneficial effects on fertilization and early embryo development. We hypothesized that estrus stage-specific hamster oviductin (eHamOVGP1) can potentiate the enhancement of tyrosine phosphorylation of sperm proteins during capacitation. Immunofluorescent staining and confocal microscopy as well as immunocytochemistry and surface replica technique localized tyrosine-phosphorylated proteins to the equatorial segment and midpiece after incubation of hamster sperm in capacitation medium in the presence or absence of eHamOVGP1. Increase of tyrosine phosphorylation level in the equatorial segment occurred as early as 5 min after incubation in the presence of eHamOVGP1. Immunostaining for eHamOVGP1 further increased upon prolonged incubation of sperm in medium containing the glycoprotein. Regardless of the presence or absence of eHamOVGP1, phosphotyrosine expression was observed along the tail, particularly at the midpiece. Western blotting of NP40-extracted sperm proteins (25, 37, and 44 kDa) and NP40-non-extractable sperm proteins (70, 83, 90 kDa) showed increased immunolabeling intensity after 5, 60, 120, and 180 min of capacitation in the presence of eHamOVGP1. Mass spectrometric analysis identified several proteins of functions known to be involved in metabolic pathways responsible for enhancement of tyrosine phosphorylation in its presence. The present investigation provides evidence that eHamOVGP1 regulates the expression of protein tyrosine phosphorylation in sperm capacitated in vitro, further supporting an important role of the presence of OVGP1 in the oviductal milieu during the process of fertilization.

  2. Yolk protein is expressed in the insect testis and interacts with sperm

    Directory of Open Access Journals (Sweden)

    Joachimiak Ewa

    2008-06-01

    Full Text Available Abstract Background Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, Spodoptera littoralis, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation. Results We collected seminal fluid proteins from males before and after daily sperm release. These samples were separated by 2-D gel electrophoresis, and gels were treated with a glycoprotein-detecting probe. We observed a group of abundant glycoproteins in the sample collected after sperm release, which was absent in the sample collected before sperm release. Sequencing of these glycoproteins by mass spectroscopy revealed peptides bearing homology with components of yolk, which is known to accumulate in developing oocytes. This unexpected result was confirmed by Western blotting demonstrating that seminal fluid contains protein immunoreactive to antibody against yolk protein YP2 produced in the follicle cells surrounding developing oocytes. We cloned the fragment of yp2 cDNA from S. littoralis and determined that it is expressed in both ovaries and testes. yp2 mRNA and YP2 protein were detected in the somatic cyst cells enveloping sperm inside the testis. During the period of sperm

  3. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    Science.gov (United States)

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads; Ballescà, Josep Lluís; Tommerup, Niels; Oliva, Rafael; Vavouri, Tanya

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline. PMID:25904136

  4. Comparative analysis of Mafriwal (Bos taurus × Bos indicus) and Kedah Kelantan (Bos indicus) sperm proteome identifies sperm proteins potentially responsible for higher fertility in a tropical climate.

    Science.gov (United States)

    Ashrafzadeh, Ali; Nathan, Sheila; Karsani, Saiful Anuar

    2013-07-30

    The fertility of zebu cattle (Bos indicus) is higher than that of the European purebred (Bos taurus) and crossbred (Bos taurus × Bos indicus) cattle in tropical areas. To identify proteins related to the higher thermo-tolerance and fertility of Zebu cattle, this study was undertaken to identify differences in sperm proteome between the high fertile Malaysian indigenous zebu cattle (Kedah Kelantan) and the sub-fertile crossbred cattle (Mafriwal). Frozen semen from three high performance bulls from each breed were processed to obtain live and pure sperm. Sperm proteins were then extracted, and two-dimensional gel electrophoresis performed to compare proteome profiles. Gel image analysis identified protein spots of interest which were then identified by liquid chromatography mass spectrometry quadrupole time-of-flight (LC MS/MS Q-TOF). STRING network analysis predicted interactions between at least 20 of the identified proteins. Among the identified proteins, a number of motility and energy related proteins were present in greater abundance in Kedah Kelantan. Sperm motility evaluation by Computer Assisted Semen Analysis (CASA) confirmed significantly higher motility in Kedah Kelantan. While results from this study do identify proteins that may be responsible for the higher fertility of Kedah Kelantan, functional characterization of these proteins is warranted to reinforce our understanding of their roles in sperm fertility.

  5. Heterogenous turnover of sperm and seminal vesicle proteins in the mouse revealed by dynamic metabolic labeling.

    Science.gov (United States)

    Claydon, Amy J; Ramm, Steven A; Pennington, Andrea; Hurst, Jane L; Stockley, Paula; Beynon, Robert

    2012-06-01

    Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.

  6. Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa.

    Science.gov (United States)

    Zhen, Linqing; Wang, Lirui; Fu, Jieli; Li, Yuhua; Zhao, Na; Li, Xinhong

    2016-01-01

    Hexavalent chromium reportedly induces reproductive toxicity and further inhibits male fertility in mammals. In this study, we investigated the molecular mechanism by which hexavalent chromium affects motility signaling in boar spermatozoa in vitro. The results indicated that Cr(VI) decreased sperm motility, protein phosphorylation, mitochondrial membrane potential (ΔΨm) and metabolic enzyme activity starting at 4μmol/mL following incubation for 1.5h. Notably, all parameters were potently inhibited by 10μmol/mL Cr, while supplementation with the dibutyryl-cAMP (dbcAMP) and the 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibition of protein phosphorylation. Interestingly, high concentrations of Cr (>10μmol/mL) increased the tyrosine phosphorylation of some high-molecular-weight proteins in the principle piece but decreased that in the middle piece associated with an extreme reduction of sperm motility. These results suggest that chromium affects boar sperm motility by impairing tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro.

  7. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

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    Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

    2015-07-01

    Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

  8. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

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    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC. SP22, a rat sperm membrane protein that is highly-correlated w...

  9. Boar sperm quality in relation to presence of sp32-like protein in spermatozoa - preliminary studies

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    Orzołek Aleksandra

    2015-06-01

    Full Text Available The aim of the study was to analyse sperm proteomes of ejaculates from Polish Large White (PLW and Polish Landrace (PL boars and to identify differences which putatively influence semen quality. Spermatozoa protein profiles were analysed by electrophoretic methods followed by selected techniques to evaluate semen quality on the following factors: sperm motility, lipid peroxidation levels (MDA production, ATP content, activities of superoxide dismutase (SOD and catalase (CAT, total antioxidant status (TAS, and total oxidant status (TOS of seminal plasma. A protein with an estimated molecular weight of 30 kDa was found in spermatozoa of selected ejaculates. Mass spectrometry demonstrated that this polypeptide is most similar to proacrosin binding protein (sp32. The presence of the protein was more frequently observed in sperm extracts obtained in spring-summer period. Ejaculates containing sp32-like protein demonstrated significantly higher spermatozoa motility, lower inhibition of MDA production by seminal plasma, and higher SOD activity in seminal plasma. Boar semen which included sp32-like protein also demonstrated lower ATP levels in spermatozoa as well as higher TAS and lower TOS of seminal plasma, though the differences were not statistically significant. Ejaculates from PLW boars, with sp32-like protein present in sperm, were characterised by significantly higher sperm motility, lower ATP content in spermatozoa, and higher TAS of seminal plasma. The diminished parameters of semen quality were observed in ejaculates from PL boars that also contained the discussed protein, but the differences were not statistically significant. These findings suggest that the presence of sp32-like protein in boar spermatozoa could influence semen quality

  10. Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma.

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    Intasqui, Paula; Camargo, Mariana; Del Giudice, Paula T; Spaine, Deborah M; Carvalho, Valdemir M; Cardozo, Karina H M; Zylbersztejn, Daniel S; Bertolla, Ricardo P

    2013-10-01

    To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post-genomic pathways associated with sperm DNA fragmentation. A cross-sectional study including 89 subjects from a human reproduction service was carried out. All semen samples were assessed for sperm DNA fragmentation using a comet assay. Results from 60 sperm were analysed using Komet 6.0.1 software and the 'Olive tail moment' variable was used to stratify these into low and high sperm DNA fragmentation groups. Seminal plasma proteins from the two groups were pooled and used for proteomic analysis. Quantitative data were used for functional enrichment studies. Seventy-two proteins were identified or quantified in seminal plasma. Of these, nine were differentially expressed in the low group and 21 in the high group. Forty-two proteins were conserved between these groups. Functional enrichment analysis indicated that sperm DNA fragmentation was related to functions such as lipoprotein particle remodelling and regulation, fatty acid binding and immune response. Proteins found exclusively in the low group may be involved in correcting spermatogenesis and/or improving sperm function. Proteins in the high group were associated with increased innate immune response, sperm motility and/or maturation and inhibition of mitochondrial apoptosis. Protein expression and post-genomic pathways of seminal plasma differ according to the rate of sperm DNA integrity. © 2013 The Authors. BJU International © 2013 BJU International.

  11. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

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    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  12. Tyrosine Phosphorylation Pattern in Sperm Proteins Isolated from Normospermic and Teratospermic Men

    Science.gov (United States)

    Jabbari, Sepideh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mahdi; Ebrahim Habibi, Azadeh; Amirjanati, Naser; Lakpour, Niknam; Asgharpour, Lima; Ardekani, Ali M.

    2009-01-01

    Introduction In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Materials and Methods Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the spermatozoa were incubated up to 6h at 37°C with 5% CO2 in 3% Bovine Serum Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. Results The results upon western blotting showed: 1) at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2) The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. Conclusion For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished

  13. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein.

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    Miwa, Naofumi

    2015-05-22

    Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP), which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

  14. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein

    Directory of Open Access Journals (Sweden)

    Naofumi Miwa

    2015-05-01

    Full Text Available Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP, which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

  15. Histones and nucleosomes in Cancer sperm (Decapod: Crustacea) previously described as lacking basic DNA-associated proteins: a new model of sperm chromatin.

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    Kurtz, Kathryn; Martínez-Soler, Fina; Ausió, Juan; Chiva, Manel

    2008-10-01

    To date several studies have been carried out which indicate that DNA of crustacean sperm is neither bound nor organized by basic proteins and, contrary to the rest of spermatozoa, do not contain highly packaged chromatin. Since this is the only known case of this type among metazoan cells, we have re-examined the composition, and partially the structure, of the mature sperm chromatin of Cancer pagurus, which has previously been described as lacking basic DNA-associated proteins. The results we present here show that: (a) sperm DNA of C. pagurus is bound by histones forming nucleosomes of 170 base pairs, (b) the ratio [histones/DNA] in sperm of two Cancer species is 0.5 and 0.6 (w/w). This ratio is quite lower than the proportion [proteins/DNA] that we found in other sperm nuclei with histones or protamines, whose value is from 1.0 to 1.2 (w/w), (c) histone H4 is highly acetylated in mature sperm chromatin of C. pagurus. Other histones (H3 and H2B) are also acetylated, though the level is much lower than that of histone H4. The low ratio of histones to DNA, along with the high level of acetylation of these proteins, explains the non-compact, decondensed state of the peculiar chromatin in the sperm studied here. In the final section we offer an explanation for the necessity of such decondensed chromatin during gamete fertilization of this species. (c) 2008 Wiley-Liss, Inc.

  16. Heat Shock Protein A2 (HSPA2): Regulatory Roles in Germ Cell Development and Sperm Function.

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    Nixon, Brett; Bromfield, Elizabeth G; Cui, Jinwei; De Iuliis, Geoffry N

    2017-01-01

    Among the numerous families of heat shock protein (HSP) that have been implicated in the regulation of reproductive system development and function, those belonging to the 70 kDa HSP family have emerged as being indispensable for male fertility. In particular, the testis-enriched heat shock 70 kDa protein 2 (HSPA2) has been shown to be critical for the progression of germ cell differentiation during spermatogenesis in the mouse model. Beyond this developmentally important window, mounting evidence has also implicated HSPA2 in the functional transformation of the human sperm cell during their ascent of the female reproductive tract. Specifically, HSPA2 appears to coordinate the remodelling of specialised sperm domains overlying the anterior region of the sperm head compatible with their principle role in oocyte recognition. The fact that levels of the HSPA2 protein in mature spermatozoa tightly correlate with the efficacy of oocyte binding highlight its utility as a powerful prognostic biomarker of male fertility. In this chapter, we consider the unique structural and biochemical characteristics of HSPA2 that enable this heat shock protein to fulfil its prominent roles in orchestrating the morphological differentiation of male germ cells during spermatogenesis as well as their functional transformation during post-testicular sperm maturation.

  17. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

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    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  18. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

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    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  19. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

    Directory of Open Access Journals (Sweden)

    Pinki Nandi

    Full Text Available Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14 detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  20. Chronic cyclophosphamide exposure alters the profile of rat sperm nuclear matrix proteins.

    Science.gov (United States)

    Codrington, Alexis M; Hales, Barbara F; Robaire, Bernard

    2007-08-01

    Chronic exposure of male rats to the alkylating agent cyclophosphamide, a well-known male-mediated developmental toxicant, alters gene expression in male germ cells as well as in early preimplantation embryos sired by cyclophosphamide-exposed males. Sperm DNA is organized by the nuclear matrix into loop-domains in a sequence-specific manner. In somatic cells, loop-domain organization is involved in gene regulation. Various structural and functional components of the nuclear matrix are targets for chemotherapeutic agents. Consequently, we hypothesized that cyclophosphamide treatment would alter the expression of sperm nuclear matrix proteins. Adult male rats were treated for 4 wk with saline or cyclophosphamide (6.0 mg kg(-1) day(-1)), and the nuclear matrix was extracted from cauda epididymal sperm. Proteins were analyzed by two-dimensional gel electrophoresis. Identified proteins within the nuclear matrix proteome were mainly involved in cell structure, transcription, translation, DNA binding, protein processing, signal transduction, metabolism, cell defense, or detoxification. Interestingly, cyclophosphamide selectively induced numerous changes in cell defense and detoxification proteins, most notably, in all known forms of the antioxidant enzyme glutathione peroxidase 4, in addition to an uncharacterized 54-kDa form; an overall increase in glutathione peroxidase 4 immunoreactivity was observed in the nuclear matrix extracts from cyclophosphamide-exposed spermatozoa. An increase in glutathione peroxidase 4 expression suggests a role for this enzyme in maintaining nuclear matrix stability and function. These results led us to propose that a change in composition of the nuclear matrix in response to drug exposure was a factor in altered sperm function and embryo development.

  1. LYZL6, an acidic, bacteriolytic, human sperm-related protein, plays a role in fertilization

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    Huang, Peng; Li, Wenshu; Yang, Zhifang; Zhang, Ning; Xu, Yixin; Bao, Jianying; Jiang, Deke; Dong, Xianping

    2017-01-01

    Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/α-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity against Micrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent manner in vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract. PMID:28182716

  2. Heterologous recombinant protein with decapacitating activity prevents and reverts cryodamage in ram sperm: An emerging biotechnological tool for cryobiology.

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    Zalazar, L; Ledesma, A; Hozbor, F; Cesari, A

    2016-01-01

    During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize.

  3. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

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    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  4. Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

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    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-03-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome

  5. Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity.

    Science.gov (United States)

    Vilagran, Ingrid; Castillo, Judit; Bonet, Sergi; Sancho, Sílvia; Yeste, Marc; Estanyol, Josep M; Oliva, Rafael

    2013-09-15

    Sperm cryopreservation is the most efficient method for storing boar sperm samples for a long time. However, one of the inconveniences of this method is the large variation between and within boars in the cryopreservation success of their sperm. The aim of the present work was thus to find reliable and useful predictive biomarkers of the good and poor capacity to withstand the freeze-thawing process in boar ejaculates. To find these biomarkers, the amount of proteins present in the total proteome in sperm cells were compared between good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) using the two-dimensional difference gel electrophoresis technique. Samples were classified as GFE and PFE using progressive motility and viability of the sperm at 30 and 240 minutes after thawing, and the proteomes from each group, before starting cryopreservation protocols, were compared. Because two proteins, acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI), presented the highest significant differences between GFE and PFE groups in two-dimensional difference gel electrophoresis assessment, Western blot analyses for ACRBP and TPI were also performed for validation. ACRBP normalized content was significantly lower in PFE than in GFE (P sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ACRBP and TPI can be used as markers of boar sperm freezability before starting the cryopreservation procedure, thereby avoiding unnecessary costs involved in this practice.

  6. Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

    Institute of Scientific and Technical Information of China (English)

    Hao-fei WANG; Zhu-qiong XIANG; Yi-xing WANG

    2003-01-01

    Objective To identify the sperm membrane proteins that are associated with antisperm antibodyMethods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody.Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5~6.2, 4.6,5.1,5.5~5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher.Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two-dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

  7. Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

    Science.gov (United States)

    Liu, Y; Qu, F; Cao, X; Chen, G; Guo, Q; Ying, X; Guo, W; Lu, L; Ding, Z

    2012-04-01

    Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

  8. Epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit

    Institute of Scientific and Technical Information of China (English)

    Robert Sullivan; Gilles Frenette; Julie Girouard

    2007-01-01

    During epididymal transit, spermatozoa acquire new proteins. Some of these newly acquired proteins behave as integral membrane proteins, including glycosylphosphatidylinositol (GPI)-anchored proteins. This suggests that the secreted epididymal proteins are transferred to spermatozoa by an unusual mechanism. Within the epididymal lumen,spermatozoa interact with small membranous vesicles named epididymosomes. Many proteins are associated with epididymosomes and the protein composition of these vesicles varies along the excurrent duct and differs from soluble intraluminal proteins. Some epididymosome-associated proteins have been identified and their functions in sperm maturation hypothesized. These include P25b, a zona pellucida binding protein, macrophage migration inhibitory factor, enzymes of the polyol pathway, HE5/CD52, type 5 glutathione peroxidase, and SPAM1 or PH-20. The electrophoretic patterns of proteins associated to epididymosomes are complex and some of these proteins are transferred to defined surface domains of epididymal spermatozoa. Epididymosomes collected from different epididymal segments interact differently with spermatozoa. This protein transfer from epididymosomes to spermatozoa is timedependent, temperature-dependent and pH-dependent, and is more efficient in the presence of zinc. Some proteins are segregated to lipid raft domains of epididymosomes and are selectively transferred to raft domains of the sperm plasma membrane. Some evidence is presented showing that epididymosomes are secreted in an apocrine manner by the epididymal epithelial cells. In conclusion, epididymosomes are small membranous vesicles secreted in an apocrine manner in the intraluminal compartment of the epididymis and play a major role in the acquisition of new proteins by the maturing spermatozoa.

  9. Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa.

    Science.gov (United States)

    Mariappa, Daniel; Aladakatti, Ravindranath H; Dasari, Santosh K; Sreekumar, Arun; Wolkowicz, Michael; van der Hoorn, Frans; Seshagiri, Polani B

    2010-02-01

    In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

  10. Expression of α-gustducin and α-transducin, G proteins coupled with taste receptors, in boar sperm.

    Science.gov (United States)

    Spinaci, M; Bucci, D; Mazzoni, M; Giaretta, E; Bernardini, C; Vallorani, C; Tamanini, C; Clavenzani, P; Galeati, G

    2014-07-01

    During the transit in the female genital tract, spermatozoa are exposed to an environment that varies in composition from the vagina to the oviduct. Because G proteins, α-gustducin and α-transducin, are accepted as markers of chemosensitive cells, this study was aimed at assessing whether these proteins are expressed in boar germ cells. Ejaculated sperm extracts were analyzed by Western blot, and indirect immunofluorescence was performed on testis sections, smears of epididymal and ejaculated spermatozoa, sperm cells after in vitro induction of capacitation and acrosome reaction (IVAR), and in sperm cells bound to zona pellucida during IVF. Based on immunoblot results, both G proteins are present in boar sperm. In the testicular tissue sections, α-gustducin and α-transducin positivity was recorded in the germinal cells near the tubular lumen, whereas no positive signal was evident in spermatogonia located in the outer region of the seminiferous tubules. α-Gustducin expression in epididymal and ejaculated spermatozoa was mainly detectable in both the acrosome and the principal piece of the tail, whereas α-transducin was confined to the acrosome and the midpiece. No changes after in vitro induction of capacitation and IVAR were observed, except for the disappearance of acrosomal positivity in reacted spermatozoa. In sperm bound to zona pellucida, the G protein signal was congruent with that observed in IVAR cells. To the best of our knowledge, this is the first description of α-transducin in mammalian sperm and the first description of α-gustducin in boar sperm. Further studies are needed to clarify the possible role of these G proteins in sperm physiology.

  11. Differential analysis of two-dimensional gel electrophoresis profiles of spermatozoa protein in human normal motility sperm and idiopathic asthenospermia

    Institute of Scientific and Technical Information of China (English)

    SHEN Shu-lin; HE Da-lin; LUO Yong; NING Liang

    2006-01-01

    Objective: To evaluate the application of two-dimensional electrophoresis in the research of differentially expressed proteins in the human asthenospermia. Methods: Two-dimensional gel electrophoresis was performed on 4 normal sperm samples from healthy men and 4 sperm samples from 4 asthenospermia patients. After silver staining, the differential expression proteins were analyzed by PDQuest 2D analysis software. Results: Six differential protein spots were identified. Four spots showed increased expression in the control gels compared with the patient gels. Conclusion: The protein profiles of differential expression between the normal spermatozoa and idiopathic asthenospermia were established and some differential proteins were found. The data of this study would establish the better fundament for further isolation and identification of differentially expressed proteins in human asthenospermia sperm.

  12. Mammalian sperm-egg fusion: the development of rat oolemma fusibility during oogenesis involves the appearance of binding sites for sperm protein "DE".

    Science.gov (United States)

    Cohen, D J; Munuce, M J; Cuasnicú, P S

    1996-07-01

    Rat epididymal protein DE mediates gamete fusion through complementary sites localized on the egg surface. To investigate whether these egg components are involved in the development of rat oolemma fusibility, both the presence of DE-binding components and the ability of the oolemma to fuse with sperm during oogenesis were examined. Localization of DE-complementary sites by indirect immunofluorescence revealed the absence of fluorescent labeling on growing oocytes with a diameter 50 microns. This localization of oolemma components changed progressively to a patchy distribution during maturation. Whereas sperm incorporation was observed only in maturing oocytes, the development of the Hoechst transfer technique to evaluate membrane fusion revealed that germinal vesicle oocytes with a diameter > 50 microns were already competent to fuse with sperm. The involvement of the DE-complementary sites in the oolemma fusibility of these oocytes was confirmed by the fact that the presence of DE during gamete coincubation significantly (p rat oolemma occurs during the growth period and involves the appearance of DE-binding components on the oocyte surface. This study provides novel information on the molecular mechanism by which the mammalian egg plasma membrane becomes competent to fuse with sperm during oogenesis.

  13. A Novel Cysteine Knot Protein for Enhancing Sperm Motility That Might Facilitate the Evolution of Internal Fertilization in Amphibians

    Science.gov (United States)

    Yokoe, Misato; Takayama-Watanabe, Eriko; Saito, Yoko; Kutsuzawa, Megumi; Fujita, Kosuke; Ochi, Haruki; Nakauchi, Yuni; Watanabe, Akihiko

    2016-01-01

    Internal fertilization ensures successful reproduction of tetrapod vertebrates on land, although how this mode of reproduction evolved is unknown. Here, we identified a novel gene encoding sperm motility-initiating substance (SMIS), a key protein for the internal fertilization of the urodele Cynops pyrrhogaster by Edman degradation of an isolated protein and subsequent reverse transcription polymerase chain reaction. The SMIS gene encoded a 150 amino-acid sequence including the cysteine knot (CK) motif. No gene with substantial similarity to the SMIS was in the data bank of any model organisms. An active site of the SMIS was in the C-terminal region of the 2nd loop of CK motif. A synthetic peptide including the active site sequence bound to the midpiece and initiated/enhanced the circular motion of C. pyrrhogaster sperm, which allows penetration of the egg jelly specialized for the internal fertilization of this species. The synthetic peptide bound to whole sperm of Rhacophorus arboreus and enhanced the rotary motion, which is adapted to propel the sperm through egg coat matrix specialized for arboreal reproduction, while it bound to the tip of head and tail of Bufo japonicus sperm, and enhanced the vibratory motion, which is suited to sperm penetration through the egg jelly specialized for the reproduction of that species in freshwater. The polyclonal antibody against the active site of the SMIS specifically bound to egg coat matrix of R. arboreus. These findings suggest that diversification of amphibian reproductive modes accompanies the specialization of egg coat and the adaptation of sperm motility to penetrate the specialized egg coat, and SMIS acts as the sperm motility enhancer of anurans and urodeles that might facilitate to adaptively optimize sperm motility for allowing the establishment of internal fertilization. PMID:27579691

  14. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro.

    Science.gov (United States)

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Zhao, Na; Zhen, Linqing; Fu, Jieli; Yang, Qiangzhen

    2016-09-01

    Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism.

  15. Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production

    Science.gov (United States)

    Honda, Keiichi; Takase, Hiroyuki; Ômura, Hisashi; Honda, Hiroshi

    2012-09-01

    How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with 15N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.

  16. Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

    Science.gov (United States)

    Kwon, Jun Tae; Ham, Sera; Jeon, Suyeon; Kim, Youil; Oh, Seungmin; Cho, Chunghee

    2017-01-01

    The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

  17. Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa.

    Science.gov (United States)

    Yamaguchi, Ryo; Fujihara, Yoshitaka; Ikawa, Masahito; Okabe, Masaru

    2012-07-01

    Eight kinds of gene-disrupted mice (Clgn, Calr3, Pdilt, Tpst2, Ace, Adam1a, Adam2, and Adam3) show impaired sperm transition into the oviducts and defective sperm binding to the zona pellucida. All of these knockout strains are reported to lack or show aberrant expression of a disintegrin and metallopeptidase domain 3 (ADAM3) on the sperm membrane. We performed proteomic analyses of the proteins of these infertile spermatozoa to clarify whether the abnormal function is caused exclusively by a deficiency in ADAM3 expression. Two proteins, named PMIS1 and PMIS2, were missing in spermatozoa from Clgn-disrupted mice. To study their roles, we generated two gene-disrupted mouse lines. Pmis1-knockout mice were fertile, but Pmis2-knockout males were sterile because of a failure of sperm transport into the oviducts. Pmis2-deficient spermatozoa also failed to bind to the zona pellucida. However, they showed normal fertilizing ability when eggs surrounded with cumulus cells were used for in vitro fertilization. Further analysis revealed that these spermatozoa lacked the ADAM3 protein, but the amount of PMIS2 was also severely reduced in Adam3-deficient spermatozoa. These results suggest that PMIS2 might function both as the ultimate factor regulating sperm transport into the oviducts and in modulating sperm-zona binding.

  18. The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition.

    Science.gov (United States)

    Nixon, Brett; Bromfield, Elizabeth G; Dun, Matthew D; Redgrove, Kate A; McLaughlin, Eileen A; Aitken, R John

    2015-01-01

    One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI), recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2), as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF) success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

  19. The role of the molecular chaperone heat shock protein A2 (HSPA2 in regulating human sperm-egg recognition

    Directory of Open Access Journals (Sweden)

    Brett Nixon

    2015-01-01

    Full Text Available One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI, recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2, as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

  20. Opisthorchis viverrini: analysis of the sperm-specific rhophilin associated tail protein 1-like.

    Science.gov (United States)

    Rattanachan, Sitthichon; Grams, Rudi; Tesana, Smarn; Smooker, Peter M; Grams, Suksiri Vichasri

    2014-12-01

    Concurrent deficiency of rhophilin associated tail protein (ROPN1) and ROPN1-like (ROPN1L) in mice causes structural abnormalities and immotility of sperm and thereby infertility. In the present research, ROPN1L of the human liver fluke Opisthorchis viverrini was molecularly characterized and showed unexpected potential as a diagnostic tool. ROPN1L transcripts were detected in 2-week-old juveniles by RT-PCR. Immunohistochemical analysis of the adult worm localized the protein in testis lobes, seminal vesicle and receptacle and immunoelectron microscopic analysis revealed its location on the tail of spermatozoa. Interestingly, sera of experimentally infected hamsters and sera of individuals suffering from opisthorchiasis showed reactivity to recombinant OvROPN1L (rOvROPN1L). The protein shows modest conservation to the human homolog at 47.2% sequence identity and a mouse anti-rOvROPN1L antiserum was not reactive with sperm protein extracts from hamsters, mice and rats. Unsurprisingly, conservation is higher in trematodes, e.g. 78.4% and 71.2% identity to Fasciola gigantica and Schistosoma haematobium, respectively and evaluation of diagnostic specificity is required using sera of individuals suffering from different trematodiases in Thailand.

  1. Evaluation of an animal protein-free semen extender for cryopreservation of epididymal sperm from North American bison (Bison bison).

    Science.gov (United States)

    Krishnakumar, S; Whiteside, D P; Elkin, B; Thundathil, J C

    2011-07-15

    The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation (tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ± 9.0, 60.5 ± 17.4, and 77.3 ± 4.6%; overall mean ± SD) as well as for wood bison (11.7 ± 8.1, 13.7 ± 5.6, and 73.4 ± 4.2%). Levels of tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ significantly between extenders for plains bison (84.16 ± 9.92%, overall mean ± SD) and for wood bison (59.53 ± 19.99%). In conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed (animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison.

  2. Green spermatozoa illuminate a 30-year-old model:sperm-egg adhesion involves intra-acrosomal proteins

    Institute of Scientific and Technical Information of China (English)

    Steve Tardif

    2011-01-01

    @@ Fertilisation in mammals involves many synchronized steps including spermegg adhesion.Prior to sperm-oolemma fusion,spermatozoa need to undergo the acrosome reaction (AR) or exocytosis.The universal belief,for many years,has been that the AR was initiated upon binding to the zona pellucida (ZP).As such acrosomal proteins were not thought to be involved in the primary contact with the ZP.These proteins were only suggested to be biologically relevant once the sperm were attached to the ZP and during subsequent events.However,recent data in the mouse have unequivocally demonstrated that spermatozoa can begin exocytosis before contact with ZP.1 It is a remarkable finding as not only will the interpretation of the interaction between sperm and cumulus cells need to be revised,but the processes of capacitation,vesiculation and exposure of acrosomal content need reexamination.

  3. The 5'-AMP-Activated Protein Kinase (AMPK Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm.

    Directory of Open Access Journals (Sweden)

    Thi Mong Diep Nguyen

    Full Text Available Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5'-AMP activated protein kinase (AMPK is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET] or inhibitor (Compound C [CC] and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS production, lipid peroxidation (LPO and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD, Glutathione Peroxidase (GPx and Glutathione Reductase (GR, increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm as well as AR (+ 100%. MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation

  4. Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis▿ †

    Science.gov (United States)

    Lin, Yi-Nan; Roy, Angshumoy; Yan, Wei; Burns, Kathleen H.; Matzuk, Martin M.

    2007-01-01

    Zona pellucida binding protein 1 (ZPBP1), a spermatid and spermatozoon protein that localizes to the acrosome, was originally identified in pigs and named for its binding to the oocyte zona pellucida. In an in silico search for germ cell-specific genes, Zpbp1 and its novel paralog, Zpbp2, were discovered and confirmed to be expressed only in the testes in both mice and humans. To study the in vivo functions of both ZPBP proteins, we disrupted Zpbp1 and Zpbp2 in mice. Males lacking ZPBP1 were sterile, with abnormal round-headed sperm morphology and no forward sperm motility. Ultrastructural studies demonstrated that absence of ZPBP1 prevents proper acrosome compaction, resulting in acrosome fragmentation and disruption of the Sertoli-spermatid junctions. Males null for ZPBP2 were subfertile, demonstrated aberrant acrosomal membrane invaginations, and produced dysmorphic sperm with reduced ability to penetrate zona pellucida. Molecular phylogenetic analysis of ZPBPs from amphibians, birds, and mammals suggests that these paralogous genes coevolved to play cooperative roles during spermiogenesis. Whereas ZPBP1 was discovered for an in vitro role in sperm-egg interactions, we have shown that both ZPBP proteins play an earlier structural role during spermiogenesis. PMID:17664285

  5. The Expression of Sperm Membrane Peptide-Hepatitis B Surface Antigen Fusion Protein with Recombinant Vaccinia Virus

    Institute of Scientific and Technical Information of China (English)

    杨晓鸣; 赵峰; 严缘昌; 李光地; 汪垣

    1998-01-01

    A synthetic oligonucleotide, HSD-2a, encoding a peptide segment of the extracellular domain of a human sperm membrane protein, YWK-Ⅱ, was fused with hepatitis B surface antigen gene (HBs gene). The fused gene was then cloned to pUC18 plasmid.

  6. Gamete therapeutics: recombinant protein adsorption by sperm for increasing fertility via artificial insemination.

    Directory of Open Access Journals (Sweden)

    Horacio Alvarez-Gallardo

    Full Text Available A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs, specifically the fertility associated antigen (FAA and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2, act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system's response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA and recombinant TIMP-2 (rTIMP-2 to bovine semen before cryopreservation for use in an artificial insemination (AI program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus heifers were selected based on their estrus cycle, body condition score (BCS, of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal measurements. Heifers were synchronized using estradiol benzoate (EB, Celosil® (PGF2α (Shering-Plough and a controlled internal drug release (CIDR device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL and rTIMP-2 (25 µg/mL. In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037. Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.

  7. New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs.

    Directory of Open Access Journals (Sweden)

    Edith Serrano

    Full Text Available Seminal plasma (SP proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1 and 2 (BSPH2. The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2, which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b.

  8. New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs)

    Science.gov (United States)

    Arruga, Diana; Pérez-Pé, Rosaura; Sánchez-Ferrer, Álvaro; Muiño-Blanco, Teresa; Cebrián-Pérez, José A.

    2015-01-01

    Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b). PMID:26333091

  9. Electrophoretic and zymographic characterization of proteins isolated by various extraction methods from ejaculated and capacitated boar sperms.

    Science.gov (United States)

    Zigo, Michal; Jonáková, Věra; Maňásková-Postlerová, Pavla

    2011-06-01

    The presented work focuses on electrophoretic and zymographic characterization of boar sperm proteins isolated by various extraction methods and on comparison of the protein profiles obtained from ejaculated and in vitro capacitated spermatozoa. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated with the following agents: 1% v/v Triton X-100, 1% v/v Triton X-114, 2% v/v acetic acid, 1% m/v sodium dodecyl sulphate (SDS), 30 mM N-octyl-β-D-glucopyranoside (OBG), rehydration buffer (RHB) for isoelectric focusing and finally by the freezing-thawing approach. The extracts were characterized in terms of 1-DE, 2-DE protein profiles, 1-DE glycoprotein staining and proteinase and hyaluronidase substrate zymographic profiles. The results have shown quantitative and qualitative differences in 1-DE protein and glycoprotein profiles with respect to the employed isolation approach. These differences were seen even more clearly in 2-DE protein profiles, where it was possible to distinguish the presence/absence, changes in relative abundance and pI/M(r) shifts of various protein spots. Proteinase and hyaluronidase zymograms supported the prediction that various isolation protocols result in various profiles of enzymatically active molecules.

  10. Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins.

    Science.gov (United States)

    Kalra, Shalini; Pradeep, Mangottil Ayyappan; Mohanty, Ashok K; Kaushik, Jai K

    2016-01-01

    Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as

  11. Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol-9.

    Science.gov (United States)

    Nithya, R S; Anuja, M M; Rajamanickam, C; Indira, M

    2012-12-01

    Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62-kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol-9 (N-9) in vitro. The sperm immobilisation studies showed that 100 μg ml(-1) of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in Rp and N-9 treated groups in comparison with the control. In Rp and N-9 treated groups, the number of acrosome-reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9. © 2012 Blackwell Verlag GmbH.

  12. Extracellular regulation of sperm transmembrane adenylyl cyclase by a forward motility stimulating protein.

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    Souvik Dey

    Full Text Available Forward motility stimulating factor (FMSF, a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tm

  13. Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

    Science.gov (United States)

    Kennedy, Chelsey E; Krieger, Kari Beth; Sutovsky, Miriam; Xu, Wei; Vargovič, Peter; Didion, Bradley A; Ellersieck, Mark R; Hennessy, Madison E; Verstegen, John; Oko, Richard; Sutovsky, Peter

    2014-05-01

    Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility.

  14. Sperm postacrosomal WW domain-binding protein is not required for mouse egg activation.

    Science.gov (United States)

    Satouh, Yuhkoh; Nozawa, Kaori; Ikawa, Masahito

    2015-10-01

    To begin embryonic development, the zygote must resume the cell cycle correctly after stimulation by sperm-borne oocyte-activating factors (SOAFs). The postacrosomal WW domain-binding protein (PAWP) is one of the strongest SOAF candidates and is widely conserved among eutherian mammals. It has been reported that the microinjection of recombinant PAWP protein can trigger not only Ca(2+) oscillations in mammalian eggs but also intracellular Ca(2+) release in amphibian eggs. It was also suggested that PAWP is involved in the formation of high-quality spermatozoa. On the other hand, negligible SOAF activity for PAWP cRNA has also been reported. In this study, we generated PAWP null mice and examined the fertilizing ability of male mice. Electron microscopy showed no aberrant morphology in spermatogenesis. Intracytoplasmic injection of a single spermatozoon from the null mouse line showed that depletion of PAWP elicited no quantitative differences in Ca(2+) oscillations or in subsequent development of the embryos. We conclude that PAWP does not play an essential role in mouse fertilization.

  15. Expression of Human Sperm Membrane Protein in the Recombinant Salmonella Typhimurium Vaccine

    Institute of Scientific and Technical Information of China (English)

    匡颖; 胡菁华; 翟玉梅; 缨时英; 王琳芳; 严缘昌

    1999-01-01

    A 550 bp cDNA fragment of HSD-Ⅰ coding for an extracellular domain of hu-man sperm membrane protein(hSMP-1)was ligated with an Adapter containing the universal stop codon,and the ligated fragment cDNA was then cloned into the MAS of pUC19.The desired plasmid with correct open reading frame was obtained, and was cut with EcoR Ⅰ.The insert was purified and then cloned into the two asd+ Salmonella expression vectors(the low copy number plasmid-pYA292 and the high copy number plasmid-pYA3137).The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recom-binant plasmids were transferred into the non-pathogenic Salmonella typhimurium X4550,which was deletion of the △cya, △crp and △asd genes.Western-blot analysis of the whole cell lysate of the two recombinants of S.typhimurium showed a predomi-nant protein band at 21 KD,which reacted with the anti-hSMP-1 antiserum. The re-sult indicated that two recombinants of S.typhimurium containing the 550 bp cDNA of HSD-Ⅰ were constructed and the characteristics of their growth in vitro were deter-mined. They may be used as new potential mucosalanti fertility.

  16. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs Effects on AMP-Activated Protein Kinase (AMPK Regulation of Chicken Sperm Functions.

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    Thi Mong Diep Nguyen

    Full Text Available Sperm require high levels of energy to ensure motility and acrosome reaction (AR accomplishment. The AMP-activated protein kinase (AMPK has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+/calmodulin-dependent protein kinase kinases (CaMKKs mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+, or of CaMKKs inhibitor (STO-609. Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β, CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+. Our results show for the first time the presence of CaMKKs (α and β and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+ entry in sperm through the Ca(2+/CaM/CaMKKs/CaMKI pathway. The Ca(2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2

  17. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

    Institute of Scientific and Technical Information of China (English)

    Debby Ickowicz; Maya Finkelstein; Haim Breitbart

    2012-01-01

    Mammalian sperm must undergo a series of biochemical and physiological modifications,collectively called capacitation,in the female reproductive tract prior to the acrosome reaction (AR).The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca2+ during both capacitation and the AR.In the present review,we summarize some of the signaling events that are involved in capacitation.During the capacitation process,phosphatidyl-inositol-3-kinase (P13K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade,and downregulated by protein kinase C α (PKCα).PKCα is active at the beginning of capacitation,resulting in P13K inactivation.During capacitation,PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism,allowing the activation of P13K.The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase.This activation of PKA leads to an increase in actin polymerization,an essential process for the development of hyperactivated motility,which is necessary for successful fertilization.Actin polymerization is mediated by PIP2 in two ways:first,P(I)P2 acts as a cofactor for phospholipase D (PLD) activation,and second,as a molecule that binds and inhibits actin-severing proteins such as gelsolin.Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation.Prior to the AR,gelsolin is released from P(I)P2 and undergoes dephosphorylation/activation,resulting in fast F-actin depolymerization,leading to the AR.

  18. Duplicate abalone egg coat proteins bind sperm lysin similarly, but evolve oppositely, consistent with molecular mimicry at fertilization.

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    Jan E Aagaard

    Full Text Available Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis, some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone (Haliotis spp., a model system of reproductive protein evolution. We test the evolutionary rates (d(N/d(S of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14, and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL.

  19. Regulation of sperm-specific proteins by IFE-1, a germline-specific homolog of eIF4E, in C. elegans.

    Science.gov (United States)

    Kawasaki, Ichiro; Jeong, Myung-Hwan; Shim, Yhong-Hee

    2011-02-01

    ABSTEACT: IFE-1 is one of the five C. elegans homologs of eIF4E, which is the mRNA 5' cap-binding component of the translation initiation complex eIF4F. Depletion of IFE-1 causes defects in sperm, suggesting that IFE-1 regulates a subset of genes required for sperm functions. To further understand the molecular function of IFE-1, proteomic analysis was performed to search for sperm proteins that are downregulated in ife-1(ok1978); fem-3(q20) mutants relative to the fem-3(q20) control. The fem-3(q20) mutant background was used because it only produces sperm at restrictive temperature. Total worm proteins were subjected to 2D-DIGE, and differentially expressed protein spots were further identified by MALDI-TOF mass spectrometry. Among the identified proteins, GSP-3 and Major Sperm Proteins (MSPs) were found to be significantly down-regulated in the ife-1(ok1978) mutant. Moreover, RNAi of gsp-3 caused an ife-1-like phenotype. These results suggest that IFE-1 is required for efficient expression of some sperm-specific proteins, and the fertilization defect of ife-1 mutant is caused mainly by a reduced level of GSP-3.

  20. Growth, testis size, spermatogenesis, semen parameters and seminal plasma and sperm membrane protein profile during the reproductive development of male goats supplemented with de-oiled castor cake.

    Science.gov (United States)

    Oliveira, C H A; Silva, A M; Silva, L M; van Tilburg, M F; Fernandes, C C L; Velho, A L M C; Moura, A A; Moreno, F B M B; Monteiro-Moreira, A C O; Moreira, R A; Lima, I M T; Rondina, D

    2015-06-01

    The present study was conducted to evaluate the effect of de-oiled castor cake on reproductive traits of crossbreed goats. Fourteen males were grouped into two lots (n = 7/group), as described: group without de-oiled castor cake (WCC) and group fed with de-oiled castor cake (CC). Goats received two diets containing a mixture of Bermudagrass hay and concentrates with the same energy (73% total digestive nutrients) and protein content (15% crude protein) during 150 days, corresponding to ages from 40 (puberty) to 60 weeks. Blood plasma concentrations of urea, albumin, lactate dehydrogenase, creatinine, alanine aminotransferase and testosterone were determined. We also evaluated scrotal circumference, sperm parameters, quantitative aspects of spermatogenesis and daily sperm production (DSP), as well as the proteome of seminal plasma and sperm membrane. Seminal fluid and sperm proteins were analyzed by 2D SDS-PAGE and mass spectrometry. After 150 days of castor cake feeding, animals had no changes in the biochemical composition of blood plasma, suggesting the absence of intoxication by ingestion of ricin. There were no alterations in dry mater intake, weight gain, testis size, peripheral concentrations of testosterone, sperm concentration, motility and morphology. Sertoli and germ cell populations in the testis and DSP were not affected either. However, there were significant variations in the expression of five seminal plasma proteins and four sperm membrane proteins. In conclusion, the replacement of soybean meal by castor cake (with ricin concentrations of 50mg/kg) did not interfere with the growth and core reproductive development of male goats. However, the diet with ricin altered the expression of certain seminal plasma and sperm membrane proteins, which play roles in sperm function and fertilization. Lower expression of these proteins may impair the ricin-fed animals to perform as high-fertility sires.

  1. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

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    Rumbley Jon N

    2011-02-01

    Full Text Available Abstract Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.

  2. Male Age Affects Female Mate Preference, Quantity of Accessory Gland Proteins, and Sperm Traits and Female Fitness in D. melanogaster.

    Science.gov (United States)

    Rezaei, Abolhasan; Krishna, Mysore Siddaiah; Santhosh, Hassan T

    2015-01-01

    For species in which mating is resource-independent and offspring do not receive parental care, theoretical models of age-based female mate preference predict that females should prefer to mate with older males as they have demonstrated ability to survive. Thus, females should obtain a fitness benefit from mating with older males. However, male aging is often associated with reductions in quantity of sperm. The adaptive significance of age-based mate choice is therefore unclear. Various hypotheses have made conflicting predictions concerning this issue, because published studies have not investigated the effect of age on accessory gland proteins and sperm traits. D. melanogaster exhibits resource-independent mating, and offspring do not receive parental care, making this an appropriate model for studying age-based mate choice. In the present study, we found that D. melanogaster females of all ages preferred to mate with the younger of two competing males. Young males performed significantly greater courtship attempts and females showed least rejection for the same than middle-aged and old males. Young males had small accessory glands that contained very few main cells that were larger than average. Nevertheless, compared with middle-aged or old males, the young males transferred greater quantities of accessory gland proteins and sperm to mated females. As a result, females that mated with young male produced more eggs and progeny than those that mated with older males. Furthermore, mating with young male reduced female's lifespan. These studies indicate that quantity of accessory gland proteins and sperm traits decreased with male age and females obtain direct fitness benefit from mating with preferred young males.

  3. Data on endogenous chicken sperm peptides and small proteins obtained through Top-Down High Resolution Mass Spectrometry

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    L. Soler

    2016-09-01

    Full Text Available The endogenous peptides and small proteins present in chicken sperm were identified in the context of the characterization of a fertility-diagnostic method based on the use of ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The interpretation and description of these data can be found in a research article, “Intact cell MALDI-TOF MS on sperm: a molecular test for male fertility diagnosis” (Soler et al., 2016 [1], and raw data derived from this analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002768. Here, we describe the inventory of all the molecular species identified, along with their biochemical features and functional analysis. This peptide/protein catalogue can be further employed as reference for other studies and reveal that the use of proteomics allows for a global evaluation of sperm cells functions.

  4. EFFECT OF PROTEINE CONTENT IN BOAR SEMINAL PLASMA ON THE SPERM MOTILITY IN DILUTED SEMEN STORED FOR 3 DAYS

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    Jelena Apić

    2015-02-01

    Full Text Available Recently, it was frequently demonstrated that fertility of sows after artificially inseminated is lower than after mating. This is associated with a reduced fertilization capacity of overdiluted insemination doses. The aim of this study was to investigate the sperm motility in the semen samples, forming from the ejaculates with high or low protein content, stored in vitro on 17oC for 3 days. Progressive motility was significantly higher (p<0.01 in the ejaculates with high, compared to the ejaculates with low protein content (82% vs. 76%. After 3 days of storage, in the1:4 dilution proportion, the average progressive motility was significantly (p<0.01 decreased in relation to this value in native semen from the boars with high (82% to64%, as well from the boars with low protein content in seminal plasma (76% to48%. However, the average diluted semen progressive motility was significantly greater (p<0.01 in the boars with high (64%, compared to the boars with low protein content in seminal plasma (48%. The number of good diluted semen samples (≥65% progressive motility, was also significantly (p<0.01 greater in the boars with high (41%, compared to the boars with low protein content in seminal plasma (12%. These results show that seminal plasma proteins play an important role in maintaining the sperm progressive motility of diluted semen in vitro stored for 3 days.

  5. KIFC1-like motor protein associates with the cephalopod manchette and participates in sperm nuclear morphogenesis in Octopus tankahkeei.

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    Wei Wang

    Full Text Available BACKGROUND: Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. METHODOLOGY/PRINCIPAL FINDINGS: We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. CONCLUSIONS/SIGNIFICANCE: The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod.

  6. Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis.

    Science.gov (United States)

    Cabrillana, María E; Monclus, María A; Sáez Lancellotti, Tania E; Boarelli, Paola V; Clementi, Marisa A; Vincenti, Amanda E; Yunes, Roberto F M; Fornés, Miguel W

    2011-09-01

    Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc.

  7. Role of PUF-8/PUF protein in stem cell control, sperm-oocyte decision and cell fate reprogramming.

    Science.gov (United States)

    Datla, Udaya Sree; Scovill, Natasha Carol; Brokamp, Austin J; Kim, Eunsuk; Asch, Adam S; Lee, Myon-Hee

    2014-10-01

    Pumilio and FBF (PUF) proteins are conserved stem cell regulators that maintain germline stem cells (GSCs) in worms and flies. Moreover, they are also present in vertebrate stem cells. The nematode Caenorhabditis elegans has multiple PUF proteins with specialized roles. Among them, PUF-8 protein controls multiple cellular processes, including proliferation, differentiation, sperm-oocyte decision, and cell fate reprogramming, depending on the genetic context in the C. elegans germline. In this review, we describe the possible mechanisms of how PUF-8 protein systematically controls multiple cellular processes in the C. elegans germline. Since PUF proteins are evolutionarily conserved, we suggest that a similar mechanism may be involved in controlling stem cell regulation and differentiation in other organisms, including humans.

  8. Characterizing mouse male germ cell-specific actin capping protein α3 (CPα3): dynamic patterns of expression in testicular and epididymal sperm

    Institute of Scientific and Technical Information of China (English)

    Keizo Tokuhiro; Yasushi Miyagawa; Hiromitsu Tanaka

    2008-01-01

    Aim: To characterize mouse capping protein α3 (CPα3) during spermatogenesis and sperm maturation. Methods: We produced rat anti-CPα3 antiserum and examined the expression of CPα3 in various mouse tissues using Western blot analysis and the localization of CPα3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPα3 and β-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPα3 antiserum and anti-actin antibody. Results: Western blot analysis using specific antiserum revealed that CPα3 was expressed specifically in testes. Interestingly, the molecular weight of CPα3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPα3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPα3 and ACTB localization was virtually identical to the localization before the acrosomal reaction.Conclusion: CPα3 might play an important role in sperm morphogenesis and/or sperm function.

  9. Semen variables and sperm membrane protein profile of Saanen bucks ( Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S)

    Science.gov (United States)

    van Tilburg, M. F.; Salles, M. G. F.; Silva, M. M.; Moreira, R. A.; Moreno, F. B.; Monteiro-Moreira, A. C. O.; Martins, J. A. M.; Cândido, M. J. D.; Araújo, A. A.; Moura, A. A. A.

    2015-05-01

    The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks ( n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant ( p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher ( p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

  10. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    DEFF Research Database (Denmark)

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes......-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed...... that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm...

  11. Seminal plasma proteins and their relationship with percentage of morphologically normal sperm in 2-year-old Brahman (Bos indicus) bulls.

    Science.gov (United States)

    Boe-Hansen, G B; Rego, J P A; Crisp, J M; Moura, A A; Nouwens, A S; Li, Y; Venus, B; Burns, B M; McGowan, M R

    2015-11-01

    The objective was to determine the relationship between seminal plasma proteins and sperm morphology in Bos indicus bulls of the Brahman breed. Fifty-six 24-month-old Australian Brahman bulls were electroejaculated and samples were examined to determine the percentage of morphologically normal sperm (PNS24) and the seminal plasma protein composition was identified and quantified by 2-D gel electrophoresis. The total integrated optical density of 152 seminal plasma protein spots (SPPs) across all gels was determined using the PDQuest software version 8.0 (Bio Rad, USA). Using a single regression mixed model with the density of individual spots as a covariate for PNS24, 17 SPPs were significantly associated with PNS24 (pBrahman bulls and sperm morphology. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Ultrastructural immunogold localization of major sperm protein (MSP) in spermatogenic cells of the nematode Acrobeles complexus (Nematoda, Rhabditida).

    Science.gov (United States)

    Yushin, Vladimir V; Claeys, Myriam; Bert, Wim

    2016-10-01

    The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as "major sperm protein", or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of

  13. Yolk protein is expressed in the insect testis and interacts with sperm

    OpenAIRE

    Joachimiak Ewa; Kotwica Joanna; Bebas Piotr; Giebultowicz Jadwiga M

    2008-01-01

    Abstract Background Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm ...

  14. Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

    Energy Technology Data Exchange (ETDEWEB)

    Liang Liang; Dou Peng; Dong Mingming; Ke Xiaokang; Bian Ningsheng [School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China); Liu Zhen, E-mail: zhenliu@nju.edu.cn [School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China)

    2009-09-14

    Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 {+-} 0.83 x 10{sup -3} s{sup -1}. The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

  15. Characterization and comparison of proteins in the sperm storage tubules of female chickens to bovine epididymal fluid

    Science.gov (United States)

    Female birds are able to store sperm in crypts called sperm storage tubules (SSTs) in their reproductive tracts for between two and six weeks. Comparatively, sperm in a cow’s reproductive tract remain viable for between 18 and 24 hours. The objective of this experiment was to try to identify and co...

  16. The acrosomal matrix from guinea pig sperm contains structural proteins, suggesting the presence of an actin skeleton.

    Science.gov (United States)

    Zepeda-Bastida, Armando; Chiquete-Felix, Natalia; Uribe-Carvajal, Salvador; Mujica, Adela

    2011-01-01

    The mammalian sperm acrosome contains a large number of hydrolytic enzymes. When the acrosomal reaction and fertilization occur, these enzymes are released in an orderly fashion, suggesting that the acrosomal matrix is highly organized. It was decided to determine the identity of the structural scaffold underlying the organization of the acrosome. In permeabilized acrosomes and in the Triton X-100-extracted acrosomal matrices from guinea pig sperm, we used indirect immunofluorescence, immunogold labeling, and Western blotting to identify F-actin, spectrin, myosin, calmodulin, and gelsolin. These proteins were detected in the acrosomal matrix for the first time. In noncapacitated, intact spermatozoa the addition of the F-actin monomerizing agent cytochalasin D resulted in loss of the acrosome, suggesting that F-actin is needed to preserve an intact acrosome. Our results suggest that the acrosomal architecture is supported by a dynamic F-actin skeleton, which probably regulates the differential rate of release of the acrosomal enzymes during acrosomal reaction and fertilization.

  17. Association of mutations in the zona pellucida binding protein 1 (ZPBP1) gene with abnormal sperm head morphology in infertile men

    Science.gov (United States)

    Yatsenko, Alexander N.; O'Neil, Derek S.; Roy, Angshumoy; Arias-Mendoza, Paola A.; Chen, Ruihong; Murthy, Lata J.; Lamb, Dolores J.; Matzuk, Martin M.

    2012-01-01

    Nearly 7% of men are afflicted by male infertility worldwide, and genetic factors are suspected to play a significant role in the majority of these patients. Although sperm morphology is an important parameter measured in the semen analysis, only a few genetic causes of teratozoospermia are currently known. The objective of this study was to define the association between alterations in the genes encoding the Golgi-associated PDZ- and coiled-coil motif containing protein (GOPC), the protein interacting with C kinase 1 (PICK1) and the acrosomal protein zona pellucida binding protein 1 (ZPBP1/sp38) with abnormal sperm head morphology in infertile men. Previous reports demonstrated that mice lacking Gopc, Pick1 and Zpbp1 are infertile due to abnormal head morphology. Herein, using our validated RNA-based method, we studied spermatozoal cDNA encoding the human GOPC, PICK1 and ZPBP1 genes in 381 teratozoospermic and 240 controls patients via direct sequencing. Among these genes, we identified missense and splicing mutations in the sperm cDNA encoding ZPBP1 in 3.9% (15/381) of men with abnormal sperm head morphology. These mutations were not observed in 240 matched controls and the dbSNP database (χ2 = 9.3, P = 0.002). In contrast, statistically significant and functionally relevant mutations were not discovered in the GOPC and PICK1 genes. In our study ZPBP1 mutations are associated with abnormal sperm head morphology, defined according to strict criteria, resembling the mouse Zpbp1 null phenotype. We hypothesize that missense mutations exert a dominant-negative effect due to altered ZPBP1 protein folding and protein:protein interactions in the acrosome. PMID:21911476

  18. Toona sinensis Leaf Aqueous Extract Improves the Functions of Sperm and Testes via Regulating Testicular Proteins in Rats under Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Bu-Chin Yu

    2012-01-01

    Full Text Available Toona sinensis leaf (TSL is commonly used as a vegetable and in spice in Asia. In this study, feeding with aqueous extract of TSL (TSL-A alleviated oxidative stress and recovered the motility and functions of sperm in rats under oxidative stress. Protein expressions in testes identified by proteomic analysis and verified by Western blot demonstrated that TSL-A not only downregulated the level of glutathione transferase mu6 (antioxidant system, heat shock protein 90 kDa-β (protein misfolding repairing system, cofilin 2 (spermatogenesis, and cyclophilin A (apoptosis but also upregulated crease3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (steroidogenesis, heat shock glycoprotein 96, and pancreatic trypsin 1 (sperm-oocyte interaction. These results indicate that TSL-A promotes the functions of sperm and testes via regulating multiple testicular proteins in rats under oxidative stress, suggesting that TSL-A is a valuable functional food supplement to improve functions of sperm and testes for males under oxidative stress.

  19. Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine.

    Science.gov (United States)

    D'Amours, Olivier; Frenette, Gilles; Bordeleau, Louis-Jean; Allard, Nancy; Leclerc, Pierre; Blondin, Patrick; Sullivan, Robert

    2012-10-01

    Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit.

  20. Identification and Characterization of a Bovine Sperm Acrosomal Matrix Protein and its Mechanism of Interaction with Acrosomal Hydrolases

    Science.gov (United States)

    Nagdas, Subir K; Smith, Linda; Mcnamara, Allen; Hernandez–Encarnacion, Luisa; Medina-Ortiz, Ilza

    2015-01-01

    Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads is comprised of 54, 50, 45, and 38–19kDa polypeptides. The objective of this study is to identify and to characterize the 45kDa (OMC45) polypeptide and to define its role in binding acrosomal hydrolases and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI–TOF–TOF yielded 8 peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X–100–permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti–OMC45 and anti–TEKT3. The OMC45 polypeptide was solubilized by RIPA (radio-immunoprecipitation assay) buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti–OMC45 immunoprecipitation pellet. An identical blot stained with anti–TEKT3 exhibited the presence of TEKT3 polypeptide in the anti–OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide; that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N–linked and O–linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N–acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid

  1. Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection.

    Science.gov (United States)

    Choi, Young-Ho; Gibbons, John R; Canesin, Heloísa S; Hinrichs, Katrin

    2016-10-15

    Prospective studies were conducted to help define procedural factors affecting in vitro embryo production via intracytoplasmic sperm injection (ICSI) of equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P zinc supplementation (0, 0.5, 1, or 1.5 μg/mL) during IVM was examined. There were no significant differences in rates of cleavage or blastocyst development (20%-31%). However, the proportion of blastocysts that developed on Day 7 for the added-zinc treatments was significantly higher than that for the control treatment (45% vs. 8%). In experiment 3, we tested whether use of high-pH medium (pH 8.0-8.4) during ICSI procedures would improve blastocyst rate when sperm with low cleavage rates after ICSI was used. When high-pH conditions were used for sperm preparation and also for the first 2 hours of incubation of injected oocytes after ICSI, the cleavage rate was unaffected but no blastocysts developed (0% vs. 24% for control). When high-pH conditions were used for sperm preparation only, the blastocyst rate was 37%. This was repeated using sperm from a second stallion; there was no significant difference in cleavage or blastocyst rates between sperm preparation in high pH vs. control medium. These findings add to our knowledge of factors affecting in vitro production of equine embryos.

  2. Actin related protein complex subunit 1b controls sperm release, barrier integrity and cell division during adult rat spermatogenesis.

    Science.gov (United States)

    Kumar, Anita; Dumasia, Kushaan; Deshpande, Sharvari; Gaonkar, Reshma; Balasinor, N H

    2016-08-01

    Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis.

  3. [Updated detection of the function of sperm plasma membrane].

    Science.gov (United States)

    Zhou, Xin; Xia, Xin-Yi; Huang, Yu-Feng

    2010-08-01

    The sperm plasma membrane is rich in polyunsaturated fatty acids and a variety of proteins, and its function is associated with sperm capacitation, acrosome reaction and sperm-egg fusion. Sperm fertilizability can be predicted by detecting the function of the sperm plasma membrane, which is performed mainly with the following five techniques: sperm hypoosmotic swelling test, Eosin gamma water test, sperm membrane lipid peroxidation determination, seminal plasma superoxide dismutase determination, and flow cytometry. The evaluation of the function of sperm plasma membrane can be applied in detecting semen quality, selecting semen centrifugation, assessing the quality and fertilizability of sex-sorted sperm, improving cryopreservation, and guiding the optimization of intracytoplasmic sperm injection. This review presents an update on the principles, methods and steps of the detection of sperm plasma membrane function, as well as an overview of its status quo and application.

  4. Sperm-egg interaction.

    Science.gov (United States)

    Evans, Janice P

    2012-01-01

    A crucial step of fertilization is the sperm-egg interaction that allows the two gametes to fuse and create the zygote. In the mouse, CD9 on the egg and IZUMO1 on the sperm stand out as critical players, as Cd9(-/-) and Izumo1(-/-) mice are healthy but infertile or severely subfertile due to defective sperm-egg interaction. Moreover, work on several nonmammalian organisms has identified some of the most intriguing candidates implicated in sperm-egg interaction. Understanding of gamete membrane interactions is advancing through characterization of in vivo and in vitro fertilization phenotypes, including insights from less robust phenotypes that highlight potential supporting (albeit not absolutely essential) players. An emerging theme is that there are varied roles for gamete molecules that participate in sperm-egg interactions. Such roles include not only functioning as fusogens, or as adhesion molecules for the opposite gamete, but also functioning through interactions in cis with other proteins to regulate membrane order and functionality.

  5. Use of heterospermic inseminations and paternity testing to evaluate the relative contributions of common sperm traits and seminal plasma proteins in boar fertility.

    Science.gov (United States)

    Flowers, W L; Deller, F; Stewart, K R

    2016-11-01

    The objective of this study was to evaluate relationships between common semen quality estimates including sperm motility, sperm morphology, spontaneous capacitation status and seminal plasma proteins and boar fertility using heterospermic inseminations and subsequent paternity testing. All boars (n=12) used in the study had excellent semen quality (≥70% normal sperm) that resulted in average farrowing rates and litter sizes of 88.9±0.7% and 11.7±0.1 pigs, respectively. Their ejaculates were combined to make heterospermic insemination doses in such a way that each boar was tested against all of his contemporaries. The proportion of piglets sired by each individual was used to separate boars into three fertility groups: High (71.6±4.8%; n=3); Medium (51.6±3.8%; n=6); and Low (25.2%±5.3%; n=3). Ejaculates from High fertility boars had more motile sperm with normal acrosomes that moved faster in a straight-line and were more likely to undergo an acrosome reaction (p≤0.05) compared with their counterparts in the Low fertility group. Ejaculates from High fertility boars contained the greatest concentrations of three seminal plasma proteins (25.9kD/5.9pI; 55.1kD/4.8pI; and 70.1kD/5.2pI; p≤0.05), whereas concentrations of a 19.1kD/6.8pI were highest in semen from Low fertility boars (p≤0.05). Multiple regression analyses indicated that concentrations of the 25.9kD/5.9pI seminal plasma protein explained 66% of the variation observed in the proportion of pigs sired within a litter among boars (p≤0.00001). These results demonstrate that heterospermic inseminations and subsequent paternity testing is an effective technique for defining relationships between common semen quality tests and fertility, especially in situations where reproductive performance of all the boars is high. Motility, normal acrosome morphology, average linear velocity of motile sperm, and the proportion of sperm capable of an acrosome reaction were all positively associated with boar

  6. Identification of New Epididymal Luminal Fluid Proteins Involved in Sperm Maturation in Infertile Rats Treated by Dutasteride Using iTRAQ

    Directory of Open Access Journals (Sweden)

    Shu-Wu Xie

    2016-05-01

    Full Text Available Background: Spermatozoa become mature and acquire fertilizing capacity during their passage through the epididymal lumen. In this study, we identified new epididymal luminal fluid proteins involved in sperm maturation in infertile rats by dutasteride, a dual 5α-reductase inhibitor, in order to provide potential epididymal targets for new contraceptives and infertility treatment. Methods: Male rats were treated with dutasteride for 28 consecutive days. We observed the protein expression profiles in the epididymal luminal fluids in infertile and normal rats using isobaric tags for relative and absolute quantitation (iTRAQ technique. The confidence of proteome data was validated by enzyme-linked immunosorbent assays. Results: 1045 proteins were tested, and 23 of them presented different expression profiling in the infertile and normal rats. The seven proteins were down-regulated, and 16 proteins were up-regulated. Among the seven proteins which were significantly down-regulated by dutasteride in the epididymal luminal fluids, there were three β-defensins (Defb2, Defb18 and Defb39, which maybe the key proteins involved in epididymal sperm maturation and male fertility. Conclusions: We report for the first time that dutasteride influences the protein expression profiling in the epididymal luminal fluids of rats, and this result provides some new epididymal targets for male contraception and infertility therapy.

  7. Overexpression of human sperm protein 17 increases migration and decreases the chemosensitivity of human epithelial ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Huang Wen-bin

    2009-09-01

    Full Text Available Abstract Background Most deaths from ovarian cancer are due to metastases that are resistant to conventional therapies. But the factors that regulate the metastatic process and chemoresistance of ovarian cancer are poorly understood. In the current study, we investigated the aberrant expression of human sperm protein 17 (HSp17 in human epithelial ovarian cancer cells and tried to analyze its influences on the cell behaviors like migration and chemoresistance. Methods Immunohistochemistry and immunocytochemistry were used to identify HSp17 in paraffin embedded ovarian malignant tumor specimens and peritoneal metastatic malignant cells. Then we examined the effect of HSp17 overexpression on the proliferation, migration, and chemoresistance of ovarian cancer cells to carboplatin and cisplatin in a human ovarian carcinoma cell line, HO8910. Results We found that HSp17 was aberrantly expressed in 43% (30/70 of the patients with primary epithelial ovarian carcinomas, and in all of the metastatic cancer cells of ascites from 8 patients. The Sp17 expression was also detected in the metastatic lesions the same as in ovarian lesions. None of the 7 non-epithelial tumors primarily developed in the ovaries was immunopositive for HSp17. Overexpression of HSp17 increased the migration but decreased the chemosensitivity of ovarian carcinoma cells to carboplatin and cisplatin. Conclusion HSp17 is aberrantly expressed in a significant proportion of epithelial ovarian carcinomas. Our results strongly suggest that HSp17 plays a role in metastatic disease and resistance of epithelial ovarian carcinoma to chemotherapy.

  8. Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody%抗人精子膜蛋白抗体1对小鼠精子顶体反应和精子-透明带结合的抑制作用

    Institute of Scientific and Technical Information of China (English)

    G.Y.Cheng; J.L.Shi; M.Wang; Y.Q.Hu; C.M.Liu; Y.F.Wang; C.Xu

    2007-01-01

    Aim:To investigate the possible functions of human sperm membrane protein (hSMP-1) in the process of fertilization.Methods: A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 (anti-rhSMP-1) antibodies. Sperm acrosome reaction and spermzona pellucida (ZP) binding assay were carried out in 10-week-old BALB/c mice. Results: Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 (P < 0.05). Conclusion: The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

  9. Low seminal zinc bound to high molecular weight proteins in asthenozoospermic patients: evidence of increased sperm zinc content in oligoasthenozoospermic patients.

    Science.gov (United States)

    Carpino, A; Siciliano, L; Petroni, M F; De Stefano, C; Aquila, S; Andó, S; Petrone, M F

    1998-01-01

    Total seminal zinc concentration, seminal zinc fraction bound to high molecular weight proteins (HMW-Zn%) and zinc content in spermatozoa were assayed in the ejaculates of 90 asthenozoospermic patients subdivided into two study groups: normoasthenozoospermics (group I: n = 50) and oligoasthenozoospermics (group II: n = 40). The zinc concentrations of patients were compared with those of a control group of donors showing normal semen parameters. All samples were also investigated for their sperm membrane functional integrity by the hypo-osmotic swelling test (HOS). The results showed normal total zinc concentrations but very low HMW-Zn% values (P spermatozoa of oligoasthenozoospermic patients compared to group I and to the control group. Oligoasthenozoospermics also displayed a lower HOS score (P spermatozoa. The higher intrasperm zinc content in these patients could be a reflection of their low sperm membrane functionality.

  10. Sperm competition, sperm numbers and sperm quality in muroid rodents.

    Directory of Open Access Journals (Sweden)

    Laura Gómez Montoto

    Full Text Available Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass, showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic

  11. Transgenic expression of green fluorescent protein in caprine embryos produced through electroporation-aided sperm-mediated gene transfer.

    Science.gov (United States)

    Kumar Pramod, R; Kumar, Rakesh; Mitra, Abhijit

    2016-01-15

    Current methods of transgenic animal production are afflicted by low efficiency and high cost. Recently, the electroporation aided sperm-mediated gene transfer (SMGT) emerges as a promising alternative with variable success rate. Among the domestic animal species, the electroporation-aided SMGT is less investigated in goats, except a few reports in which attempts have been made using the auto-uptake method of SMGT. In this study, we report an optimized electroporation condition for SMGT of caprine sperm cells. Results of this study demonstrated that electroporation of caprine sperm cells at 300 V for 200 mS in TALP medium allowed the maximum uptake of foreign DNA with minimum adverse effects on the vital semen parameters viz., progressive motility, viability, and membrane and acrosome integrity. Further, DNA binding assay revealed DNA uptake by 81.3% sperm cells when 1.0 μg of DNA was used under optimum electroporation conditions as compared to 16.5% on simple incubation. The qPCR analysis showed four-fold more (Pelectroporation than incubation. A similar cleavage rate was observed after IVF using either electroporated (23.20 ± 1.20) or non-electroporated (25.20 ± 2.41) sperm cells suggesting the absence of adverse effect of electroporation on the fertilizing ability. Out of the 116 embryos produced by electroporated sperm, five (4.31%) embryos showed the expression of the foreign gene. In conclusion, our results confirm that using optimized electroporation conditions, the caprine sperm cells can uptake foreign DNA effectively with minimum negative effect on the semen parameters and could produce transgenic embryos.

  12. The quest for the sea urchin egg receptor for sperm.

    Science.gov (United States)

    Vacquier, Victor D

    2012-08-31

    This review discusses identification, isolation and characterization of proteins mediating species-selective sperm-to-egg adhesion during sea urchin fertilization. Bindin is the only sea urchin sperm protein known to mediate species-selective sperm attachment to eggs. Two completely different egg surface proteins, 350-kDa and EBR1, have affinity for bindin and each one meets all the criteria to be a species-selective sperm receptor. Experiments suggest that sperm bindin recognizes both the sulfated O-linked oligosaccharides on the egg 350-kDa glycoprotein, and also the repeated protein sequence modules of EBR1.

  13. Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

    Science.gov (United States)

    Cataldo, L; Baig, K; Oko, R; Mastrangelo, M A; Kleene, K C

    1996-11-01

    The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

  14. Influence of macrophage migration inhibitory factor (MIF) on the zinc content and redox state of protein-bound sulphydryl groups in rat sperm: indications for a new role of MIF in sperm maturation.

    Science.gov (United States)

    Eickhoff, Regina; Baldauf, Christina; Koyro, Hans-Werner; Wennemuth, Gunther; Suga, Yasushi; Seitz, Jürgen; Henkel, Ralf; Meinhardt, Andreas

    2004-08-01

    The function of macrophage migration inhibitory factor (MIF) in sperm maturation was studied by investigating its role in the biochemical maturation of the outer dense fibres. Rat sperm obtained from the caput and cauda epididymis were stimulated overnight with either recombinant MIF or MIF-containing vesicles originating from epididymal fluid at various concentrations. The zinc content of both the sperm and the medium was determined by means of atomic absorption spectrometry. Incubation in both recombinant MIF and vesicular MIF resulted in a statistically significant decrease of the zinc content in stimulated caput sperm of approximately 50%. In parallel, the conditioned media showed a clear increase in the concentration of this trace metal. The effect of MIF was less marked in cauda sperm. In addition, we demonstrated a statistically significant increase of detectable free thiol groups in the sperm mid- and principle piece in isolated rat sperm after stimulation with MIF at concentrations of 25 and 50 ng/ml. Our data suggest that MIF plays an important role in the maturation process of rat sperm during epididymal transit by inducing the elimination of zinc and affecting the amount of free sulphydryl groups in the sperm flagella.

  15. An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana

    Science.gov (United States)

    In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, p...

  16. Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

    OpenAIRE

    Chai, Ran‐Ran; Chen, Guo‐Wu; Shi, Hui‐Juan; O, Wai‐Sum; Martin‐DeLeon, Patricia A.; Chen, Hong

    2016-01-01

    Abstract Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoas...

  17. Junctate, an inositol 1,4,5-triphosphate receptor associated protein, is present in rodent sperm and binds TRPC2 and TRPC5 but not TRPC1 channels.

    Science.gov (United States)

    Stamboulian, Séverine; Moutin, Marie-Jo; Treves, Susan; Pochon, Nathalie; Grunwald, Didier; Zorzato, Francesco; De Waard, Michel; Ronjat, Michel; Arnoult, Christophe

    2005-10-01

    The acrosome reaction, the first step of the fertilization, is induced by calcium influx through Canonical Transient Receptor Potential channels (TRPC). The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. In this study, we demonstrate that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, we show by pull-down experiments that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. We focused on the interaction between TRPC2 and junctate, and we show that the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. We show that junctate binds to TRPC2 independently of the calcium concentration and that the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. In the present study, we investigated whether DAG could promote the acrosome reaction. We found that 100 microM OAG, a permeant DAG

  18. Hypercholesterolemia Impaired Sperm Functionality in Rabbits

    Science.gov (United States)

    Monclus, Maria A.; Cabrillana, Maria E.; Clementi, Marisa A.; Espínola, Leandro S.; Cid Barría, Jose L.; Vincenti, Amanda E.; Santi, Analia G.; Fornés, Miguel W.

    2010-01-01

    Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events. PMID:20976152

  19. Hypercholesterolemia impaired sperm functionality in rabbits.

    Directory of Open Access Journals (Sweden)

    Tania E Saez Lancellotti

    Full Text Available Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR. Sperm cell morphology was seriously affected, displaying primarily a "folded head"-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events.

  20. Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation

    Institute of Scientific and Technical Information of China (English)

    Debora J. Cohen; Vanina G. Da Ros; Dolores Busso; Diego A. Ellerman; Julieta A. Maldera; Nadia Goldweic; Patricia S. Cuasnicú

    2007-01-01

    Rat protein DE is an androgen-dependent cysteine-rich secretory protein (CRISP) synthesized by proximal epididymal regions. DE, also known as CRISP- 1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target.Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 (S2). The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx-1 (CRISP-2) was capable of binding to rodent eggs,human epididymal AEG-related protein (ARP) and helothermine (from lizard saliva) were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx-1 and anti-Tpx-1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods.

  1. Functional interaction of phospholipid hydroperoxide glutathione peroxidase with sperm mitochondrion-associated cysteine-rich protein discloses the adjacent cysteine motif as a new substrate of the selenoperoxidase.

    Science.gov (United States)

    Maiorino, Matilde; Roveri, Antonella; Benazzi, Louise; Bosello, Valentina; Mauri, Pierluigi; Toppo, Stefano; Tosatto, Silvio C E; Ursini, Fulvio

    2005-11-18

    The mitochondrial capsule is a selenium- and disulfide-rich structure enchasing the outer mitochondrial membrane of mammalian spermatozoa. Among the proteins solubilized from the sperm mitochondrial capsule, we confirmed, by using a proteomic approach, the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as a major component, and we also identified the sperm mitochondrion-associated cysteine-rich protein (SMCP) and fragments/aggregates of specific keratins that previously escaped detection (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohé, L. (1999) Science 285, 1393-1396). The evidence for a functional association between PHGPx, SMCP, and keratins is further supported by the identification of a sequence motif of regularly spaced Cys-Cys doublets common to SMCP and high sulfur keratin-associated proteins, involved in bundling hair shaft keratin by disulfide cross-linking. Following the oxidative polymerization of mitochondrial capsule proteins, catalyzed by PHGPx, two-dimensional redox electrophoresis analysis showed homo- and heteropolymers of SMCP and PHGPx, together with other minor components. Adjacent cysteine residues in SMCP peptides are oxidized to cystine by PHGPx. This unusual disulfide is known to drive, by reshuffling oxidative protein folding. On this basis we propose that oxidative polymerization of the mitochondrial capsule is primed by the formation of cystine on SMCP, followed by reshuffling. Occurrence of reshuffling is further supported by the calculated thermodynamic gain of the process. This study suggests a new mechanism where selenium catalysis drives the cross-linking of structural elements of the cytoskeleton via the oxidation of a keratin-associated protein.

  2. KIFC1-Like Motor Protein Associates with the Cephalopod Manchette and Participates in Sperm Nuclear Morphogenesis in Octopus tankahkeei

    OpenAIRE

    Wei Wang; Jun-Quan Zhu; He-Ming Yu; Fu-Qing Tan; Wan-Xi Yang

    2010-01-01

    BACKGROUND: Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. METH...

  3. 曙红与鲑鱼精蛋白显色反应的研究%Study on Color Reaction of Eosin and Salmon Sperm Protein

    Institute of Scientific and Technical Information of China (English)

    韩照祥; 马丽丽

    2012-01-01

    [Objective] The research aimed to study the color reaction of eosin (gliadin) and salmon sperm protein. [Method] Conjugation reaction of eosin (gliadin) and salmon sperm protein which occurred in the presence of polyvinyl alcohol (PVA) was studied by spectrophotometry. The color reaction conditions and the factors which could affect the sensitivity were studied,and the best reactive conditions were determined. [ Result] When reagent blank was used as a reference,the maximum absorption wavelength of the compound which included eosin and protamine sulfate was 542 nm. Absorbance at the maximum absorption wavelength showed good linear relationship with the protein concentration. [ Conclusion ] If it was used for the quantitative determination of protein, high sensitivity, good linearity, reagent stability and low toxicity could be gained. And eosin and salmon sperm protein compound wasn't easy to adhere to the vessel wall. The operation is simple,rapid and accurate.%[目的]对曙红与鲑鱼精蛋白显色的反应进行研究.[方法]采用分光光度法,研究曙红(醇溶)与鲑鱼精蛋白在聚乙烯醇(PVA)存在时的结合反应,并且研究了显色反应的条件和影响灵敏度的因素,确定了最佳反应条件.[结果]以试剂空白为参比,曙红-硫酸鱼精蛋白复合物的最大吸收波长为542 nm,最大吸收波长处的吸光度与蛋白质的浓度呈良好的线性关系,可用于蛋白质的定量测定.[结论]该方法灵敏度高、线性关系好、试剂稳定、毒性小,且曙红、硫酸鱼精蛋白复合物不易黏附在容器壁上,操作简便、快速、结果准确.

  4. HDAC6 deacetylates alpha tubulin in sperm and modulates sperm motility in Holtzman rat.

    Science.gov (United States)

    Parab, Sweta; Shetty, Omshree; Gaonkar, Reshma; Balasinor, Nafisa; Khole, Vrinda; Parte, Priyanka

    2015-02-01

    Histone deacetylase 6 (HDAC6) is an alpha (α)-tubulin deacetylase and its over-expression has been demonstrated to promote chemotactic cell movement. Motility in sperm is driven by the flagella, the cytoskeletal structure comprising the microtubules, which are heterodimers of α- and β-tubulins. We have hypothesized that HDAC6, by virtue of being an α-tubulin deacetylase, might modulate sperm motility. However, the presence of HDAC6 on sperm has hitherto not been reported. In this study, we have demonstrated, for the first time, the presence of HDAC6 transcript and protein in the testicular and caudal sperm of rat. We have observed a significantly overlapping expression of HDAC6 with acetyl α-tubulin (Ac α-tubulin) in the mid-piece and principal piece of sperm flagella, and the co-precipitation of α-tubulin and Ac α-tubulin together with HDAC6 and vice versa in sperm lysates. This indicates that HDAC6 interacts with α-tubulin. The HDAC6 activity of sperm, sperm motility and status of Ac α-tubulin investigated in the presence of HDAC inhibitors Trichostatin A, Tubastatin A and sodium butyrate demonstrate that HDAC6 in sperm is catalytically active and that inhibitors of HDAC6 increase acetylation and restrict sperm motility. Thus, we show that (1) active HDAC6 enzyme is present in sperm, (2) HDAC6 in sperm is able to deacetylate α-tubulin, (3) inhibition of HDAC6 results in increased Ac α-tubulin expression and (4) HDAC6 inhibition affects sperm motility. This evidence suggests that HDAC6 is involved in modulating sperm movement.

  5. Molecular Cloning of Spergen-4, Encoding a Spermatogenic Cell-Specific Protein Associated with Sperm Flagella and the Acrosome Region in Rat Spermatozoa.

    Science.gov (United States)

    Howida, Ali; Salaheldeen, Elsaid; Iida, Hiroshi

    2016-04-01

    We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.

  6. Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation.

    Science.gov (United States)

    Intasqui, Paula; Camargo, Mariana; Del Giudice, Paula T; Spaine, Deborah M; Carvalho, Valdemir M; Cardozo, Karina H M; Cedenho, Agnaldo P; Bertolla, Ricardo P

    2013-09-01

    Sperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation. Sperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MS(E)). Differentially expressed proteins were used for a functional enrichment study. Two hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells. Sperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

  7. Fibronectin type II-module proteins in the bovine genital tract and their putative role in cell volume control during sperm maturation.

    Science.gov (United States)

    Sahin, Evrim; Petrunkina, Anna M; Ekhlasi-Hundrieser, Mahnaz; Hettel, Christiane; Waberski, Dagmar; Harrison, Robin A P; Töpfer-Petersen, Edda

    2009-01-01

    The male reproductive tract of ungulates contains two protein families bearing tandemly arranged fibronectin II (Fn2) modules; one (small Fn2 proteins) bears two modules (e.g. BSP-A1/2), the other (long Fn2 proteins) bears four (e.g. epididymal sperm-binding protein 1 (ELSPBP1)). While it is well known that small Fn2 proteins are present in bull semen, nothing is known about long Fn2 proteins. In the present study, the presence of ELSPBP1 proteins in the bull epididymis and their association with maturing spermatozoa were investigated using a specific antibody against canine ELSPBP1. Analysis of western blots showed ELSPBP1 to be present in the caput, corpus and cauda regions of the epididymis. The protein, which bound phosphorylcholine (PC) strongly, appeared to associate with the spermatozoa during maturation because it was absent from caput spermatozoa but present on cauda spermatozoa. Immunocytochemistry of cauda spermatozoa showed the protein to be bound to the post-acrosomal and midpiece regions. ELSPBP1 could not be detected on freshly ejaculated spermatozoa but was revealed after a capacitating treatment. Our previous studies have shown differences between bovine caput and cauda spermatozoa in terms of their ability to control cell volume. Because of the close homology of BSP-A1/2 PC binding regions with Fn2 regions in ELSPBP1, BSP-A1/2 was used as a model to investigate the effect of a PC-binding Fn2 protein on cell volume control. While the protein had no effect on cauda spermatozoa, it caused caput spermatozoa to swell more in response to hypotonic stress, similarly to untreated cauda spermatozoa.

  8. Redox regulation of mammalian sperm capacitation

    Directory of Open Access Journals (Sweden)

    Cristian O′Flaherty

    2015-01-01

    Full Text Available Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P H for sperm capacitation. Peroxiredoxins (PRDXs are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility.

  9. Molecular cloning and partial characterization of a plant VAP33 homologue with a major sperm protein domain

    NARCIS (Netherlands)

    Laurent, F.; Labesse, G.; Wit, de P.

    2000-01-01

    In a search for proteins interacting with the resistance protein Cf9 from tomato, a new cDNA was cloned and characterized. Protein sequence database searches suggested that the 120 residue-N terminal domain of the encoded protein (named VAP27) is highly similar to the VAP33 protein family from anima

  10. Sperm release pathway

    Science.gov (United States)

    ... called seminiferous tubules, which are the sites of sperm production. The structure on top of the seminiferous tubules in the testes is the epididymis. The sperm migrate from of the seminiferous tubules to the ...

  11. Intracytoplasmic sperm injection

    Science.gov (United States)

    Intracytoplasmic sperm injection, or ICSI, is a form of in vitro fertilization in which fertilization occurs outside of the ... laboratory dish. Within a few hours, a single sperm is injected through a fine needle into the ...

  12. Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration

    Institute of Scientific and Technical Information of China (English)

    Qi Chen; Hongying Peng; Li Lei; Ying Zhang; Haibin Kuang; Yujing Cao; Qi-xian Shi; Tonghui Ma; Enkui Duan

    2011-01-01

    In the journey from the male to female reproductive tract,mammalian sperm experience a natural osmotic decrease (e.g.,in mouse,from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation,meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however,no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here,we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling,characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet,forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct,resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the "trade-off" between sperm motility and cell swelling upon physiological hypotonicity,thereby optimizing postcopulatory sperm behavior.

  13. Etiologies of sperm oxidative stress

    Directory of Open Access Journals (Sweden)

    Parvin Sabeti

    2016-04-01

    Full Text Available Sperm is particularly susceptible to reactive oxygen species (ROS during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions

  14. Intracytoplasmic Sperm Injection (ICSI)

    Science.gov (United States)

    ... sperm must attach to the outside of the egg. Once attached, the sperm pushes through the outer layer to the inside ... in vitro fertilization (IVF) to help fertilize the egg. During ICSI, a single sperm is injected directly into the cytoplasm the egg. ...

  15. Sperm preparation for fertilization

    NARCIS (Netherlands)

    Gadella, B.M.

    2014-01-01

    Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen cry

  16. Cryoprotective effect of resveratrol on DNA damage and crucial human sperm messenger RNAs, possibly through 5' AMP-activated protein kinase activation.

    Science.gov (United States)

    Shabani Nashtaei, Maryam; Nekoonam, Saeid; Naji, Mohammad; Bakhshalizadeh, Shabnam; Amidi, Fardin

    2017-07-26

    This work aimed at investigating the effect of resveratrol on (1) DNA integrity and (2) fertilizing capacity of sperm by quantifying the presence of key paternal transcripts considered as markers for male fertility (protamine 1 [PRM1] and protamine 2 [PRM2]) and pregnancy success (adducin 1 alpha [ADD1]) in cryopreserved human spermatozoa through modulation of AMP-activated protein kinase (AMPK). The study populations was drawn from 22 normozoospermic healthy volunteers which were incubated with or without AMPK activator (resveratrol [RSV], 15 µM) or inhibitor (Compound C [CC], 30 µM) for 1 h and were then cryopreserved. Untreated frozen-thawed spermatozoa served as controls. The RSV-induced AMPK activation decreased the level of DNA fragmentation in comparison with the control (21.18 ± 0.92 vs. 22.50 ± 0.40; p < 0.01). The relative mRNA expression levels of protamines (1 and 2) and ADD1 in RSV pretreated frozen-thawed human spermatozoa were also improved significantly compared to the control (p < 0.05). Conversely, the inhibitory effect of CC on AMPK activity deteriorated the deleterious effects of cryopreservation on these parameters (p < 0.01). In conclusion, these results demonstrated the cryoprotective effect of the RSV-induced increase in AMPK activity on DNA integrity and key paternal transcripts of cryopreserved human spermatozoa. These findings are of great importance for improving the available cryopreservation protocols in terms of the number of lesions that produced over key genes and the dramatic effects on sperm DNA fragmentation.

  17. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  18. Comparative Sperm Proteomics in Mouse Species with Divergent Mating Systems

    Science.gov (United States)

    Vicens, Alberto; Borziak, Kirill; Karr, Timothy L.; Roldan, Eduardo R.S.

    2017-01-01

    Abstract Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete. PMID:28333336

  19. Gamete evolution and sperm numbers: sperm competition versus sperm limitation.

    Science.gov (United States)

    Parker, Geoff A; Lehtonen, Jussi

    2014-09-22

    Both gamete competition and gamete limitation can generate anisogamy from ancestral isogamy, and both sperm competition (SC) and sperm limitation (SL) can increase sperm numbers. Here, we compare the marginal benefits due to these two components at any given population level of sperm production using the risk and intensity models in sperm economics. We show quite generally for the intensity model (where N males compete for each set of eggs) that however severe the degree of SL, if there is at least one competitor for fertilization (N - 1 ≥ 1), the marginal gains through SC exceed those for SL, provided that the relationship between the probability of fertilization (F) and increasing sperm numbers (x) is a concave function. In the risk model, as fertility F increases from 0 to 1.0, the threshold SC risk (the probability q that two males compete for fertilization) for SC to be the dominant force drops from 1.0 to 0. The gamete competition and gamete limitation theories for the evolution of anisogamy rely on very similar considerations: our results imply that gamete limitation could dominate only if ancestral reproduction took place in highly isolated, small spawning groups. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  20. Daily sperm production

    DEFF Research Database (Denmark)

    Kyjovska, Zdenka Orabi; Boisen, Anne Mette Zenner; Jackson, Petra

    2013-01-01

    instillation with Printex90. Body and testicle weight, sperm content per g testicular parenchyma and daily sperm production (DSP) were assessed. The protocol for assessment of DSP was optimized for application in mice (C57BL/6J) and the influence of different parameters was studied. Maternal particulate...... exposure did not affect DSP statistically significantly in the F1 generation, although TiO2 tended to reduce sperm counts. Overall, time-to-first F2 litter increased with decreasing sperm production. There was no effect on sperm production in the F2 generation originating after TiO2 exposure. F2 offspring......, whose fathers were prenatally exposed to Printex90, showed lowered sperm production. Furthermore, we report statistically significant differences in sperm production between mouse strains....

  1. Expression and Purification of Sperm Whale Myoglobin

    Science.gov (United States)

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline

    2010-01-01

    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  2. Expression and Purification of Sperm Whale Myoglobin

    Science.gov (United States)

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline

    2010-01-01

    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  3. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle.

    Science.gov (United States)

    Zhong, Wenbin; Zhou, You; Li, Jiwei; Mysore, Raghavendra; Luo, Wei; Li, Shiqian; Chang, Mau-Sun; Olkkonen, Vesa M; Yan, Daoguang

    2014-04-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle.

  4. Sperm navigation in complex environments

    Science.gov (United States)

    Olson, Sarah

    2016-11-01

    Sperm can swim in a variety of environments, interacting with chemicals and other proteins in the fluid. Some of these extra proteins or cells may act as friction, possibly preventing or enhancing forward progression of swimmers. The homogenized fluid flow is assumed to be governed by the incompressible Brinkman equation, where a friction term with a resistance parameter represents a sparse array of obstacles. Representing the swimmers with a centerline approximation, we employ regularized fundamental solutions to investigate swimming speeds, trajectories, and interactions of swimmers. Asymmetric waveforms due to an increase in flagellar calcium is known to be important for sperm to reach and fertilize the egg. The trajectories of hyperactivated swimmers are found to have a decreased path curvature. Although attraction of two swimmers is more efficient in the Stokes regime, we find that attraction does not occur for larger resistance. Additionally, we study interactions of swimmers in a channel. NSF DMS 1413110.

  5. Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.

    Science.gov (United States)

    Coonrod, S A; Naaby-Hansen, S; Shetty, J; Shibahara, H; Chen, M; White, J M; Herr, J C

    1999-03-15

    The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster

  6. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Wenbin [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Zhou, You [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Li, Jiwei [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Mysore, Raghavendra [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Luo, Wei; Li, Shiqian [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Chang, Mau-Sun [Institute of Biochemical Sciences, National Taiwan University, No. 1, Taipei, Taiwan (China); Olkkonen, Vesa M. [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Yan, Daoguang, E-mail: tydg@jnu.edu.cn [Department of Biotechnology, Jinan University, Guangzhou 510632 (China)

    2014-04-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle.

  7. Sperm macromolecules associated with bull fertility.

    Science.gov (United States)

    Kaya, Abdullah; Memili, Erdoğan

    2016-06-01

    Bull fertility, ability of the sperm to fertilize and activate the egg that sustain embryo development, is vitally important for effective and efficient production of cattle. Fertility is a complex trait with low heritability. Despite recent advances in genomic selection and possibility of enormous paternal benefits to profitable cattle production, there exist no reliable tests for evaluating semen quality and predicting bull fertility. This review focuses on sperm macromolecules such as transcripts, proteins and the epigenome, i.e., the functional genome that are associated with bull fertility. Generating new information in these systems is important beyond agriculture because such progress advances the fundamental science of the mammalian male gamete while at the same time introduces biotechnology into livestock production. Sperm macromolecules and epigenome markers associated with bull fertility can be used alone or in combination with the current SNP microarrays to determine sperm quality and to indicate bull fertility.

  8. Korean red ginseng extract rejuvenates testicular ineffectiveness and sperm maturation process in aged rats by regulating redox proteins and oxidative defense mechanisms.

    Science.gov (United States)

    Kopalli, Spandana Rajendra; Hwang, Seock-Yeon; Won, Yu-Jin; Kim, Sung-Won; Cha, Kyu-Min; Han, Chang-Kyun; Hong, Jae-Yup; Kim, Si-Kwan

    2015-09-01

    Distortion of intracellular oxidant and antioxidant balances appears to be a common feature that underlies in age-related male sexual impairment. Therefore regulating oxidative defense mechanisms might be an ideal approach in improving male sexual dysfunctions. In the present study, the effect of Korean red ginseng aqueous extract (KRG) on age-induced testicular dysfunction in rats was investigated. KRG (200mg/kg) mixed with regular pellet diet was administered orally for six months and the morphological, spermatogenic and antioxidant enzyme status in testis of aged rats (18months) were evaluated. Data indicated a significant change in morphology and decrease in spermatogenesis-related parameters in aged rats (AC) compared with young rats (YC). Sperm number, germ cell count, Sertoli cell count and Sertoli cell index were significantly (pglutathione peroxidase, glutathione S-transferase, glutathione reductase and catalase) and non-enzymatic (reduced glutathione, ascorbic acid and α-tocopherol) antioxidants (ptesticular ineffectiveness in rats by modulating redox proteins and oxidative defense mechanisms.

  9. Production and glycosylation of sperm constitutive proteins in the lizard Lacerta vivipara. Evolution during the reproductive period.

    Science.gov (United States)

    Depeiges, A; Force, A; Dufaure, J P

    1987-01-01

    From epididymal fluid samples taken at three different times during the reproductive period (early April, late April, mid-May), the soluble proteins were separated with one dimensional electrophoresis on polyacrylamide gel. Their evolution was studied: firstly quantitatively, after staining with Coomassie blue, or, for one protein (the "L" protein), by immunodetection; secondly, according to their glycosylation after transfer to nitrocellulose and treatment with a set of labelled lectins: from Wheat germ, Ricinus communis, Lens culinaris, Asparagus pea or Canavalia ensiformis, with or without use of their specific inhibitor sugars. At least 15 proteins underwent a quantitative and/or qualitative evolution, mainly during the month of April. Protein "L" (19 kDa), which is androgen dependent and which fixates on to spermatozoa during their epididymal transit, appears to be little or not glycosylated. By contrast its accumulation in the epididymal canal increases considerably during the month of April. Five other proteins proved to be especially interesting because of their evolution during this same period, notably the MW 94, 67, 35, 29 and 25.5 kDa proteins. With the exception of the 67 kDa all the others increased quantitatively. All were decisively enriched in mannose or in methyl-mannoside residues. The proteins of MW 29 and 25.5 kDa were also enriched in galactose or N-acetyl galactosamine residues. These findings are of physiological significance since they are set up concomitantly with the acquisition of maximum motility of spermatozoa in the distal segment of the epididymis, and they coincide with a very great increase in testosteronemia.

  10. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    Directory of Open Access Journals (Sweden)

    Shawn W Zimmerman

    Full Text Available Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL, a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and

  11. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    Science.gov (United States)

    Zimmerman, Shawn W; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K; Sutovsky, Miriam; Odhiambo, John F; Powell, Michael D; Miller, David J; Sutovsky, Peter

    2011-02-23

    Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

  12. An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm

    Directory of Open Access Journals (Sweden)

    Shiv Kumar Yadav

    2017-05-01

    Full Text Available Separation of X- and Y-chromosome bearing sperm has been practiced for selection of desired sex of offspring to increase the profit in livestock industries. At present, fluorescence-activated cell sorter is the only successful method for separation of X- and Y-chromosome bearing sperm. This technology is based on the differences in DNA content between these two types of sperm and has been commercialized for bovine sperm. However, this technology still has problems in terms of high economic cost, sperm damage, and lower pregnancy rates compared to unsorted semen. Therefore, an inexpensive, convenient, and non-invasive approach for sperm sexing would be of benefit to agricultural sector. Within this perspective, immunological sperm sexing method is one of the attractive choices to separate X- and Y-chromosome bearing sperm. This article reviews the current knowledge about immunological approaches, viz., H-Y antigen, sex-specific antigens, and differentially expressed proteins for sperm sexing. Moreover, this review also highlighted the different methods for identification of X- and Y-sperm.

  13. IgSF8 (EWI-2) and CD9 in fertilization: Evidence of distinct functions for CD9 and a CD9-associated protein in mammalian sperm-egg interaction

    OpenAIRE

    Glazar, Amanda I.; Evans, Janice P.

    2009-01-01

    On the mouse egg, the tetraspanin CD9 is nearly essential for sperm-egg fusion, with another tetraspanin, CD81, playing a complementary role. Based on what is known about these proteins, egg tetraspanins are likely to be involved in regulation of membrane order through associations with other egg membrane proteins. Here we identify a first-level interaction (stable in 1% Triton X-100) between CD9 and the Immunoglobulin Superfamily member IgSF8 (also known as EWI-2), the first evidence in eggs...

  14. Lifestyle influences human sperm functional quality

    Institute of Scientific and Technical Information of China (English)

    Mnica Ferreira; Joana Vieira Silva; Vladimiro Silva; Antnio Barros; Margarida Fardilha

    2012-01-01

    Objective:To investigate the impact of acute lifestyle changes on human sperm functional quality.Methods:In the academic festivities week, young and apparently healthy male students who voluntarily submit themselves to acute lifestyle alterations(among the potentially important variations are increase in alcohol, caffeine, and tobacco consumption and circadian rhythm shifts) were used as a model system.Sperm samples were obtained before and after the academic week and compared by traditional semen analysis(n=54) and also tested for cleavedPolyADP-ribose polymerase(PARP) protein, an apoptotic marker(n=35).Results:Acute lifestyle changes that occurred during the academic week festivities(the study model) resulted both in a significant reduction in sperm quality, assessed by basic semen analysis(decrease in sperm concentration, total number of spermatozoa, progressive and non-progressive motility and increase in sperm morphological abnormalities) and by an increase in the expression of the apoptotic marker, cleavedPARP, in the ejaculate.Conclusions:Acute lifestyle changes have clear deleterious effects on sperm quality.We propose cleavedPARP as a novel molecular marker, valuable for assessing spermquality in parallel with the basic semen analysis method.

  15. Major regulatory mechanisms involved in sperm motility.

    Science.gov (United States)

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

  16. Role of the epididymis in sperm competition

    Institute of Scientific and Technical Information of China (English)

    Russell C. Jones; Jean-Louis Dacheux; Brett Nixon; Heath W. Ecroyd

    2007-01-01

    Although it is generally understood that the testes recruited kidney ducts for reproductive function during the evolution of vertebrates, little is understood of the biological significance of the adaptation. In the context of the evolution of the mammalian epididymis, this report provides evidence that a major role of the epididymis is to enhance a male's chance of achieving paternity in a competitive mating system. A unique example of sperm cooperation in monotremes is used as evidence that the epididymis produces sperm competition proteins to form groups of 100 sperm into bundles that have a forward motility nearly thrice that of individual spermatozoa. As it required 3-h incubation in vitro under capacitation conditions to release motile sperm from the bundles, it is suggested that the monotremes provide an example of capacitation that is quite different from capacitation in higher mammals. It is Suggested that variation between species in the intensity of sperm competition could explain the variation that occurs between species in the amount of post-testicular sperm maturation and storage in the epididymis, an explanation of why the human epididymis does not play as important a role in reproduction as the epididymis of most mammals.

  17. Sperm-egg adhesion and fusion in mammals.

    Science.gov (United States)

    Sutovsky, Peter

    2009-04-01

    Fertilisation is an orchestrated, stepwise process during which the participating male and female gametes undergo irreversible changes, losing some of their structural components while contributing others to the resultant zygote. Following sperm penetration through the egg coat, the sperm plasma membrane fuses with its oocyte counterpart, the oolemma. At least two plasma membrane proteins essential for sperm-oolemma fusion--IZUMO and CD9 on the male and female gametes, respectively--have been identified recently by classical cell biology approaches and confirmed by gene deletion. Oolemma-associated tetraspanin CD81, closely related to CD9, also appears to have an essential role in fusion. Additional proteins that may have nonessential yet still facilitating roles in sperm-oolemma adhesion and fusion include oolemma-anchored integrins and oocyte-expressed retroviral envelope proteins, sperm disintegrins, and sperm-borne proteins of epididymal origin such as CRISP1 and CRISP2. This review discusses these components of the gamete fusion mechanism within the framework of gamete structure, membrane biology, cell signalling and cytoskeletal dynamics, and revisits the topic of antipolyspermy defence at the oolemma level. Harnessing the mechanisms of sperm-egg fusion is of importance to animal biotechnology and to human assisted fertilisation, wherein male patients with reduced sperm fusibility have been identified.

  18. Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage

    DEFF Research Database (Denmark)

    Den Boer, Susanne Petronella A; Boomsma, Jacobus Jan; Baer, Boris

    2009-01-01

    Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here...... we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect...... on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion...

  19. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    Science.gov (United States)

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  20. Presence and function of dopamine transporter (DAT in stallion sperm: dopamine modulates sperm motility and acrosomal integrity.

    Directory of Open Access Journals (Sweden)

    Javier A Urra

    Full Text Available Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT, serotonin (SERT and norepinephrine (NET transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylaminostyryl]-N-methylpyridinium iodide (ASP(+, as substrate. In addition, we also showed that dopamine (1 mM treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909 and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.

  1. Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity.

    Science.gov (United States)

    Urra, Javier A; Villaroel-Espíndola, Franz; Covarrubias, Alejandra A; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I

    2014-01-01

    Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP(+)), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.

  2. The human sperm proteome 2.0: An integrated resource for studying sperm functions at the level of posttranslational modification.

    Science.gov (United States)

    Wang, Ying; Wan, Jinyuan; Ling, Xiufeng; Liu, Mingxi; Zhou, Tao

    2016-10-01

    Various types of PTMs play important roles in the regulation of sperm proteins. However, most large-scale proteomic studies only focused on a single type of modification due to the limitation of enrichment methods. To investigate the complex composition of modified sperm proteins, we constructed the human sperm proteome 2.0 that integrated lysine acetylated, phosphorylated, N-linked glycosylated, and protein N-terminal acetylated proteins from previously published proteomic datasets. A total of 6069 modified sites on 2132 proteins were annotated. Functional enrichment analyses showed that different types of modified sperm proteins displayed different functional distributions. We found that acetylated, phosphorylated, and glycosylated proteins are more directly involved in sperm functions. While N-termnial acetylated proteins and nonmodified proteins appear to be more associated with the basic cellular functions. Thus, it is efficient to search for fertility-associated biomarkers in acetylated, phosphorylated, and glycosylated proteins. We also predicted modification cross-talks within the same proteins or between different proteins that provided potential hotspot targets for understanding the regulation of sperm functions via multiple modifications.

  3. Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage

    DEFF Research Database (Denmark)

    Den Boer, Susanne Petronella A; Boomsma, Jacobus Jan; Baer, Boris

    2009-01-01

    Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. He...

  4. Sperm length, sperm storage and mating system characteristics in bumblebees

    DEFF Research Database (Denmark)

    Baer, Boris; Schmid-Hempel, Paul; Høeg, Jens Thorvald

    2003-01-01

    of ejaculated sperm that was stored in a queen's spermatheca. Both longer sperm and shorter sperm could be preferentially stored, depending on the colony in which the males and queens were born and raised. These results indicate that the genotype of males may affect sperm length and that cryptic female choice...

  5. Sperm competition and the evolution of sperm design in mammals

    Directory of Open Access Journals (Sweden)

    Gomendio Montserrat

    2011-01-01

    Full Text Available Abstract Background The influence of sperm competition upon sperm size has been a controversial issue during the last 20 years which remains unresolved for mammals. The hypothesis that, when ejaculates compete with rival males, an increase in sperm size would make sperm more competitive because it would increase sperm swimming speed, has generated contradictory results from both theoretical and empirical studies. In addition, the debate has extended to which sperm components should increase in size: the midpiece to accommodate more mitochondria and produce more energy to fuel motility, or the principal piece to generate greater propulsion forces. Results In this study we examined the influence of sperm competition upon sperm design in mammals using a much larger data set (226 species than in previous analyses, and we corrected for phylogenetic effects by using a more complete and resolved phylogeny, and more robust phylogenetic control methods. Our results show that, as sperm competition increases, all sperm components increase in an integrated manner and sperm heads become more elongated. The increase in sperm length was found to be associated with enhanced swimming velocity, an adaptive trait under sperm competition. Conclusions We conclude that sperm competition has played an important role in the evolution of sperm design in mammals, and discuss why previous studies have failed to detect it.

  6. Sperm competition and the evolution of sperm design in mammals.

    Science.gov (United States)

    Tourmente, Maximiliano; Gomendio, Montserrat; Roldan, Eduardo R S

    2011-01-13

    The influence of sperm competition upon sperm size has been a controversial issue during the last 20 years which remains unresolved for mammals. The hypothesis that, when ejaculates compete with rival males, an increase in sperm size would make sperm more competitive because it would increase sperm swimming speed, has generated contradictory results from both theoretical and empirical studies. In addition, the debate has extended to which sperm components should increase in size: the midpiece to accommodate more mitochondria and produce more energy to fuel motility, or the principal piece to generate greater propulsion forces. In this study we examined the influence of sperm competition upon sperm design in mammals using a much larger data set (226 species) than in previous analyses, and we corrected for phylogenetic effects by using a more complete and resolved phylogeny, and more robust phylogenetic control methods. Our results show that, as sperm competition increases, all sperm components increase in an integrated manner and sperm heads become more elongated. The increase in sperm length was found to be associated with enhanced swimming velocity, an adaptive trait under sperm competition. We conclude that sperm competition has played an important role in the evolution of sperm design in mammals, and discuss why previous studies have failed to detect it.

  7. Nuclear Sm antigens in the sperm of different organisms.

    Science.gov (United States)

    Delgado, F; Brito, M; Concha, I I; Schroeder, R; Burzio, L O

    1994-08-01

    Immunoblot analysis of sperm protein from several species revealed the presence of polypeptides recognised by anti-Sm sera obtained from patients with systemic lupus erythematosus. Immunoreactive polypeptides in human, bull, mouse and rat sperm were identified as protein B', B and D as compared with the Sm polypeptides of HeLa cells. In the sperm of rooster, the teleost fish Cyprinus carpio and the mussel Choromytilus chorus, the immunoreactive polypeptide profile was more complex. To ascertain the sperm origin of the Sm antigens, immunolocalisation with anti-Sm serum was carried out. The results demonstrated that in all the species studied staining was confined to the sperm nucleus, confirming that some polypeptides of the small nuclear ribonucleoprotein complex are present in the gamete.

  8. Aquaporins in sperm osmoadaptation: an emerging role for volume regulation

    Institute of Scientific and Technical Information of China (English)

    Qi CHEN; En-kui DUAN

    2011-01-01

    Upon ejaculation, mammalian sperm experience a natural osmotic decrease during male to female reproductive tract transition. This hypo-osmotic exposure not only activates sperm motility, but also poses potential harm to sperm structure and function by inducing unwanted cell swelling. In this physiological context, regulatory volume decrease (RVD) is the major mechanism that protects cells from detrimental swelling, and is essential to sperm survival and normal function. Aquaporins are selective water channels that enable rapid water transport across cell membranes. Aquaporins have been implicated in sperm osmoregulation. Recent discoveries show that Aquaporin-3 (AQP3), a water channel protein, is localized in sperm tail membranes and that AQP3 mutant sperm show defects in volume regulation and excessive cell swelling upon physiological hypotonic stress in the female reproductive tract, thereby highlighting the importance of AQP3 in the postcopulatory sperm RVD process. In this paper, we discuss current knowledge, remaining questions and hypotheses about the function and mechanismic basis of aquaporins for volume regulation in sperm and other cell types.

  9. Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm-oviduct binding.

    Science.gov (United States)

    Leemans, Bart; Gadella, Bart M; Stout, Tom A E; Sostaric, Edita; De Schauwer, Catharina; Nelis, Hilde; Hoogewijs, Maarten; Van Soom, Ann

    2016-04-01

    In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been characterized as a key molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca(2+) and HCO3(-) on sperm-oviduct binding in the horse. Carbohydrate-specific lectin staining indicated that N-acetylgalactosamine, N-acetylneuraminic acid (sialic acid) and D-mannose or D-glucose were the most abundant carbohydrates on equine oviduct epithelia, whereas D-galactose moieties were not detected. However, in a competitive binding assay, sperm-oviduct binding density was not influenced by any tested carbohydrates, glycosaminoglycans, lectins or D-penicillamine, nor did the glycosaminoglycans induce sperm tail-associated protein tyrosine phosphorylation. Furthermore, N-glycosidase F (PNGase) pretreatment of oviduct explants and APM did not alter sperm-oviduct binding density. By contrast, a combination of the sperm-capacitating factors albumin and HCO3(-) severely reduced (>10-fold) equine sperm-oviduct binding density by inducing rapid head-to-head agglutination, both of which events were independent of Ca(2+) and an elevated pH (7.9). Conversely, neither albumin and HCO3(-) nor any other capacitating factor could induce release of oviduct-bound sperm. In conclusion, a combination of albumin and HCO3(-) markedly induced sperm head-to-head agglutination which physically prevented stallion sperm to bind to oviduct epithelium.

  10. The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, 31P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition

    Directory of Open Access Journals (Sweden)

    Müller Karin

    2009-11-01

    Full Text Available Abstract Background Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. Results Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. Conclusion The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the

  11. Direct visualization of sperm competition and sperm storage in Drosophila.

    Science.gov (United States)

    Civetta, A

    Drosophila females engage in multiple matings [1] [2] [3] [4] even though they can store sperm in specialized organs for most of their life [5]. The existence of sperm competition in Drosophila has been inferred from the proportion of progeny sired by the second male in double-mating experiments [6] [7] [8]. Investigators have used this approach to quantify genetic variation underlying sperm competition [8] [9] [10], to elucidate its genetic basis [11], to identify the dependence of different male competitive ability on the genotype of the females with which they mate [12] and to discern the potential role of sperm competition in species isolation [13] [14]. This approach assumes that the sperm from two males stored in a female compete to fertilize the eggs. The mechanism by which sperm competition is accomplished is still unknown, however. Here, fluorescence microscopy, cytometry, and differently labeled sperm were used to analyze the fate of sperm inside the female's sperm storage organs, to quantify sperm competition, and to assess how closely paternity success corresponds to the appearance and location of the sperm. The results show that the first male's sperm is retained for a shortened period if the female remates, and that the second males that sire more progeny either induce females to store and use more of their sperm or strongly displace resident sperm.

  12. 金黄地鼠腹侧前列腺在体修饰精子膜蛋白作用研究%Ventral Prostate Glands Modification of Sperm Membrane Proteins in Golden Hamster

    Institute of Scientific and Technical Information of China (English)

    骆建民; 郑玉鸾; 周白菡

    2008-01-01

    [Objective] To investigate the effect of the secretory proteins of the ventral prostate glands on the sperm membrane proteins in golden hamsters. [Methods] The sperm was collected from female hamsters uteri after mated with the males with or without ventral prostate gland. Male golden hamsters were divided into four experimental groups: (i) all accessory sex glands (ASG) removed; (ii) ventral prostate gland removed; (iii) all ASG removed except ventral prostate gland and (iv) sham-operated controls. Each group contained 6 hamsters. The sperm membrane proteins were extracted from uterine sperm and analyzed by SDS-PAGE and two dimension electrophoresis. [Results] The SDS-PAGE results of sperm membrane showed that the ventral prostate glands added up some proteins or increased the quantity of some proteins, which contained molecular mass (MM)15 k, 29 k, 38 k, 55 k, and 91 k proteins, to the sperm membrane. 2D-electrophoresis of sperm membrane showed that some extra protein spots were appeared in with ventral prostate gland groups, their MM and isoelectric point (IP) corresponding to 16 k/8.60, 16.6 k/9.20, 28 k/5.88, 28 k/6.10, 29 k/5.98, 32 k/6.35, 32 k/6.50, 32 k/7.20, 61 k/5.90,and 83 k/6.40, respectively. The quantity of some protein spots increased in sperm membrane of ventral prostate glands group, their MM/IP coresponding to 17 k/5.95, 17.5 k/6.50, 24 k/7.20, 26 k/5.40, 26 k/5.60, 27 k/7.20, 27 k/7.50, 28 k/5.70, 29 k/8.50, 42 k/6.50, and 42 k/7.00, respectively. [Conclusion] The ventral prostate gland secretion plays a role in regulating the sperm membrane proteins and the latter may in turn affect the male fertility or embryo development.%[目的]在整体水平探讨前列腺分泌蛋白对金黄地鼠精子膜蛋白的影响.[方法]雄性鼠手术分为4组,分别为附属性腺全去组,腹前列腺摘除组,去除其他附属性腺仅保留腹前列腺组和假手术组,每组动物6只.收集与保留前列腺及去除组的雄性鼠交配后的子宫

  13. Chemotactic Maneuverability of Sperm

    CERN Document Server

    Guasto, Jeffrey S; Zimmer, Richard K; Stocker, Roman

    2011-01-01

    In this fluid mechanics video, we explore the kinematics of chemotaxing sperm cells (sea urchin, \\textit{Arbacia punctulata}) swimming in a chemoattractant gradient. We demonstrate that the complex swimming trajectories resulting in chemotactic behavior (`turn-and-run' motility) are comprised of several distinct flagellar maneuvers. These motility patterns likely play an important role optimizing chemotaxic motility and navigation, when the sperm cells are subjected external fluid flows.

  14. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

    Directory of Open Access Journals (Sweden)

    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  15. Accessory sperm: a biomonitor of boar sperm fertilization capacity.

    Science.gov (United States)

    Ardón, Florencia; Evert, Meike; Beyerbach, Martin; Weitze, Karl-Fritz; Waberski, Dagmar

    2005-04-15

    The number of accessory sperm found in the zona pellucida of porcine embryos was correlated to their individual quality and to the embryo quality range found within a single sow. Our goal was to determine whether accessory sperm counts provide semen evaluation with additional, useful information. Accessory sperm count was highest when only normal embryos were found in a given sow and diminished if oocytes or degenerated embryos were present (P<0.01). Within a given sow, normal embryos had higher (P<0.05) accessory sperm counts than degenerated embryos, although not when oocytes were also present. Fertilization capacity of sperm is optimal when only normal embryos are found in a given sow; this capacity is indicated by high accessory sperm counts. A decrease in fertilization capacity is reflected in diminishing accessory sperm counts. The boar had a significant effect (P<0.01) on accessory sperm count, but not on the percentage of normal embryos; this suggests that accessory sperm may be more sensitive indicators of the fertilization capacity of sperm than the percentage of normal embryos. We conclude that accessory sperm count can be used for the detection of compensable defects in sperm and is a valid parameter for assessing sperm fertilization capacity.

  16. Sperm Meets Egg: The Genetics of Mammalian Fertilization.

    Science.gov (United States)

    Bianchi, Enrica; Wright, Gavin J

    2016-11-23

    Fertilization is the culminating event of sexual reproduction, which involves the union of the sperm and egg to form a single, genetically distinct organism. Despite the fundamental role of fertilization, the basic mechanisms involved have remained poorly understood. However, these mechanisms must involve an ordered schedule of cellular recognition events between the sperm and egg to ensure successful fusion. In this article, we review recent progress in our molecular understanding of mammalian fertilization, highlighting the areas in which genetic approaches have been particularly informative and focusing especially on the roles of secreted and cell surface proteins, expressed in a sex-specific manner, that mediate sperm-egg interactions. We discuss how the sperm interacts with the female reproductive tract, zona pellucida, and the oolemma. Finally, we review recent progress made in elucidating the mechanisms that reduce polyspermy and ensure that eggs normally fuse with only a single sperm.

  17. A Systematic Analysis of a Deep Mouse Epididymal Sperm Proteome

    Energy Technology Data Exchange (ETDEWEB)

    Chauvin, Theodore; Xie, Fang; Liu, Tao; Nicora, Carrie D.; Yang, Feng; Camp, David G.; Smith, Richard D.; Roberts, Kenneth P.

    2012-12-21

    Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It is therefore theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end we have performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and have confidently identified 2,850 proteins, which is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, β-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

  18. Separating Subcellular Proteins in Uncapacitation and Capacitation of Boar Sperm Cells and Identifying the Tyrosine Phosphoprotein%猪精子获能前后细胞亚组分蛋白分离及酪氨酸磷酸化蛋白的鉴定

    Institute of Scientific and Technical Information of China (English)

    胡启蒙; 张媛媛; 陈振亮; 王亮亮; 李新红

    2012-01-01

    The aim of this study was to separate subsets proteins in uncapacitation and capacitation of boar sperm cells and to identify the protein tyrosine phosphorylation, and to establish the theoretical foundation for the mammalian sperm fertilization biology research. In vitro , the boar sperms were cultivated for capacitation and separated subsets proteins. The tyrosine phosphoryla-ted proteins in uncapacitation and capacitation sperms were identified by using SDS-PAGE and Western blot. In capacitation boar sperm, the degree of tyrosine phosphorylation on 126, 108, 79 ku proteins were significantly higher than them on uncapacition boar sperm. The vigor indexes and the results of SDS-PAGE of uncapacitation and capacitation sperms were different,all the vigor indexes of capacitation sperms were significantly improved and these changes were related with tyrosine phosphorylation. The molecular weight of tyrosine phosphorylated proteins on membrane were approximately 25, 47 and 50 ku and on cytoplasm is about 47 ku. The degree of tyrosine phosphoproteins of 25 and 47 ku in capacitation sperm were significantly higher than them in the uncapacitation sperm(P<0. 05). This phenomenon also existed in cell nucleus. In sperm nucleus,these proteins' molecular weight were 23, 37 and 42-50 ku. The degree of tyrosine phos-phrylated 23 ku protein in nucleus of capacitation sperms are higher. The results suggests that the main tyrosine phosphorylated proteins were on the nucleus and membrane proteins, and a few of them are plasma proteins.%本研究对猪精子获能前后细胞亚组分蛋白进行分离以及对酪氨酸磷酸化蛋白进行鉴定,旨在为哺乳动物精子受精生物学研究奠定理论基础.利用动物精子体外获能培养、细胞亚组分分离技术及蛋白免疫印迹的方法,分离猪精子细胞亚组分蛋白及酪氨酸磷酸化蛋白鉴定.结果表明,猪精子经过获能培养后各项活力指标均得到显著提高,且与精子蛋白发生

  19. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    Energy Technology Data Exchange (ETDEWEB)

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  20. Subversive practices of sperm donation - globalizing Danish sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    During the past two decades, Denmark has developed in to an important destination for fertility travellers in need of donor sperm. Furthermore, two of the largest sperm banks in Europe have been established in Denmark, exporting sperm globally. This development has taken place at the same time...

  1. Sperm Motility in Flow

    Science.gov (United States)

    Guasto, Jeffrey; Juarez, Gabriel; Stocker, Roman

    2012-11-01

    A wide variety of plants and animals reproduce sexually by releasing motile sperm that seek out a conspecific egg, for example in the reproductive tract for mammals or in the water column for externally fertilizing organisms. Sperm are aided in their quest by chemical cues, but must also contend with hydrodynamic forces, resulting from laminar flows in reproductive tracts or turbulence in aquatic habitats. To understand how velocity gradients affect motility, we subjected swimming sperm to a range of highly-controlled straining flows using a cross-flow microfluidic device. The motion of the cell body and flagellum were captured through high-speed video microscopy. The effects of flow on swimming are twofold. For moderate velocity gradients, flow simply advects and reorients cells, quenching their ability to cross streamlines. For high velocity gradients, fluid stresses hinder the internal bending of the flagellum, directly inhibiting motility. The transition between the two regimes is governed by the Sperm number, which compares the external viscous stresses with the internal elastic stresses. Ultimately, unraveling the role of flow in sperm motility will lead to a better understanding of population dynamics among aquatic organisms and infertility problems in humans.

  2. Identification of γ-aminobutyric acid transporter (GAT1) on the rat sperm

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Some recent studies indicated that GABAergic sys tem is involved in mammalian sperm acrosome reaction (AR), but direct evidence pertaining to the expression of gat1 in mammalian sperm is not yet demonstrated. In this study, we evaluated the presence of 67kDa GAT1 protein and mRNA in rat testis by Western blotting and reverse transcription-polymerase chain reaction. Meanwhile, im munohistochemical and immunofluorescent analyses also identified GAT1 protein on the elongated spermatid and sperm. These results indicated that rat testis is a novel site of gat1 expression. Further studies should be taken to explore the role of GAT1 protein on sperm acrosome reaction.

  3. Sorting of Sperm by Morphology

    Science.gov (United States)

    Koh, James; Marcos, Marcos

    2016-11-01

    Many studies have proven that the percentage of morphologically normal sperm is a significant factor in determining the success of assisted reproduction. The velocity of sperm in a microchannel with shear flow subjected to an external field will be explored theoretically. The difference in response between morphologically normal and abnormal sperm will be computed from a statistical approach, to study the feasibility and effectiveness of sorting by an external field to remove abnormal sperm. The full name of this author is Marcos.

  4. Enhancement of mouse sperm motility by trophinin-binding peptide

    Directory of Open Access Journals (Sweden)

    Park Seong

    2012-11-01

    Full Text Available Abstract Background Trophinin is an intrinsic membrane protein that forms a complex in the cytoplasm with bystin and tastin, linking it microtubule-associated motor dynein (ATPase in some cell types. Previously, we found that human sperm tails contain trophinin, bystin and tastin proteins, and that trophinin-binding GWRQ (glycine, tryptophan, arginine, glutamine peptide enhanced motility of human sperm. Methods Immunohistochemistry was employed to determine trophinin protein in mouse spermatozoa from wild type mouse, by using spermatozoa from trophinin null mutant mice as a negative control. Multivalent 8-branched GWRQ (glycine, tryptophan, arginine, glutamine peptide or GWRQ-MAPS, was chemically synthesized, purified by HPLC and its structure was confirmed by MALDI-TOF mass spectrometry. Effect of GWRQ-MAPS on mouse spermatozoa from wild type and trophinin null mutant was assessed by a computer-assisted semen analyzer (CASA. Results Anti-trophinin antibody stained the principal (central piece of the tail of wild type mouse sperm, whereas the antibody showed no staining on trophinin null sperm. Phage particles displaying GWRQ bound to the principal piece of sperm tail from wild type but not trophinin null mice. GWRQ-MAPS enhanced motility of spermatozoa from wild type but not trophinin null mice. CASA showed that GWRQ-MAPS enhanced both progressive motility and rapid motility in wild type mouse sperm. Conclusions Present study established the expression of trophinin in the mouse sperm tail and trophinin-dependent effect of GWRQ-MAPS on sperm motility. GWRQ causes a significant increase in sperm motility.

  5. Role of the oviduct in sperm capacitation.

    Science.gov (United States)

    Rodriguez-Martinez, H

    2007-09-01

    Following insemination of spermatozoa pre-ovulation, the mammalian oviduct ensures, by the formation of a functional sperm reservoir (SR), that suitable (low) numbers of viable and potentially fertile spermatozoa are available for fertilization at the ampullary isthmic junction (AIJ). As ovulation approaches, a proportion of the SR-stored spermatozoa is continuously distributed towards the AIJ and individually activated leading to step-wise capacitation and the attainment of hyperactivated motility. This paper reviews in vivo changes in the intra-luminal milieu of the oviduct of pigs and cows, in particular the SR and the AIJ which relate to the modulation of sperm capacitation around spontaneous ovulation. In vivo, most viable spermatozoa in the pre-ovulatory SR are uncapacitated. Capacitation rates significantly increase after ovulation, apparently not massively but concurrent with the individual, continuous sperm dislocation from the SR. Bicarbonate, whose levels differ between the SR and the AIJ, appears as the common primary effector of the membrane destabilizing changes that encompasses the first stages of capacitation. Sperm activation can be delayed or even reversed by co-incubation with membrane proteins of the tubal lining, isthmic fluid or specific tubal glycosaminoglycans, such as hyaluronan. Although the pattern of response to in vitro induction of sperm activation - capacitation in particular - is similar for all spermatozoa, the capacity and speed of the response is very individual. Such diversity in responsiveness among spermatozoa insures full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation at the AIJ, thus maximizing the chances of normal fertilization.

  6. Subversive practices of sperm donation - globalizing Danish sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    During the past two decades, Denmark has developed in to an important destination for fertility travellers in need of donor sperm. Furthermore, two of the largest sperm banks in Europe have been established in Denmark, exporting sperm globally. This development has taken place at the same time...... into how the bending of boundaries by “inappropriate parents”, fertility travellers, private sperm banks and fertility clinics have been part in negotiating the changes of the legislation in practice, and thus been part of developing a Danish industry of sperm banking. The presentation is based on a multi...

  7. TRPM8, a versatile channel in human sperm.

    Directory of Open Access Journals (Sweden)

    Gerardo A De Blas

    Full Text Available BACKGROUND: The transient receptor potential channel (TRP family includes more than 30 proteins; they participate in various Ca(2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+ ([Ca(2+]i. Ca(2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. PRINCIPAL FINDINGS: Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature and antagonists (BCTC and capsazepine. Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM and 80% by BCTC (1.6 microM. Activation of TRPM8 either by temperature or menthol induced [Ca(2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM and BCTC (1.6 microM. However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. CONCLUSIONS: This is the first report dealing with the presence of a thermo sensitive channel (TRPM8 in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

  8. TRPM8, a Versatile Channel in Human Sperm

    Science.gov (United States)

    Ocampo, Ana Y.; Serrano, Carmen J.; Castellano, Laura E.; Hernández-González, Enrique O.; Chirinos, Mayel; Larrea, Fernando; Beltrán, Carmen; Treviño, Claudia L.

    2009-01-01

    Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis. PMID:19582168

  9. Subversive practices of sperm donation - globalizing Danish sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    During the past two decades, Denmark has developed in to an important destination for fertility travellers in need of donor sperm. Furthermore, two of the largest sperm banks in Europe have been established in Denmark, exporting sperm globally. This development has taken place at the same time...... as the use of donated sperm continuously has been debated as an ethical issue, and increasingly been regulated. In this presentation I will discuss how Denmark became a destination for fertility travelling (sperm donation) as a result of various subversive strategies of family making. The article inquires...... into how the bending of boundaries by “inappropriate parents”, fertility travellers, private sperm banks and fertility clinics have been part in negotiating the changes of the legislation in practice, and thus been part of developing a Danish industry of sperm banking. The presentation is based on a multi...

  10. Subversive practices of sperm donation - globalizing Danish sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    During the past two decades, Denmark has developed in to an important destination for fertility travellers in need of donor sperm. Furthermore, two of the largest sperm banks in Europe have been established in Denmark, exporting sperm globally. This development has taken place at the same time...... as the use of donated sperm continuously has been debated as an ethical issue, and increasingly been regulated. In this presentation I will discuss how Denmark became a destination for fertility travelling (sperm donation) as a result of various subversive strategies of family making. The article inquires...... into how the bending of boundaries by “inappropriate parents”, fertility travellers, private sperm banks and fertility clinics have been part in negotiating the changes of the legislation in practice, and thus been part of developing a Danish industry of sperm banking. The presentation is based on a multi...

  11. Polyandry in the medfly - shifts in paternity mediated by sperm stratification and mixing.

    Science.gov (United States)

    Scolari, Francesca; Yuval, Boaz; Gomulski, Ludvik M; Schetelig, Marc F; Gabrieli, Paolo; Bassetti, Federico; Wimmer, Ernst A; Malacrida, Anna R; Gasperi, Giuliano

    2014-01-01

    In the Mediterranean fruit fly (medfly), Ceratitis capitata, a highly invasive agricultural pest species, polyandry, associated with sperm precedence, is a recurrent behaviour in the wild. The absence of tools for the unambiguous discrimination between competing sperm from different males in the complex female reproductive tract has strongly limited the understanding of mechanisms controlling sperm dynamics and use. Here we use transgenic medfly lines expressing green or red fluorescent proteins in the spermatozoa, which can be easily observed and unambiguously differentiated within the female fertilization chamber. In twice-mated females, one day after the second mating, sperm from the first male appeared to be homogenously distributed all over the distal portion of each alveolus within the fertilization chamber, whereas sperm from the second male were clearly concentrated in the central portion of each alveolus. This distinct stratified sperm distribution was not maintained over time, as green and red sperm appeared homogeneously mixed seven days after the second mating. This dynamic sperm storage pattern is mirrored by the paternal contribution in the progeny of twice-mated females. Polyandrous medfly females, unlike Drosophila, conserve sperm from two different mates to fertilize their eggs. From an evolutionary point of view, the storage of sperm in a stratified pattern by medfly females may initially favour the fresher ejaculate from the second male. However, as the second male's sperm gradually becomes depleted, the sperm from the first male becomes increasingly available for fertilization. The accumulation of sperm from different males will increase the overall genetic variability of the offspring and will ultimately affect the effective population size. From an applicative point of view, the dynamics of sperm storage and their temporal use by a polyandrous female may have an impact on the Sterile Insect Technique (SIT). Indeed, even if the female's last

  12. Microfluidic single sperm analysis

    NARCIS (Netherlands)

    Wagenaar, de Bjorn

    2016-01-01

    Microfluidic technology has been occasionally used for in vitro analysis and separation of cells. The small dimensions of microfluidic chips are very suitable to study cells on the single cell level rather than in whole populations. Also sperm cells have been studied and manipulated using microfluid

  13. Effects of Synthetic Serum Supplementation in Sperm Preparation Media on Sperm Capacitation and Function Test Results

    Directory of Open Access Journals (Sweden)

    Ying-Fu Shih

    2016-01-01

    Full Text Available Albumin supplementation of culture media induces sperm capacitation in assisted reproduction technique cycles. Synthetic serum supplementation is clinically used to replace albumin for preventing transmission of infectious agents. However, the effects of synthetic serum supplementation on sperm capacitation have rarely been investigated. Spermatozoa from 30 men with normal basic semen analysis results were collected, divided into five aliquots, and cultured in capacitating conditions in four combinations of two synthetic serum supplements, serum substitute supplement (SSS and serum protein substitute (SPS, and two fertilization media, Quinns Advantage™ Fertilization (QF and human tubular fluid (HTF media. Reactive oxygen species (ROS levels in spermatozoa were measured through chemiluminescence. Furthermore, acrosome reaction and western blotting for tyrosine phosphorylation were used to evaluate sperm capacitation. HTF+SSS had significantly higher ROS levels than QF+SPS did (11,725 ± 1,172 versus 6,278 ± 864 relative light units. In addition, the spermatozoa cultured in QF+SPS had lower motility, acrosome reaction rates, and tyrosine phosphorylation levels compared with those cultured in HTF+SSS. In conclusion, the effects of synthetic serum supplementation on sperm capacitation varied according to the combination of media. These differences may lead to variations in spermatozoon ROS levels, thus affecting sperm function test results.

  14. Cryptic choice of conspecific sperm controlled by the impact of ovarian fluid on sperm swimming behavior.

    Science.gov (United States)

    Yeates, Sarah E; Diamond, Sian E; Einum, Sigurd; Emerson, Brent C; Holt, William V; Gage, Matthew J G

    2013-12-01

    Despite evidence that variation in male-female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species' ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species' identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior.

  15. Expression of SEPT4 protein in the ejaculated sperm of idiopathic asthenozoospermic men%SEPT4蛋白在特发性弱精子症患者精子中的表达

    Institute of Scientific and Technical Information of China (English)

    李玉山; 冯晓霞; 吉晓菲; 王全先; 高学敏; 杨险峰; 潘周辉; 孙琳; 马奎

    2011-01-01

    Objective: To investigate the role of the SEPT4 protein in the pathogenesis of idiopathic asthenozoospermia.Methods: Samples of ejaculated sperm from idiopathic asthenozoospermia patients and normozoospermic men were separated and purified by Percoll discontinuous density gradients, the distribution and expression of SEPT4 in the sperm samples were determined by immunocytochemistry, and the expressions of SEPT4 mRNA and SEPT4 protein were detected by RT-PCR and Western blot.Results:Immunocytochemistry showed that the expression of SEPT4, located in the annulus, was significantly reduced in the sperm of the idiopathic asthenozoospermia patients (t = 3.452, P < 0.01 ).RT-PCR revealed that the expression of SEPT4 mRNA was significantly lower in the sperm of the idiopathic asthenozoospermia patients than in those of the normozoospermic men ( t = 3.521, P < 0.05 ).Western blot confirmed the results of RT-PCR (t = 5.872, P < 0.05).Conclusion: The expression of SEPT4 is significantly decreased in the ejaculated sperm of idiopathic asthenozoospennia patients, which might be one of the causes of idiopathic aathenozoospermia.%目的:探讨SEPT4蛋白在特发性弱精子症发生机制中的作用.方法:采用非连续密度梯度离心分离和纯化精子,免疫细胞化学技术检测SEPT4在特发性弱精子症患者和正常男性精子中的定位及表达变化,同时用RT-PCR和Western印迹技术,从基因和蛋白水平检测SEPT4的表达及差异.结果:免疫细胞化学表明,SEPT4定位于精子环并且在特发性弱精子症患者精子中的表达显著低于正常男性(94.75±19.95 vs 111.51±17.57,t=3.452,P<0.01);RT-PCR显示,特发性弱精子症患者精子中SEPT4 mRNA的表达水平与正常男性相比显著降低(0.56±0.15 vs 0.71±0.16,t=3.521,P<0.05);Western印迹结果与RT-PCR结果一致,SEPT4蛋白在特发性弱精子症患者精子中的表达亦显著低于正常男性(0.48±0.20 vs 0.77±0.18,t=5.872,P<0.05).结论:SEPT4在特

  16. The evolution of sperm morphometry in pheasants.

    Science.gov (United States)

    Immler, S; Saint-Jalme, M; Lesobre, L; Sorci, G; Roman, Y; Birkhead, T R

    2007-05-01

    Post-copulatory sexual selection is thought to be a potent evolutionary force driving the diversification of sperm shape and function across species. In birds, insemination and fertilization are separated in time and sperm storage increases the duration of sperm-female interaction and hence the opportunity for sperm competition and cryptic female choice. We performed a comparative study of 24 pheasant species (Phasianidae, Galliformes) to establish the relative importance of sperm competition and the duration of sperm storage for the evolution of sperm morphometry (i.e. size of different sperm traits). We found that sperm size traits were negatively associated with the duration of sperm storage but were independent of the risk of sperm competition estimated from relative testis mass. Our study emphasizes the importance of female reproductive biology for the evolution of sperm morphometry particularly in sperm-storing taxa.

  17. Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2.

    Science.gov (United States)

    Bench, G; Corzett, M H; Kramer, C E; Grant, P G; Balhorn, R

    2000-08-01

    Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.

  18. Changes in the distribution and molecular mass of boar sperm acrosome-associated 1 proteins during the acrosome reaction; their validity as indicators for occurrence of the true acrosome reaction.

    Science.gov (United States)

    Ogura, Yukari; Takagishi, Yuki; Harayama, Hiroshi

    2016-09-01

    The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca(2+)-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca(2+)-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28kDa) increased in extracellular Ca(2+)-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions.

  19. Differential proteome association study of freeze-thaw damage in ram sperm.

    Science.gov (United States)

    He, Yuxuan; Wang, Ke; Zhao, Xingxu; Zhang, Yong; Ma, Youji; Hu, Junjie

    2016-02-01

    In this study proteomics analysis was performed to investigate damage caused to ram sperm by the freeze-thaw process. Sperm motility, viability, reactive oxygen species (ROS) and adenosine triphosphate (ATP) content were measured to evaluate sperm quality. Compared with fresh groups, motility, viability and ATP content were all lower in freeze-thawed sperm (P < 0.001), and ROS content was higher (P < 0.001). Moreover, 25 differential protein spots were detected in two-dimensional gels using PDQuest 8.0 software and the corresponding proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) coupled with searching of the NCBI protein sequence database. Among these proteins, hexokinase1 (HXK1), the enzyme that catalyzes the first step of glycolysis in the sperm glycolytic pathway, is known to be associated with sperm motility. Casein kinase II subunit alpha (CSNK2A2), a serine/threonine-selective protein kinase, is associated with sperm apoptosis. We used immunoblotting and immunofluorescence to analyze the expression and localization of these two proteins. HXK1 and CSNK2A2 expression levels in fresh sperm were significantly higher than that in freeze-thawed sperm (P < 0.001). HXK1 and CSNK2A2 were detected in the main part of the sperm flagellum, and the immunofluorescence signal from these proteins was weakened in the freeze-thawed group. Decreased expression of HXK1 and CSNK2A2 may be associated with decreased sperm motility and viability following freeze-thawing.

  20. Sperm counts and sperm sex ratio in male infertility patients

    Institute of Scientific and Technical Information of China (English)

    Michael L Eisenberg; Lata Murthy; Kathleen Hwang; Dolores J Lamb; Larry I Lipshultz

    2012-01-01

    In recent years,investigators have noted a trend toward a declining proportion of male births in many industrialized nations.While men bear the sex-determining chromosome,the role of the female partner as it pertains to fertilization or miscarriage may also alter the gender ratio.We attempted to determine a man's secondary sex ratio (F1 generation) by directly examining the sex chromosomes of his sperm.We examined our male infertility clinic database for all men who had undergone a semen fluorescence in situ hybridization (FISH).Patient demographic and semen parameters were recorded.Chi-squared analysis was used to compare gender ratios (Ychromosomes/total chromosomes).Multivariable logistic regression was used to predict the odds of possessing a Y-bearing sperm after accounting for demographic and semen parameters.A total of 185 men underwent sperm FISH.For the entire cohort,the proportion of Y chromosome-bearing sperm was 51.5%.Men with less than five million motile sperm had a significantly lower proportion of Y chromosome-bearing sperm (50.8%) compared to men with higher sperm counts (51.6%; P=0.02).After multivariable adjustment,a higher sperm concentration,total motile sperm count and semen volume significantly increased the odds of having a Y chromosome-bearing sperm (P<0.01).As a man's sperm production declines,so does the proportion of Y chromosome-bearing sperm.Thus,a man's reproductive potential may predict his ability to sire male offspring.

  1. Morphology, Structure of Dimorphic Sperm, and Reproduction in the Hermaphroditic Commensal Bivalve Pseudopythina tsurumaru (Galeommatoidea: Kellidae)

    DEFF Research Database (Denmark)

    Lützen, Jørgen; Jespersen, Åse; Takahashi, Tohru

    2004-01-01

    Galeommatoide, commensal bivalve, reproduction, dimorphic sperm, sperm ultrastructure, spermatozeugma......Galeommatoide, commensal bivalve, reproduction, dimorphic sperm, sperm ultrastructure, spermatozeugma...

  2. Prostaglandin modulation of mouse and human sperm capacitation.

    Science.gov (United States)

    Herrero, M B; Viggiano, J M; Boquet, M; Gimeno, M A

    1997-09-01

    To determine whether prostaglandins produce a capacitation and/or acrosome reaction, the effect of prostaglandins on capacitated mouse spermatozoa and the effect of prostaglandin pre-incubation on human and mouse spermatozoa were studied. Prostaglandins did not induce an acrosome reaction in capacitated mouse sperm. PGE1 pre-incubation in a protein-free medium enhanced acrosome loss of mouse sperm challenged with A-23187 or solubilized mouse zona pellucida. Human sperm were pre-incubated in media containing prostaglandins, and an acrosome reaction was induced with calcium ionophore or human follicular fluid. PGE1 pre-incubation enhanced acrosome loss by human sperm when the action was induced with calcium ionophore, but had no effect on follicular fluid induction. We conclude that PGE1 acts as a capacitating factor in vitro for mouse spermatozoa, and enhances acrosome-reaction induction with calcium ionophore in human spermatozoa.

  3. Turbulence of swarming sperm

    Science.gov (United States)

    Creppy, Adama; Praud, Olivier; Druart, Xavier; Kohnke, Philippa L.; Plouraboué, Franck

    2015-09-01

    Collective motion of self-sustained swarming flows has recently provided examples of small-scale turbulence arising where viscous effects are dominant. We report the first observation of universal enstrophy cascade in concentrated swarming sperm consistent with a body of evidence built from various independent measurements. We found a well-defined k-3 power-law decay of a velocity field power spectrum and relative dispersion of small beads consistent with theoretical predictions in 2D turbulence. Concentrated living sperm displays long-range, correlated whirlpool structures of a size that provides an integral scale of turbulence. We propose a consistent explanation for this quasi-2D turbulence based on self-structured laminated flow forced by steric interactions and alignment, a state of active matter that we call "swarming liquid crystal." We develop scaling arguments consistent with this interpretation.

  4. Do candidate genes mediating conspecific sperm precedence affect sperm competitive ability within species? A test case in Drosophila.

    Science.gov (United States)

    Civetta, Alberto; Finn, Scott

    2014-07-16

    When females mate to multiple males, the last male to mate fathers the majority of progeny. When males of different species inseminate a female, the sperm of the male conspecific to the female is favored in fertilization in a process known as conspecific sperm precedence (CSP). A large number of studies in Drosophila have assayed the genetic basis of sperm competition, with a main focus on D. melanogaster and accessory gland protein genes. Only a few studies have attempted to disentangle the genetic basis of CSP between related species of Drosophila. Although there is no a priori reason to believe that genes influencing intraspecific sperm competitive ability might also mediate conspecific sperm precedence, no study has addressed the question. Here, we test a group of candidate CSP genes between D. simulans and D. mauritiana for their effect on sperm competition in D. melanogaster. The use of P-element insertion lines identified CG14891 gene disruption as the only one causing a significant decrease in second male paternity success relative to wild-type and ebony tester males. The gene disruption affected both sperm displacement and the sperm fertilizing ability. Out of five genes tested using RNA interference, only gene knockdown of CG6864(Mst89B) [corrected] significantly reduced the male's ability to father progeny when second to mate. Our results suggest that CG14891 and CG6468 might have been co-opted from an intraspecies gene function (i.e., sperm competition) into an interspecies avoidance phenotype (i.e., CSP). Alternatively, the dual role of these genes could be a consequence of their pleiotropic roles. Copyright © 2014 Civetta and Finn.

  5. Cytometry of mammalian sperm

    Energy Technology Data Exchange (ETDEWEB)

    Gledhill, B.L.

    1983-10-11

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

  6. Sperm function test

    Directory of Open Access Journals (Sweden)

    Pankaj Talwar

    2015-01-01

    Full Text Available With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation. They should be protected from the bad effects of pus cells and reactive oxygen species (ROS (leukocyte detection test, ROS estimation. Their number should be in sufficient in terms of (count, structure normal to be able to fertilize eggs (semen morphology. Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test, should have good mitochondrial function to be able to provide energy (mitochondrial activity index test. They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test. Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test to the oocyte during fertilization.

  7. The BSA-induced Ca(2+ influx during sperm capacitation is CATSPER channel-dependent

    Directory of Open Access Journals (Sweden)

    Ren Dejian

    2009-10-01

    Full Text Available Abstract Background Serum albumin is a key component in mammalian sperm capacitation, a functional maturation process by which sperm become competent to fertilize oocytes. Capacitation is accompanied by several cellular and molecular changes including an increased tyrosine phosphorylation of sperm proteins and a development of hyperactivated sperm motility. Both of these processes require extracellular calcium, but how calcium enters sperm during capacitation is not well understood. Methods BSA-induced changes in intracellular calcium concentration were studied using Fluo-4 and Fura-2 calcium imaging with wild-type and Catsper1 knockout mouse sperm. Results We found that the fast phase of the BSA-induced rises in intracellular calcium concentration was absent in the Catsper1 knockout sperm and could be restored by an EGFP-CATSPER1 fusion protein. The calcium concentration increases were independent of G-proteins and phospholipase C but could be partially inhibited when intracellular pH was clamped. The changes started in the principal piece and propagated toward the sperm head. Conclusion We conclude that the initial phase of the increases in intracellular calcium concentration induced by BSA requires the CATSPER channel, but not the voltage-gated calcium channel. Our findings identify the molecular conduit responsible for the calcium entry required for the sperm motility changes that occur during capacitation.

  8. The sperm mitochondria-specific translocator has a key role in maternal mitochondrial inheritance.

    Science.gov (United States)

    Hayashida, Kenji; Omagari, Katsuhisa; Masuda, Jun-ichi; Hazama, Hiroaki; Kadokawa, Yoshiko; Ohba, Kazuo; Kohno, Shigeru

    2005-06-01

    The mechanism of maternal mitochondrial inheritance in animals involves the selective elimination of sperm mitochondria by the elimination factor of the egg and the sperm mitochondria-specific factor. In vitro fertilization using sperm from isogenic mice incorporating heterospecific mitochondrial DNA (mtDNA) showed that the number of PCR positives of sperm mtDNA in two-cell embryos was significantly increased following sperm incubation with anti-tetratricopeptide repeat-containing protein involved in spermatogenesis (tpis) protein, anti-translocator of mitochondrial outer membrane (Tom) 22 and anti-Tom40 antibodies. The treatment of fertilized eggs with EGTA and other endonuclease inhibitors increased the sperm mtDNA levels. We conclude that the elimination factor, which is probably an endonuclease, is selectively received by the tpis protein of the sperm mitochondrial outer membrane within the egg. It is then transported into the sperm mitochondria by Tom22 and Tom40, where it destroys the sperm mtDNA, establishing the maternal inheritance of mtDNA.

  9. Sperm Shape (Morphology): Does It Affect Fertility?

    Science.gov (United States)

    ... Website of the American Society for Reproductive Medicine Sperm morphology (shape): Does it affect fertility? How is ... semen analysis. This measures many features of the sperm and semen (the fluid in which the sperm ...

  10. Mecobalamin promotes mouse sperm maturation.

    Science.gov (United States)

    Oshio, S; Ozaki, S; Ohkawa, I; Tajima, T; Kaneko, S; Mohri, H

    1989-01-01

    The effect of Mecobalamin (alpha-(5,6-dimethyl benzimidazolyl)-Co-methyl-cobamide: Me-B 12) on sperm production in the oligozoospermic mice experimentally induced by the treatment with adriamycin (0.3 mg/kg, three times a week for 5 weeks) was evaluated quantitatively by means of equilibrium sedimentation in Percoll. After centrifugation, the distribution profile of the sperm showed two peaks, i.e. the first peak near the bottom consisting of mature sperm with good motility and the second peak containing immature and/or immotile sperm. By oral administration of Me B 12 (1.0 mg/kg/day) to the oligozoospermic mice for 10 weeks, the sperm count, sperm motility, motile sperm count, diameter of seminiferous tubules and the percentage of good motile sperm with higher apparent density were increased as compared with those of the control. These results suggest that Me-B 12 enhanced the testicular function, resulting in an increased output of mature sperm.

  11. Sperm storage in caecilian amphibians

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    Kuehnel Susanne

    2012-06-01

    Full Text Available Abstract Background Female sperm storage has evolved independently multiple times among vertebrates to control reproduction in response to the environment. In internally fertilising amphibians, female salamanders store sperm in cloacal spermathecae, whereas among anurans sperm storage in oviducts is known only in tailed frogs. Facilitated through extensive field sampling following historical observations we tested for sperm storing structures in the female urogenital tract of fossorial, tropical caecilian amphibians. Findings In the oviparous Ichthyophis cf. kohtaoensis, aggregated sperm were present in a distinct region of the posterior oviduct but not in the cloaca in six out of seven vitellogenic females prior to oviposition. Spermatozoa were found most abundantly between the mucosal folds. In relation to the reproductive status decreased amounts of sperm were present in gravid females compared to pre-ovulatory females. Sperm were absent in females past oviposition. Conclusions Our findings indicate short-term oviductal sperm storage in the oviparous Ichthyophis cf. kohtaoensis. We assume that in female caecilians exhibiting high levels of parental investment sperm storage has evolved in order to optimally coordinate reproductive events and to increase fitness.

  12. Postcoital Sperm Assessment Comparative Study.

    Science.gov (United States)

    Pelekanos, Michael J

    2015-11-01

    This postcoital sperm assessment study was performed over a 10 month time period (November 2014-August 2015). Fifteen couples enrolled in the study. The study was a non-blinded, non-randomized, single-center comparison study comparing The Stork® OTC (Rinovum Women's Health, Monroeville, PA) to natural intercourse (NI), using the subjects as their own control/baseline. This was an efficacy study designed to compare the number of sperm in the cervical mucus following the use of The Stork OTC conception aid with the number of sperm in the cervical mucus following natural intercourse. Subjects used both The Stork OTC conception system and the natural intercourse method to evaluate concentrations of sperm in the cervical mucus. Post-coital test (PCT) data was collected demonstrating higher concentrations of sperm within the cervical mucus with The Stork OTC conception system versus natural intercourse for 85% of test subjects in this study. Of the 15 couples enrolled in the study, 2 were lost to follow-up. Mean age for male subjects was 31.7 +/ 5.4 years of age and mean age for female subjects was 29.7+/- 5.4. The average sperm score value of the 85% of test subjects with higher sperm concentrations from The Stork OTC was 3.23 times the score value of sperm concentration compared to natural intercourse. The remaining 15% of test subjects showed no change in sperm score value between The Stork OTC and natural intercourse.

  13. Ubiquitination of prohibitin in mammalian sperm mitochondria: possible roles in the regulation of mitochondrial inheritance and sperm quality control.

    Science.gov (United States)

    Thompson, Winston E; Ramalho-Santos, João; Sutovsky, Peter

    2003-07-01

    Ubiquitination of the sperm mitochondria during spermatogenesis has been implicated in the targeted degradation of paternal mitochondria after fertilization, a mechanism proposed to promote the predominantly maternal inheritance of mitochondrial DNA in humans and animals. The identity of ubiquitinated substrates in the sperm mitochondria is not known. In the present study, we show that prohibitin, a highly conserved, 30- to 32-kDa mitochondrial membrane protein, occurs in a number of unexpected isoforms, ranging from 64 to greater than 185 kDa in the mammalian sperm mitochondria, which are the ubiquitinated substrates. These bands bind antiubiquitin antibodies, displaying a pattern consistent with polyubiquitinated "ladders." Immunoprecipitation of sperm extracts with antiprohibitin antibodies followed by probing of the resultant immunocomplexes with antiubiquitin yields a banding pattern identical to that observed by antiprohibitin Western blot analysis. In fact, the presumably nonubiquitinated 30-kDa prohibitin band shows no antiubiquitin immunoreactivity. We demonstrate that ubiquitination of prohibitin occurs in testicular spermatids and spermatozoa. Ubiquitinated prohibitin molecules also accumulate in the defective fractions of ejaculated spermatozoa, which are thought to undergo surface ubiquitination during epididymal passage. In such sperm fractions, ubiquitin also coprecipitates with tubulin and microtubule-associated proteins, presumably contributed by the axonemes of defective, ubiquitinated spermatozoa. The results of the present study suggest that prohibitin is one of the ubiquitinated substrates that makes the sperm mitochondria recognizable by the egg's ubiquitin-proteasome dependent proteolytic machinery after fertilization and most likely facilitates the marking of defective spermatozoa in the epididymis for degradation.

  14. Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm

    Institute of Scientific and Technical Information of China (English)

    HE Yu-xuan; LI Hong-yan; ZHANG Yong; HE Jian-hua; ZHANG Hong; ZHAO Xing-xu

    2013-01-01

    This study is first to investigate proteomic changes in sheep sperm induced by carbon ion radiation using two-dimensional electrophoresis (2-DE) analysis in the project of breeding a new variety of sheep. Differential expression proteins were detected using the PDQuest 8.0 software after staining with Coomassie blue. Valid spots were then analyzed through liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 480 total protein spots displayed in 2-D gels, 6 specific protein spots were observed in sperm gels. A search against protein sequences in the National Center for Biotechnology Information databases (NCBI) indicated that differentially expressed proteins correspond to two proteins, identified to be enolase and transcription factor AP-2-alpha (TFAP-2α). The two proteins were up-regulated in the irradiated sperm. To the best of our knowledge, this study is the first to identify proteomic changes induced by carbon ion radiation in sheep sperm. The analysis of differential expression protein may be useful in identifying new breeding markers in sheep reproduction and in clarifying the mechanisms involved in irradiation or space breeding.

  15. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane.

    Science.gov (United States)

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review.

  16. Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

    OpenAIRE

    Tian, Jingdong; Gong, Hui; Thomsen, Gerald H.; Lennarz, William J.

    1997-01-01

    A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-depe...

  17. Proteomics and the genetics of sperm chromatin condensation

    Institute of Scientific and Technical Information of China (English)

    Rafael Oliva; Judit Castillo

    2011-01-01

    Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies.

  18. PLCζ or PAWP: revisiting the putative mammalian sperm factor that triggers egg activation and embryogenesis.

    Science.gov (United States)

    Kashir, Junaid; Nomikos, Michail; Swann, Karl; Lai, F Anthony

    2015-05-01

    In mammals, egg activation is initiated by multiple cytosolic Ca(2+) transients (Ca(2+) oscillations) that are triggered following delivery of a putative sperm factor from the fertilizing sperm. The identity of this 'sperm factor' thus holds much significance, not only as a vital component in creating a new life, but also for its potential therapeutic and diagnostic value in human infertility. Recent data have emerged suggesting the sperm factor may be a post-acrosomal sheath WW domain-binding protein (PAWP). However, a significant body of research points to a testis-specific phospholipase C zeta (PLCζ) as the sperm factor. Herein, we examine the evidence presented in favour of PAWP in relation to PLCζ and the requisite physiological properties of the mammalian sperm factor.

  19. Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Satoru Sonoda

    2016-06-01

    Full Text Available Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance.

  20. The expression and putative role of brain-derived neurotrophic factor and its receptor in bovine sperm.

    Science.gov (United States)

    Li, C; Li, C; Zhu, X; Wang, C; Liu, Zhuo; Li, W; Lu, Chen; Zhou, Xu

    2012-02-01

    The neurotrophin family of proteins promote the survival and differentiation of nerve cells and are thought to play an important role in development of reproductive tissues. The objective of the present study was to detect the presence of Brain-derived neurotrophic factor (BDNF) and its receptor TrkB in bovine sperm, and explore the potential role of BDNF in sperm function. We demonstrated that both the neorotrophin BDNF and the tyrosine kinase receptor protein TrkB were expressed in ejaculated bovine sperm. Furthermore, BDNF per se was secreted by sperm. Insulin and leptin secretion by bovine sperm were increased (P BDNF, whereas insulin was decreased by K252a. Therefore, we inferred that BDNF could be a regulator of sperm secretion of insulin and leptin through the TrkB receptor. Sperm viability and mitochondrial activity were both decreased (P BDNF/TrkB signaling pathway was blocked with K252a. Furthermore, BDNF promoted apoptosis of bovine sperm through TrkB binding (P BDNF secreted by bovine sperm was important in regulation of insulin and leptin secretion in ejaculated bovine sperm. Furthermore, BDNF may affect sperm mitochondrial activity and apoptosis, as well as their viability.

  1. Make it spin: individual trapping of sperm for analysis and recovery using micro-contact printing

    NARCIS (Netherlands)

    Frimat, Jean-Philippe; Bronkhorst, M.; de Wagenaar, B.; Bomer, Johan G.; van der Heijden, Ferdinand; van den Berg, Albert; Segerink, Loes Irene

    2014-01-01

    In this article, we describe the development of a high throughput platform to spatially manipulate viable sperm for motility measurements and recovery of the best single sperm for fertilization purposes. Micro-contact printing was used to pattern islands of adhesive proteins (fibronectin) separated

  2. The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm-egg recognition.

    Science.gov (United States)

    Redgrove, Kate A; Nixon, Brett; Baker, Mark A; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R John

    2012-01-01

    A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm-egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm-zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

  3. Sperm donation in Israel.

    Science.gov (United States)

    Mor-Yosef, S; Schenker, J G

    1995-04-01

    Science and technology in the field of human reproduction present new legal, ethical and religious questions which do not always have immediate answers. The first step in the rapidly developed field of reproductive technology was the use of sperm donation (artificial insemination by donor, AID) and the establishment of sperm banks. The state of Israel faced these problems when the regulations for sperm donation were discussed. The fact that the main holy places for the three monotheistic religions are in Israel directly influences the make-up of the population constituents. Therefore, besides a majority of secular people, a high percentage of the population of Israel is very religious: Jews, Moslems and Christians. Thus any resolution relating to AID should take this demographic combination into account. The practice of AID is opposed by the different monotheistic religions. To avoid the conflict between secular and religious people, and between the different religions' perspectives, the legal problem of AID in Israel was solved not by laws but by regulations which were published by the Ministry of Health. The main idea behind this attitude is that the state and its authorities should not and do not deal with ethical or religious questions. Thus, the decision was left to the couples and to the donors. The regulations address technical requirements, health problems and confidential issues concerning the couple, the donor and the child. In this paper we present the different views relating to these problems as perceived by the different religions, and describe the solution that was accepted by the Israeli Ministry of Health.

  4. Penicillamine prevents ram sperm agglutination in media that support capacitation.

    Science.gov (United States)

    Leahy, T; Rickard, J P; Aitken, R J; de Graaf, S P

    2016-02-01

    Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7 ± 2.7% to 2.8 ± 1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

  5. Llama oviductal sperm reservoirs: involvement of bulbourethral glands.

    Science.gov (United States)

    Apichela, S A; Argañaraz, M E; Giuliano, S; Zampini, R; Carretero, I; Miragaya, M; Miceli, D C

    2014-04-01

    The aim of this study was to elucidate the role of llama seminal plasma in the formation of oviductal sperm reservoirs. Female llamas with follicles in the mature phase were mated with a bulbourethral glands-removed male. Females mated with nonbulbourethral glands-removed males were used as control. Oviducts were obtained by surgery 24 h after mating. The uterotubal junction and isthmus were examined by scanning electron microscopy, and mucopolysaccharides were identified by Alcian blue staining. To know the proteins probably involved in sperm reservoir formation, SDS-PAGE of seminal plasma (8% and 18% resolving gel) was made. Spermatozoa only adhered to the oviductal mucosa surface of uterotubal junction of females mated with nonbulbourethral glands-removed males confirming that seminal plasma and, in particular, bulbourethral secretions are related with the oviductal sperm reservoir formation. Histological sections showed sperm in the lumen, immersed in substance, positive for acid mucopolysaccharides. Alcian blue staining of seminal plasma proteins SDS-PAGE showed a band of high molecular weight containing mucopolysaccharides, only present in nonbulbourethral glands-removed males. Bulbourethral glands would secrete at least eight different proteins that most likely participate in the process of sperm storage in the oviduct.

  6. Sperm preparation for ART

    Directory of Open Access Journals (Sweden)

    Schill Wolf-Bernhard

    2003-11-01

    Full Text Available Abstract The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI. Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate

  7. RNA in human sperm

    Institute of Scientific and Technical Information of China (English)

    Rui Pires Martins; Stephen A. Krawetz

    2005-01-01

    We have yet to develop a fundamental understanding of the molecular complexities of human spermatozoa. This encompasses the unique packaging and structure of the sperm genome along with their paternally derived RNAs in preparation for their delivery to the egg. The diversity of these transcripts is vast, including several anti-sense molecules resembling known regulatory micro-RNAs. The field is still grasping with its delivery to the oocyte at fertilization and possible significance. It remains tempting to analogize them to maternally-derived transcripts active in early embryo patterning. Irrespective of their role in the embryo, their use as a means to assess male factor infertility is promising.

  8. Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Head Remodelling and Sperm Tail Development.

    Directory of Open Access Journals (Sweden)

    Nadege Vernet

    Full Text Available A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp is required for efficient completion of the second meiotic division (that generates haploid round spermatids, restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (but not Zfy1 contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head.

  9. Sperm membrane modulation by Sapindus mukorossi during sperm maturation

    Institute of Scientific and Technical Information of China (English)

    ManishNivsarkar; NeetaShrivastava

    2002-01-01

    Aim:TO observe the alterations in the biochemical and biophysical changes in the sperm membrane during sperm maturation in male rats treated with the water extract of the fruit pericarp of S.mukorossi.Methods:Adult male Sprague-Dawley rats were gavaged the aqueous extract of the fruit pericarp of S.mukorossi at a dose of 50mg/kg/dfor45days.On day46,the sperm parameters were observed in idfferent sections of the epididymis and the sperm superoxide dismutase and the lipid peroxidation was edtermined and compared with the controls.The testis and epidiymis were routinely prepared for histological examination under the light microscope.Results:No significant differences in the sperm number and morphology were observed between the control and reated groups.However,a significant inhibition(P<0.05-0.01)of sperm motility in the caput,corpus and cauda regions of the epididymis was seen in the treated group.No significant histopathological changes were found in the testis and epididymis.Theimportant finding was that in the treated animals,the spermatozoa showed an abnormal distribution of the superoxide dismutase activity,being minimum in the caput and maximum in the corpus,which was just opposite to that of the controls.Conclusion:The study provides a unique observation where the plant extract alters the sperm membrane physiology without change the testicular and epididymal morphology.

  10. Sperm DNA fragmentation is related to sperm morphological staining patterns.

    Science.gov (United States)

    Sá, Rosália; Cunha, Mariana; Rocha, Eduardo; Barros, Alberto; Sousa, Mário

    2015-10-01

    In this prospective comparative study, sperm DNA fragmentation (sDNAfrag) was compared at each step of a sequential semen preparation, with semen parameters according to their degree of severity. At each step (fractions) of the sequential procedure, sDNAfrag was determined: fresh (Raw), after gradient centrifugation, washing, and swim-up (SU) for 70 infertile men enrolled in intracytoplasmic sperm injection cycles. sDNAfrag significantly (P = 0.04; P < 0.0001) decreased throughout the steps of semen preparation, with centrifugation and washing not increasing it. A negative correlation to sperm motility was observed in Raw and SU fractions, and a higher sDNAfrag was observed in samples with lower semen quality. Our results confirm that the steps of the sequential procedure do not compromise sperm DNA integrity and progressively decreased sDNAfrag regardless of the sperm abnormality and that semen parameters with lower quality present higher sDNAfrag. Four distinct patterns were observed, of which the entire sperm head staining was the pattern most expressed in all studied fractions. Additionally, the sperm head gene-rich region staining pattern was reduced by the procedure. This suggests that pattern quantification might be a useful adjunct when performing sDNAfrag testing for male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  11. New insights into transduction pathways that regulate boar sperm function.

    Science.gov (United States)

    Hurtado de Llera, A; Martin-Hidalgo, D; Gil, M C; Garcia-Marin, L J; Bragado, M J

    2016-01-01

    Detailed molecular mechanisms mediating signal transduction cascades that regulate boar sperm function involving Ser/Thr and tyrosine phosphorylation of proteins have been reviewed previously. Therefore, this review will focus in those kinase pathways identified recently (boar spermatozoa that regulate different functional spermatozoa processes. AMP-activated protein kinase (AMPK) is a cell energy sensor kinase that was first identified in mammalian spermatozoa in 2012, and since then it has emerged as an essential regulator of boar sperm function. Signaling pathways leading to AMPK activation in boar sperm are highlighted in this review (PKA, CaMKKα/β, and PKC as well as Ca(2+) and cAMP messengers as upstream regulators). Interestingly, stimuli considered as cell stress (hyperosmotic stress, inhibition of mitochondrial activity, absence of intracellular Ca(2+)) markedly activate AMPK in boar spermatozoa. Moreover, AMPK plays a remarkable and necessary regulatory role in mammalian sperm function, controlling essential boar sperm functional processes such as motility, viability, mitochondrial membrane potential, organization and fluidity of plasma membrane, and outer acrosome membrane integrity. These mentioned processes are all required under fluctuating environment of spermatozoa when transiting through the female reproductive tract to achieve fertilization. An applied role of AMPK in artificial insemination techniques is also suggested as during boar seminal doses preservation at 17 °C, physiological levels of AMPK activity markedly increase (maximum on Day 7) and result essential to maintain the aforementioned fundamental sperm processes. Moreover, regulation of sperm function exerted by the glycogen synthase kinase 3 and Src family kinase pathways is summarized.

  12. Cholesterol-Loaded Cyclodextrin Increases the Cholesterol Content of Goat Sperm to Improve Cold and Osmotic Resistance and Maintain Sperm Function after Cryopreservation.

    Science.gov (United States)

    Salmon, Vianney M; Leclerc, Pierre; Bailey, Janice L

    2016-04-01

    The success of semen cryopreservation depends on sperm membrane integrity and function after thawing. Cholesterol-loaded cyclodextrin (CLC) is used for in vitro incorporation of cholesterol to protect cells against cold temperatures. We hypothesized that CLC treatment also enhances sperm cholesterol content to increase tolerance to osmotic shock and cryoresistance, thereby improving fertility. We confirmed the fact that treatment of goat semen with 3 mg/ml CLC increases sperm cholesterol content using both the Liebermann-Burchard approach and filipin III labeling of membrane cholesterol. Sperm were then treated with or without CLC and cryopreserved. After thawing, sperm cholesterol dramatically fell, even in the presence of CLC, which explains the mechanism of cryocapacitation. CLC treatment, however, maintained a normal prefreeze cholesterol level in sperm after cryopreservation. Furthermore, fresh sperm treated with CLC and subjected to either cold shock or incubated in hypo-, iso-, and hyperosmotic media, designed to mimic stresses associated with freezing/thawing, displayed increased temperature and osmotic tolerance. CLC treatment also improved sperm viability, motility, and acrosome integrity after thawing. Furthermore, CLC treatment did not affect the sperm's ability to undergo in vitro capacitation according to chlortetracycline fluorescence and protein tyrosine phosphorylation. A pilot field trial demonstrated that artificial insemination with sperm that underwent increased cholesterol levels following CLC treatment yielded higher fertility ( ITALIC! P< 0.1) and proliferation ( ITALIC! P< 0.05) rates in vivo than untreated semen from the same ejaculate samples. These observations suggest that CLC treatment could be used to improve cryoprotection during the freezing and thawing of goat sperm.

  13. Human Sperm Competition: A Comparative Evolutionary Analysis

    Directory of Open Access Journals (Sweden)

    Michael N. Pham

    2014-08-01

    Full Text Available Sperm competition occurs when a female copulates with two or more males within a sufficiently brief time period, resulting in sperm of the different males competing to fertilize ova. Sperm competition has been documented or inferred to occur across several species. We address the evidence for sperm competition in humans by reviewing literature indicating apparently convergent adaptations to sperm competition in humans and non-humans. We discuss future research directions, and conclude that the evidence for anatomical, biological, physiological, and behavioral adaptations to human sperm competition provides compelling evidence that sperm competition has been a recurrent feature of human evolutionary history.

  14. Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

    Directory of Open Access Journals (Sweden)

    Xiaojing Yang

    Full Text Available Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1 in human embryonic kidney 293 (HEK293 cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

  15. Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

    Science.gov (United States)

    Yang, Xiaojing; Zhao, Yuewen; Yang, Xiaolong; Kan, Frederick W K

    2015-01-01

    Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

  16. Comparative sperm ultrastructure in Nemertea.

    Science.gov (United States)

    von Döhren, J; Beckers, P; Vogeler, R; Bartolomaeus, T

    2010-07-01

    Although the monophyly of Nemertea is strongly supported by unique morphological characters and results of molecular phylogenetic studies, their ingroup relationships are largely unresolved. To contribute solving this problem we studied sperm ultrastructure of 12 nemertean species that belong to different subtaxa representing the commonly recognized major monophyletic groups. The study yielded a set of 26 characters with an unexpected variation among species of the same genus (Tubulanus and Procephalothrix species), whereas other species varied in metric values or only one character state (Ramphogordius). In some species, the sperm nucleus has grooves (Zygonemertes virescens, Amphiporus imparispinosus) that may be twisted and give a spiral shape to the sperm head (Paranemertes peregrina, Emplectonema gracile). To make the characters from sperm ultrastructure accessible for further phylogenetic analyses, they were coded in a character matrix. Published data for eight species turned out to be sufficiently detailed to be included. Comparative evaluation of available information on the sperm ultrastructure suggests that subtaxa of Heteronemertea and Hoplonemertea are supported as monophyletic by sperm morphology. However, the data do not provide information on the existing contradictions regarding the internal relationships of "Palaeonemertea." Nevertheless, our study provides evidence that sperm ultrastructure yields numerous potentially informative characters that will be included in upcoming phylogenetic analyses.

  17. Non-genetic contributions of the sperm nucleus to embryonic development

    Institute of Scientific and Technical Information of China (English)

    Yasuhiro Yamauchi; Jeffrey A Shaman; W Steven Ward

    2011-01-01

    Recent data from severallaboratories have provided evidence thatthe newly fertilized oocyte inherits epigenetic signals from the sperm chromatin that are required for proper embryonic development. For the purposes of this review, the term epigenetrc is used to describe all types of molecular information that are transmitted from the sperm cell to the embryo. There are at least six different forms of epigenetic information that have already been established as being required for proper embryogenesis in mammals or for which there is evidence thatit may do so. These are (i) DNA methylation; (ii) sperm-specific histones, (iii) other chromatin-associated proteins; (iv) the perinuclear theca proteins; (v) sperm-born RNAs and, the focus of this review; and (vi) the DNA loop domain organization by the sperm nuclear matrix. These epigenetic signals should be considered when designing protocols for the manipulation and cryopreservation of spermatozoa for assisted reproductive technology as necessary components for effective fertilization and subsequent embryo development.

  18. Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

    Science.gov (United States)

    Yi, Y-J; Zimmerman, S W; Manandhar, G; Odhiambo, J F; Kennedy, C; Jonáková, V; Maňásková-Postlerová, P; Sutovsky, M; Park, C-S; Sutovsky, P

    2012-04-01

    Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p sperm capacitation and acrosomal function during fertilization.

  19. TRY-5 is a sperm-activating protease in Caenorhabditis elegans seminal fluid.

    Directory of Open Access Journals (Sweden)

    Joseph R Smith

    2011-11-01

    Full Text Available Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction.

  20. Cigarette smoking impairs sperm bioenergetics

    Directory of Open Access Journals (Sweden)

    Kazim R. Chohan

    2010-02-01

    Full Text Available OBJECTIVE: The growing consensus on the negative impact of cigarette smoking on fertility prompted us to compare the rate of sperm respiration in smokers and non-smokers. MATERIALS AND METHODS: Semen samples from 20 smokers and 58 non-smokers consulting at the andrology laboratory for fertility evaluation were used. Smoking was defined as consumption of at least a half a pack per day. A phosphorescence analyzer that measures O2 concentration in sperm suspensions as function of time was used to determine the rate of respiration. In a sealed vial, the rate of sperm respiration (k was defined as -d[O2]/dt; where [O2] was obtained from the phosphorescence decay rate of a palladium phosphor. [O2] in solutions containing sperm and glucose declined linearly with time, showing the kinetics of O2 consumption was zero-order. Inhibition of O2 consumption by cyanide confirmed the oxidations that occurred in the sperm mitochondrial respiratory chain. RESULTS: There were no differences (p > 0.28 between smokers and non-smokers for ejaculate volume, motility, concentration, normal morphology, viability and hypo-osmotic swelling test. The rate (mean ± SD, in µM O2/min/108 sperm of sperm mitochondrial O2 consumption in the smokers was 0.96 ± 0.58 and in the non-smokers 1.39 ± 0.67 (p = 0.004. CONCLUSIONS: The rate of sperm respiration was significantly lower in smokers. This negative impact of cigarette smoking on sperm aerobic metabolism may, in part, explain the lower rate of fertility in smokers.

  1. Branchial Cilia and Sperm Flagella Recruit Distinct Axonemal Components

    Science.gov (United States)

    Konno, Alu; Shiba, Kogiku; Cai, Chunhua; Inaba, Kazuo

    2015-01-01

    Eukaryotic cilia and flagella have highly conserved 9 + 2 structures. They are functionally diverged to play cell-type-specific roles even in a multicellular organism. Although their structural components are therefore believed to be common, few studies have investigated the molecular diversity of the protein components of the cilia and flagella in a single organism. Here we carried out a proteomic analysis and compared protein components between branchial cilia and sperm flagella in a marine invertebrate chordate, Ciona intestinalis. Distinct feature of protein recruitment in branchial cilia and sperm flagella has been clarified; (1) Isoforms of α- and β-tubulins as well as those of actins are distinctly used in branchial cilia or sperm flagella. (2) Structural components, such as dynein docking complex, tektins and an outer dense fiber protein, are used differently by the cilia and flagella. (3) Sperm flagella are specialized for the cAMP- and Ca2+-dependent regulation of outer arm dynein and for energy metabolism by glycolytic enzymes. Our present study clearly demonstrates that flagellar or ciliary proteins are properly recruited according to their function and stability, despite their apparent structural resemblance and conservation. PMID:25962172

  2. Branchial cilia and sperm flagella recruit distinct axonemal components.

    Directory of Open Access Journals (Sweden)

    Alu Konno

    Full Text Available Eukaryotic cilia and flagella have highly conserved 9 + 2 structures. They are functionally diverged to play cell-type-specific roles even in a multicellular organism. Although their structural components are therefore believed to be common, few studies have investigated the molecular diversity of the protein components of the cilia and flagella in a single organism. Here we carried out a proteomic analysis and compared protein components between branchial cilia and sperm flagella in a marine invertebrate chordate, Ciona intestinalis. Distinct feature of protein recruitment in branchial cilia and sperm flagella has been clarified; (1 Isoforms of α- and β-tubulins as well as those of actins are distinctly used in branchial cilia or sperm flagella. (2 Structural components, such as dynein docking complex, tektins and an outer dense fiber protein, are used differently by the cilia and flagella. (3 Sperm flagella are specialized for the cAMP- and Ca2+-dependent regulation of outer arm dynein and for energy metabolism by glycolytic enzymes. Our present study clearly demonstrates that flagellar or ciliary proteins are properly recruited according to their function and stability, despite their apparent structural resemblance and conservation.

  3. Human sperm rheotaxis: a passive physical process

    OpenAIRE

    Zhuoran Zhang; Jun Liu; Jim Meriano; Changhai Ru; Shaorong Xie; Jun Luo; Yu Sun

    2016-01-01

    A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long–range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense an...

  4. Involvement of cAMP-dependent unique signaling cascades in the decrease of serine/threonine-phosphorylated proteins in boar sperm head.

    Science.gov (United States)

    Isono, Ayane; Tate, Shunsuke; Nakamura-Mori, Kazumi; Noda, Taichi; Ishikawa, Sho; Harayama, Hiroshi

    2016-04-01

    We previously suggested that protein phosphatase-dependent decrease of postacrosomal phosphorylated proteins may be necessary for the occurrence of acrosome reaction in livestock spermatozoa (Adachi et al., J Reprod Dev 54, 171-176, 2008; Mizuno et al., Mol Reprod Dev 82, 232-250, 2015). The aim of this study was to examine the involvement of the intracellular cAMP signaling cascades in the regulation of the decrease of postacrosomal phosphorylated proteins in boar spermatozoa. Boar ejaculated spermatozoa were incubated with cAMP analogs and then used for the immunodetection of serine/threonine-phosphorylated proteins and assessment of acrosome morphology. The protein phosphatase-dependent decrease of postacrosomal phosphorylated proteins was greatly promoted by the incubation with a cAMP analog Sp-5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS). This decrease was induced before the initiation of acrosome reaction and did not require the millimolar concentration of extracellular Ca(2+) which was necessary for the initiation of acrosome reaction. Moreover, suppression of protein kinase A activity with an inhibitor (H89) had almost no influence on both decrease of phosphorylated proteins and occurrence of acrosome reaction in the spermatozoa incubated with cBiMPS. In addition, the prolonged incubation with a potentially exchange protein directly activated by cAMP-selective cAMP analog (8pM) could only partially mimic effects of cBiMPS on these events. These results indicate that the cAMP-dependent signaling cascades which are less dependent on protein kinase A may regulate the decrease of postacrosomal phosphorylated proteins in boar spermatozoa before the extracellular Ca(2+)-triggered initiation of acrosome reaction. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Daily Sperm Production, Gonadal and Extra-Gonadal Sperm ...

    African Journals Online (AJOL)

    SH

    The control diet 1 had neither probiotic nor prebiotic while prebiotic, probiotic and symbiotic (prebiotic + ... live microbial feed supplement that has beneficial effects on ... Four diets were formulated including the control ... same trend. The sperm ...

  6. Sperm Dynamics in Spiders (Araneae): Ultrastructural Analysis of the Sperm Activation Process in the Garden Spider Argiope bruennichi (Scopoli, 1772)

    OpenAIRE

    Oliver Vöcking; Gabriele Uhl; Peter Michalik

    2013-01-01

    Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that...

  7. Sperm fractions obtained following density gradient centrifugation in human ejaculates show differences in sperm DNA longevity

    Directory of Open Access Journals (Sweden)

    Jaime Gosálvez

    2014-06-01

    Conclusion: 1 Unnecessary incubation of spermatozoa prior to artificial insemination or in vitro fertilization, should be avoided, since sperm DNA longevity is significantly reduced after ex vivo sperm handling and 2 Although sperm selection by DCG significantly reduces the baseline levels of SDF of sperm in Fraction 3, sperm DNA longevity in this fraction was ultimately lower following 24 h incubation when compared to sperm recovered from non-centrifuged NSS.

  8. Physiological roles of semenogelin I and zinc in sperm motility and semen coagulation on ejaculation in humans.

    Science.gov (United States)

    Yoshida, Kaoru; Kawano, Natsuko; Yoshiike, Miki; Yoshida, Manabu; Iwamoto, Teruaki; Morisawa, Masaaki

    2008-03-01

    At ejaculation, human sperm are considered to be mechanically trapped and become immotile in the semen coagulum by binding to semenogelins (Sgs) from the seminal vesicle and zinc ions from the prostate. However, the physiological combined roles of the protein and heavy metal on sperm motility are unknown. Here, we have first demonstrated that Sg I alone, which does not form the semen coagulum without zinc, is an inhibitor of the motility of intact human sperm at physiological concentration. On the other hand, zinc ions alone had no effect on sperm motility, but confer recovery of sperm motility that has been inhibited by Sg I at a concentration equal to or less than 1 mg/ml. These observations suggest that the roles played by Sg I and zinc on sperm motility are not mechanical but physiological. Quartz crystal microbalance analysis suggests that the sperm extract first bind to Sg I and then zinc ions which subsequently increase the protein accumulation, suggesting that Sgs inhibit sperm motility by directly binding to the sperm surface. Further accumulation of Sg I mediated by zinc ions may entrap the quiescent sperm at semen ejaculation.

  9. 抗细胞核自身抗原精子蛋白的抗体具有抗生育效果%The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Jian-Li Shi; Guo-Yan Cheng; Yan-Qing Hu; Chen Xu

    2009-01-01

    为了研究抗睾丸型细胞核自身抗原精子蛋白(tNASP)的抗体是否会影响生育,我们成功克隆表达了一段339 bp小鼠睾丸型细胞核自身抗原精子蛋白(mtNASP)的cDNA.本实验以小鼠为动物模型,着重研究重组mtNASP蛋白和合成肽htNASP393-408(人睾丸型细胞核自身抗原精子蛋白第393-408氨基酸序列)的抗生育效果.用mtNASP和htNASP393-408分别主动免疫小鼠,二者都能在小鼠体内产生高效价的抗体.然后用8-10周龄的小鼠进行精卵结合和精卵融合实验.结果显示在不同浓度的抗mtNASP抗体存在下,小鼠卵子与获能精子的精卵结合能力以及去透明带小鼠卵子和获能精子的精卵融合能力都有明显下降.分别免疫重组mtNASP蛋白和htNASP393-408合成肽的小鼠也都能产生明显的抗生育效果,而且用合成肽免疫的小鼠其抗生育效果是可逆的.我们的实验数据表明,用重组的mtNASP蛋白进行免疫可以产生很强的抗体反应并且抑制小鼠生育.%To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein (tNASP) could result in reproductive failure, we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP (mtNASP). Using mouse as a model, recombinant mtNASP (rmtNASP) and a synthetic peptide, human tNASP393-408 (htNASP393-408), were investigated for their antifertility effect. Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice. Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice. Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP. There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide. The effect on fertility in the mice immunized

  10. Ion channels, phosphorylation and mammalian sperm capacitation.

    Science.gov (United States)

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-05-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

  11. Ion channels, phosphorylation and mammalian sperm capacitation

    Institute of Scientific and Technical Information of China (English)

    Pablo E Visconti; Dario Krapf; José Luis de la Vega-Beltrán; Juan José Acevedo; Alberto Darszon

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

  12. Associations of hypoosmotic swelling test, relative sperm volume shift, aquaporin7 mRNA abundance and bull fertility estimates.

    Science.gov (United States)

    Kasimanickam, R K; Kasimanickam, V R; Arangasamy, A; Kastelic, J P

    2017-02-01

    Mammalian sperm are exposed to a natural hypoosmotic environment during male-to-female reproductive tract transition; although this activates sperm motility in vivo, excessive swelling can harm sperm structure and function. Aquaporins (AQPs) is a family of membrane-channel proteins implicated in sperm osmoregulation. The objective was to determine associations among relative sperm volume shift, hypoosmotic swelling test (HOST), sperm aquaporin (AQP) 7 mRNA abundances, and sire conception rate (SCR; fertility estimate) in Holstein bulls at a commercial artificial insemination center. Three or four sires for each full point SCR score from -4 to +4 were included. Each SCR estimate for study bulls (N = 30) was based on > 500 services (mean ± SEM) of 725 ± 13 services/sire). Sperm from a single collection day (two ejaculates) from these commercial Holstein bulls were used. Relative mRNA expression of AQP7 in sperm was determined by polymerase chain reaction. Mean relative sperm volume shift and percentage of sperm reacted in a HOST (% HOST) were determined (400 sperm per bull) after incubating in isoosmotic (300 mOsm/kg) and hypoosmotic (100 mOsm/kg) solutions for 30 min. There was no correlation between %HOST and SCR (r = 0.28 P > 0.1). However, there was a positive correlation between relative sperm volume shift and SCR (r = 0.65, P 2) fertility sire groups. In conclusion, bulls with higher SCR had significantly greater AQP7 mRNA abundance in frozen-thawed sperm. This plausibly contributed to greater regulation of sperm volume shift, which apparently conferred protection from detrimental swelling and impaired functions. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. MANIFESTATION OF THE EFFECT OF CRYOSELECTION IN CARP OFFSPRINGS OBTAINED FROM DEFROSTED SPERM SUSPENSION WITH MODIFIED CRYOPROTECTIVE SOLUTIONS

    OpenAIRE

    V. Cherepnin

    2016-01-01

    Purpose. The purpose of these studies was to evaluate the effect of the cryoselection of carp sperm of multiple age groups depending on the quality of thawed sperm after modification of the composite cryoprotective medium, which was used for the dilution of native sperm before freezing. Methodology. Coenzyme B12 (cobamamide), blood plasma of Prussian carp (Carassius gibelio), which was subjected to natural cold-acclimation, and purified protein antifreeze tmAFP isolated from larval mealwo...

  14. Sperm mitochondrial DNA deletion in Iranian infertiles with asthenozoospermia.

    Science.gov (United States)

    Bahrehmand Namaghi, I; Vaziri, H

    2017-04-01

    Asthenozoospermia is an important cause of male infertility. The mutations in sperm mitochondrial DNA (mtDNA) result in either functionless or malfunctioning some proteins, subsequently affecting sperm motility leading to asthenozoospermia. The purpose of this study was to investigate sperm mtDNA 4,977-bp deletion in infertile men with low sperm motility/immotile spermatozoa compared to healthy subjects with high sperm motility. Semen samples of 256 asthenozoospermic infertiles and 200 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap-polymerase chain reaction (Gap-PCR) was performed. The deletion was observed in 85.93% of patients with asthenozoospermia compared with 14% in controls [OR = 37.5397, 95% confidence interval = 12.937-108.9276, p asthenozoospermia-induced infertility in the population examined. Large-scale mtDNA deletions in spermatozoa may induce bioenergetic disorders. Nevertheless, to validate our results broader research may be needed.

  15. Morphofunctional disturbances of human sperm after incubation with organophosphorate pesticides.

    Science.gov (United States)

    Contreras, H R; Badilla, J; Bustos-Obregón, E

    1999-08-01

    The organophosphorate pesticides are highly toxic for insects and mammals, but their effects in the male reproductive tract are scarcely known. Many alterations induced by organophosphorate pesticides have been described, such as: cytogenetic alterations in germinal cells, oligozoospermia and teratozoospermia in the mouse. Parathion, the pesticide mostly utilized in Chilean agriculture, is rapidly metabolized to paraoxon, the active metabolite, in mammalian organisms. The purpose of this study is to evaluate the effect of Parathion and paraoxon on different morphological and functional parameters of the sperm. Human spermatozoa were incubated with Parathion and paraoxon at different concentrations (0.05, 0.1, 0.2, 0.4 and 0.8 mM). Vitality (tripan blue and eosin tests), acrosome reaction (triple stain test), plasma membrane integrity (HOS-test), and chromatin stability (sodium thioglycolate test) were determined. The observations were done by optical microscopy at 1000x of magnification and three hundred sperms were evaluated for each treatment. The results indicated that Parathion and paraoxon increase the percent of sperm with acrosome reaction and also increase the percentage of sperm with chromatin decondensation in a dose-dependent manner. The vitality and plasma membrane integrity decrease significantly in a dose-dependent manner. The results suggest a direct action of Parathion and paraoxon on the different parameters studied. The morphofunctionality of sperm is altered significatively, suggesting that Parathion and paraoxon, thanks to their alkylating and electrophylic properties, could act on DNA and proteins respectively, to elicit these changes.

  16. 左卡尼汀对特发性弱精子症精子质量及蛋白激酶CK2活性的影响%Effects of Levocarnitine on sperm quality and activity of protein kinase CK2 in patients with idiopathic asthenozoospermia

    Institute of Scientific and Technical Information of China (English)

    黄雪坤; 尚慧玲; 黄娟华; 刘建霞; 张四友

    2011-01-01

    目的 研究左卡尼汀对特发性弱精子症精子质量及蛋白激酶CK2活性的影响,从而探讨其可能的作用机制.方法 利用计算机辅助精子分析(CASA)系统对35例特发性弱精子症患者接受左卡尼汀(1 g/次,tid)治疗前、治疗3个月后和15例健康男性的精液进行分析;提取精子总蛋白,利用同位素掺入法检测精子蛋白激酶CK2活性.结果 左卡尼汀能显著提高特发性弱精子症患者精子的活动率、前向性运动精子百分率(a+b级)、曲线运动速度(VCL)、直线运动速度(VSL)和平均路径运动速度(VAP).左卡尼汀治疗前CK2活性每分钟计算值为1445±431,治疗后为2209±502,治疗后精液CK2活性明显升高(P<0.05).治疗前后CK2活性变化与治疗前后精子活动率(r=0.45,P<0.05)、a+b级(r=0.58,P<0.01)、VCL(r=0.43,P<0.05)、VSL(r=0.40,P<0.05)、VAP(r=0.41,P<0.05)变化均呈正相关性.结论 左卡尼汀能显著提高特发性弱精子症精子质量,其作用机制可能是增加了蛋白激酶CK2活性.%Objective To study the effects of Levocarnitine on sperm quality and activity of protein kinase CK2 in patients with idiopathic asthenozoospermia , and further explore the possible molecular mechanisms. Methods Sperms, obtained from 35 idiopathic asthenozoospermia patients before and after treated with Levocarnitine ( 1 g/time, tid) for 3 months and 15 healthy fertile men, were analyzed by computer-aid semen analysis ( CASA ) system. In addition, protein was extracted from those sperm samples and the CK2 activity was assayed by detecting incorporation of 32P of [ 7- P] ATP into the substrate. Results The sperm in idiopathic asthenozoospermia patients treated with Levocarnitine showed a significant increase in sperm progressive motility , grade a + b sperm percent, curvilinear velocity ( VCL) , straight-line velocity (VSL) and average path velocity (VAP). CK2 activity in idiopathic asthenozoospermia patients after treated with Levocarnitine

  17. Sperm-Egg Interaction: Evidence for Boar Sperm Plasma Membrane Receptors for Porcine Zona Pellucida

    Science.gov (United States)

    Peterson, Rudolph N.; Russell, Lonnie; Bundman, Donna; Freund, Matthew

    1980-01-01

    Freshly ejaculated, noncapacitated boar sperm bind rapidly and in large numbers to pig egg zona pellucida in vitro. In the present study, the number of sperm bound decreased sharply when sperm motility was lowered by energy poisons or by reducing the temperature. Highly motile sperm from humans, guinea pigs, and rats, added at concentrations ten times higher than control sperm, did not bind to the porcine zona. At the same high concentration, a small number of hamster and bull sperm bound to the zona. Binding of boar sperm to the zona pellucida was blocked almost completely by diluted whole antiserum to sperm plasma membranes and by univalent (Fab) antibody to these membranes. When antibody to sperm plasma membrane was first absorbed with plasma membrane vesicles, sperm binding was not inhibited. These results provide direct evidence for the existence of sperm plasma membrane receptors for the zona pellucida of the pig.

  18. High Quality RNA in Semen and Sperm: Isolation, Analysis and Potential Application in Clinical Testing

    Science.gov (United States)

    Georgiadis, Andrew P.; Kishore, Archana; Zorrilla, Michelle; Jaffe, Thomas M.; Sanfilippo, Joseph S.; Volk, Etta; Rajkovic, Aleksandar; Yatsenko, Alexander N.

    2015-01-01

    Purpose Male infertility is a complex health condition. To our knowledge there are no molecular biomarkers of male infertility. Sperm RNA is a potential biomarker for detecting sperm abnormalities and viability at infertility clinics. However, RNA use is hindered by its inconsistent quantity, quality, multiple cell types in semen and condensed sperm structure. Materials and Methods We tested the usefulness of high quality RNA isolated from mature sperm and whole semen by our protocol, which reduces RNA degradation by maintaining semen and protocol components at 37C and decreasing processing time. We isolated RNA from 83 whole semen samples, 18 samples of motile sperm prepared by the swim-up protocol and 18 of sperm prepared by the standard Percoll gradient method. Results Electrophoretic and spectral analysis of RNA revealed high quality 18S and 28S rRNAs in 71 of 83 whole semen samples (86%) and 15 of 18 mature sperm swim-up samples (83%). However, high quality RNA was isolated from only 7 of 18 Percoll gradient sperm samples (39%). Interestingly quantitative reverse transcriptase-polymerase chain reaction analysis of 4 somatic and 10 germ cell markers showed that whole semen and swim-up samples had similar RNA profiles. RNA sequencing revealed that most encoded proteins were involved in mature sperm function, regulation of DNA replication, transcription, translation, cell cycle and embryo development. Conclusions We believe that semen and sperm specific RNAs are highly informative biomarkers for germ cell stages and somatic cell contribution. Therefore, these RNAs could be valuable diagnostic indicators of sperm survival, fertilization and early embryogenesis, and could serve as a predictor of the in vitro fertilization prognosis. PMID:25088949

  19. A role for carbohydrate recognition in mammalian sperm-egg binding

    Energy Technology Data Exchange (ETDEWEB)

    Clark, Gary F., E-mail: clarkgf@health.missouri.edu

    2014-08-01

    Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the egg cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.

  20. The 'omics revolution and our understanding of sperm cell biology

    Institute of Scientific and Technical Information of China (English)

    Mark A Baker

    2011-01-01

    The foundations of proteomics are to study gene products and their regulatory roles within cells. Paradoxically, the only evidence that sperm cells make new proteins is through mitochondrial protein synthesis. Yet despite this, spermatozoa are the perfect candidates for mass spectrometry and hence, proteomic analysis. These enterprising cells use a plethora of post-translational modifications in order to gain functionality following their production within the testis. By using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and more recently liquid chromatography-mass spectrometry (LC-MS)/MS, recent advances in sperm cell biology, through the use of proteomics, is making unparalleled progress. The protein inventory lists being generated have shed light on transmembrane proteins, kinases and chaperones never previously recognized. In addition, the ability to isolate either phosphopeptides or glycopeptides and quantify the differences between cells of two different populations make proteomic analysis of spermatozoa a real chance to finally answer some age old questions.

  1. Sperm protein 17 targeted near-infrared fluorescence imaging of tumor in mice%精子蛋白17靶向的小鼠肿瘤近红外荧光成像

    Institute of Scientific and Technical Information of China (English)

    李芳秋; 张士新; 安联校; 顾月清

    2012-01-01

    Objective Human sperm protein 17 ( Spl7 ) is aberrantly expressed in some cancers, and can he used as a target for specific molecular diagnosis and treatment of these malignancies. The purpose of this study is to identify tumors in mire with a high-affinity prohe consisting of a near-infrared ( NIR ) and the specific monoclonal antibody against Spl7 ( anti-Sp17-mah ). Methods We used immunohistoehemistry to identify the highly abnormal expression of Spl7 in the hepatoeellular carcinoma cell line and tumor xeno-grafts in mire. By coupling a NIR fluorescent (NIRF) eyaninc dye ( indoeyaninc green derivative 2, ICG-Dcr-02 ) to anti-Sp17-mah, we designed a hio-optical high-affinity fluorescent prohc anti-Spl7-ICG-Dcr-02, which was injected into tumor-hearing nude mice through the caudal vein for evaluating its tumor-targeting effect by the near infrared imaging system. Results The overcxpression of Spl7 was demonstrated on the surface of the hcpatoecilular carcinoma cell linc; SMMC-7721. Anti-Sp17-ICG-Dcr-02 with immuno-activity was successfully synthesized. The immuno-activity and photo stability of anti-Sp17-ICG-Der-02 exhibited a high targeting capability in Sp17 expressing tumor models ( SMMC-7721 ), and its accumulation in tumors lasted for at least 7 days. Conclusion The high-affinity proble anti-Spl7-ICG-Dcr-02 has a potential application value; for the specific diagnosis of tumor in vivo.%目的 人精子蛋白17(human sperm protein 17,Sp17) 异常表达在一些恶性肿瘤细胞,可被用作肿瘤特异性分子诊断和治疗的靶点.文中将抗Sp17单克隆抗体与近红外染料偶联,制成高亲和力探针,用近红外成像技术对活体动物肿瘤靶点进行确认.方法 用免疫组化技术证明,Sp17在肝细胞癌细胞系SMMC-7721及裸鼠皮下移植瘤组织高水平异常表达.将近红外染料吲哚花箐素衍生物(ICG-Der-02)与抗Sp17单克隆抗体(anti-Sp17 mAb)偶联,获得特异性探针.静脉注射至荷瘤小鼠体内,用

  2. What use is an infertile sperm? A comparative study of sperm-heteromorphic Drosophila

    DEFF Research Database (Denmark)

    Holman, Luke; Freckleton, Robert P; Snook, Rhonda R

    2007-01-01

    Sperm size and number are important determinants of male reproductive success. The genus Drosophila exhibits a remarkable diversity of sperm production strategies, including the production of multiple sperm morphs by individual males, a phenomenon called sperm heteromorphism. Sperm-heteromorphic ......Sperm size and number are important determinants of male reproductive success. The genus Drosophila exhibits a remarkable diversity of sperm production strategies, including the production of multiple sperm morphs by individual males, a phenomenon called sperm heteromorphism. Sperm......-heteromorphic Drosophila species in the obscura group produce large numbers of infertile "parasperm" in addition to fertile eusperm. Parasperm have been hypothesized to perform a number of roles in place of fertilization, predominantly focused on their potential function in postcopulatory sexual selection. However...

  3. Involvement of redox- and phosphorylation-dependent pathways in osmotic adaptation in sperm cells of euryhaline tilapia.

    Science.gov (United States)

    Morita, Masaya; Nakajima, Ayako; Takemura, Akihiro; Okuno, Makoto

    2011-06-15

    Sperm cells involved in fertilisation must tolerate hypo-osmotic and hyper-osmotic environments. Euryhaline tilapia (Oreochromis mossambicus) can acclimatise to and reproduce in freshwater and seawater because its sperm are able to adapt to these differing osmotic environments. In this study, we found that the dephosphorylation of sperm proteins in O. mossambicus correlated with the activation of flagellar motility when sperm were exposed to hypotonic or hypertonic conditions, and that differences in phosphorylation may reflect adaptations to a given osmotic environment. Of the sperm proteins that were dephosphorylated, the phosphorylation pattern of an 18 kDa protein, identified as the superoxide anion scavenger Cu/Zn superoxide dismutase (Cu/Zn SOD), was different in freshwater- and seawater-acclimatised tilapia sperm. Cu/Zn SOD was distributed from the sperm head to the flagellum. Additionally, differences were observed between freshwater and seawater tilapia in the nitration of tyrosine residues (which might be mediated by SOD) in sperm flagellar proteins in response to osmotic shock. These results demonstrate that reactive-oxygen-species-dependent mechanisms contribute to both osmotic tolerance and the activation of flagellar motility.

  4. Bioenergetics of Mammalian Sperm Capacitation

    Directory of Open Access Journals (Sweden)

    Alessandra Ferramosca

    2014-01-01

    Full Text Available After ejaculation, the mammalian male gamete must undergo the capacitation process, which is a prerequisite for egg fertilization. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS are the two major metabolic pathways producing ATP which is the primary source of energy for spermatozoa. Since recent data suggest that spermatozoa have the ability to use different metabolic substrates, the main aim of this work is to present a broad overview of the current knowledge on the energy-producing metabolic pathways operating inside sperm mitochondria during capacitation in different mammalian species. Metabolism of glucose and of other energetic substrates, such as pyruvate, lactate, and citrate, is critically analyzed. Such knowledge, besides its obvious importance for basic science, could eventually translate into the development of novel strategies for treatment of male infertility, artificial reproduction, and sperm selection methods.

  5. Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

    OpenAIRE

    1986-01-01

    The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results i...

  6. Microfluidic single sperm entrapment and analysis

    NARCIS (Netherlands)

    Wagenaar, B. de; Berendsen, J.T.; Bomer, J.G.; Olthuis, W.; Berg, A. van den; Segerink, L.I.

    2015-01-01

    Selection of healthy spermatozoa is of crucial importance for the success rates of assisted reproduction technologies (ART) such as in vitro fertilization and intra-cytoplasmic sperm injection. Although sperm selection for ART procedures is predominantly based on sperm motility, successful fertiliza

  7. The inner perivitelline layer sperm-hole assay: use of filter paper rings for the isolation of the perivitelline layer overlying the germinal disc and new observations on its morphology

    Science.gov (United States)

    Sperm must penetrate the hen’s ovum (egg yolk) for fertilization. The inner perivitelline layer (IPL) is a fibrous protein mesh surrounding the ovum that the sperm attach to and then digest a hole through at the onset of the fertilization process. This process of sperm attachment and digestion of ...

  8. Panax ginseng induces the expression of CatSper genes and sperm hyperactivation

    Directory of Open Access Journals (Sweden)

    Eun Hwa Park

    2014-12-01

    Full Text Available The cation channel of sperm (CatSper protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca 2+ influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca 2+ levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression.

  9. Panax ginseng induces the expression of CatSper genes and sperm hyperactivation

    Science.gov (United States)

    Park, Eun Hwa; Kim, Do Rim; Kim, Ha Young; Park, Seong Kyu; Chang, Mun Seog

    2014-01-01

    The cation channel of sperm (CatSper) protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca2+ influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca2+ levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression. PMID:24969054

  10. A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation.

    Science.gov (United States)

    delBarco-Trillo, Javier; García-Álvarez, Olga; Soler, Ana Josefa; Tourmente, Maximiliano; Garde, José Julián; Roldan, Eduardo R S

    2016-03-16

    Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage. © 2016 The Author(s).

  11. A Role for the Chemokine Receptor CCR6 in Mammalian Sperm Motility and Chemotaxis

    Science.gov (United States)

    Caballero-Campo, Pedro; Buffone, Mariano G.; Benencia, Fabian; Conejo-García, José R.; Rinaudo, Paolo F.; Gerton, George L.

    2013-01-01

    Although recent evidence indicates that several chemokines and defensins, well-known as inflammatory mediators, are expressed in the male and female reproductive tracts, the location and functional significance of chemokine networks in sperm physiology and sperm reproductive tract interactions are poorly understood. To address this deficiency in our knowledge, we examined the expression and function in sperm of CCR6, a receptor common to several chemoattractant peptides, and screened several reproductive tract fluids for the presence of specific ligands. CCR6 protein is present in mouse and human sperm and mainly localized in the sperm tail with other minor patterns in sperm from mice (neck and acrosomal region) and men (neck and midpiece regions). As expected from the protein immunoblotting and immunofluorescence results, mouse Ccr6 mRNA is expressed in the testis. Furthermore, the Defb29 mRNA encoding the CCR6 ligand, β-defensin DEFB29, is expressed at high levels in the epididymis. As determined by protein chip analysis, several chemokines (including some that act through CCR6, such as CCL20/MIP-3α (formerly Macrophage Inflammatory Protein 3α) and protein hormones were present in human follicular fluid, endometrial secretions, and seminal plasma. In functional chemotaxis assays, capacitated human sperm exhibited a directional movement towards CCL20, and displayed modifications in motility parameters. Our data indicate that chemokine ligand/receptor interactions in the male and female genital tracts promote sperm motility and chemotaxis under non-inflammatory conditions. Therefore, some of the physiological reactions mediated by CCR6 ligands in male reproduction extend beyond a pro-inflammatory response and might find application in clinical reproduction and/or contraception. PMID:23765988

  12. OPTIMAL ELECTROPORATION CONDITION FOR SPERM MEDIATED GENE TRANSFER IN STRIPPED CATFISH (Pangasionodon hypophthalmus

    Directory of Open Access Journals (Sweden)

    Raden Roro Sri Pudji Sinarni Dewi

    2010-06-01

    Full Text Available The success of transgenic fish production has been achieved through eggs fertilization using electroporated sperms carrying exogenous DNA. This study was conducted in order to obtain the optimal electroporation condition for stripped catfish sperm. A plasmid containing green fluorescent protein (GFP gene driven by carp β-actin promoter was transferred into sperm using electrophoresis method towards transgenic stripped catfish (Pangasionodon hypophthalmus production. Electroporation was carried out using square wave shock with pulse length of 30 ms and pulse interval of 0.1 sec. Treatments are combination between voltage (50 V, 75 V, and 100 V and pulse number (1 and 3. Exogenous DNA concentration used was 10 μg/mL of Tris-EDTA. Results showed that increasing the voltage from 50 to 100 decreased sperm motility, while pulse number did not affect sperm motility. Voltage of 50 gave the best motility of sperm, although sperm viability relatively similar between treatments and control except at 100 V with 3 pulses number. Further, electroporation-treated sperms were able to fertilize eggs. Higher hatching rate of eggs was obtained in electroporation treatment at 50 V with pulse number of 1 and 3. The persistence of transferred GFP was detected in electroporated and incubated sperms (control. However, GFP was only detected in larvae from eggs that were fertilized by electroporated sperm. Thus, electroporation could be applied to produce transgenic stripped catfish.

  13. Sperm motility initiation by egg jelly of the anuran, Discoglossus pictus may be mediated by sperm motility-initiating substance of the internally-fertilizing newt, Cynops pyrrhogaster.

    Science.gov (United States)

    Takayama-Watanabe, Eriko; Campanella, Chiara; Kubo, Hideo; Watanabe, Akihiko

    2012-11-01

    The egg jelly of Discoglossus pictus contains sperm motility-activating activity, the molecular basis of which has not been studied. Discoglossus pictus sperm initiated motility immediately after immersion in egg-jelly extract, as well as after immersion in hyposmotic solution, which initiates sperm motility in the external fertilization of anuran amphibians. Sequential treatment of the D. pictus sperm with these two solutions revealed the predominant effect of hyposmolality in initiation of motility. The motility initiation induced by jelly extract was suppressed by a monoclonal antibody (mAb) that is specific for the 34 kDa sperm motility-initiating substance (SMIS) in the egg jelly of the newt, Cynops pyrrhogaster. Immunoblotting using the anti-SMIS mAb revealed several antigenic proteins that included major ones with sizes of 18- and 34-kDa in D. pictus jelly extract. Scanning electron microscopic observation revealed that granules of jelly matrix, in which SMIS localizes and which have a critical role in the internal fertilization of C. pyrrhogaster, were not observed near the surface of the D. pictus egg jelly. These results suggest that sperm motility-activating activity in egg jelly of D. pictus may be mediated by SMIS homologous proteins that act through a mechanism that is partially different from that of C. pyrrhogaster.

  14. RNA Interference-Mediated Downregulation ofsAC Gene Inhibits Sperm Hyperactivation in Male Rats (Rattus norvegicus)

    Institute of Scientific and Technical Information of China (English)

    YU Jing; JIANG Xiao-qiang; ZHOU Shuai; WANG Gen-lin

    2014-01-01

    Hyperactivation is one of the most critical parts for fertilization. cAMP generated by soluble adenylyl cyclase (sAC) is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs ofsAC gene were designed and separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3-30.8% and 49.1-50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR, respectively. The results showed that two siRNAs, Actb-717 and Actb-4205, were the best RNAi sites for silencing sAC. The VCL (curvilinear velocity) and ALH (amplitude of lateral head displacement) of RNA interference (RNAi)-transfected sperm were reduced. cAMP and protein phosphorylation in RNAi transfected sperm were also decreased. The hyperactivation-related genes,such as CatSper2,LDHC andPKA, were downregulated in the sperm, whichsAC was knockdown. These ifndings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation, and downregulatedsAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction. These ifndings suggest that these hyperactivation-ralated genes andsAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.

  15. No evidence for sperm priming responses under varying sperm competition risk or intensity in guppies

    Science.gov (United States)

    Evans, Jonathan P.

    2009-07-01

    Sperm competition theory predicts that males should tailor their investment in ejaculates according to the number of rival males competing to fertilize a female’s eggs. Research spanning several taxa supports this prediction by showing that males are often sensitive to the level of sperm competition and adjust their investment in sperm numbers accordingly. More recent work has revealed that males may also tailor the quality of sperm according to the number of males competing for fertilization. Here I test for both effects in guppies ( Poecilia reticulata) in an experiment that simultaneously evaluates the risk and intensity models of sperm competition. The experiment determined whether male guppies adjust the number (stripped ejaculate size) and quality (sperm velocity and viability) of sperm that are primed over a 3-day period according to experimental changes in the perceived level of sperm competition. A total of 136 focal males were initially stripped of all retrievable sperm and assayed for these sperm traits before being allocated at random to one of four treatments simulating different levels of sperm competition risk and intensity. During the 3-day treatment phase, focal males had visual and olfactory access to a sexually receptive (initially virgin) female maintained with different numbers of stimulus males to simulate variation in the risk and intensity of sperm competition. Following this, males were assayed again for the sperm traits. Contrary to predictions, there was no significant change in any of the measured variables among treatments, although qualitatively the patterns for sperm velocity and viability did conform to expectation. The lack of any trend for the number of sperm primed was unequivocal and future work examining the effects of sperm competition on sperm production should focus on whether males differentially allocate sperm numbers among matings that differ in the level of sperm competition.

  16. The relationship between sperm viability and DNA fragmentation rates

    OpenAIRE

    Mary K. Samplaski; Dimitromanolakis, Apostolos; Lo, Kirk C; Grober, Ethan D.; Mullen, Brendan; Garbens, Alaina; Jarvi, Keith A

    2015-01-01

    Background In humans, sperm DNA fragmentation rates have been correlated with sperm viability rates. Reduced sperm viability is associated with high sperm DNA fragmentation, while conversely high sperm viability is associated with low rates of sperm DNA fragmentation. Both elevated DNA fragmentation rates and poor viability are correlated with impaired male fertility, with a DNA fragmentation rate of > 30% indicating subfertility. We postulated that in some men, the sperm viability assay coul...

  17. Characterisation of Caenorhabditis elegans sperm transcriptome and proteome

    OpenAIRE

    Ma, Xuan; Zhu, Yingjie; Li, Chunfang; Xue, Peng; Zhao, Yanmei; Chen, Shilin; Yang, Fuquan; Miao, Long

    2014-01-01

    Background Although sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome. Results First, sperm transcriptome cons...

  18. Effect of male age on sperm traits and sperm competition success in the guppy (Poecilia reticulata).

    Science.gov (United States)

    Gasparini, C; Marino, I A M; Boschetto, C; Pilastro, A

    2010-01-01

    Deleterious mutations can accumulate in the germline with age, decreasing the genetic quality of sperm and imposing a cost on female fitness. If these mutations also affect sperm competition ability or sperm production, then females will benefit from polyandry as it incites sperm competition and, consequently, minimizes the mutational load in the offspring. We tested this hypothesis in the guppy (Poecilia reticulata), a species characterized by polyandry and intense sperm competition, by investigating whether age affects post-copulatory male traits and sperm competition success. Females did not discriminate between old and young males in a mate choice experiment. While old males produced longer and slower sperm with larger reserves of strippable sperm, compared to young males, artificial insemination did not reveal any effect of age on sperm competition success. Altogether, these results do not support the hypothesis that polyandry evolved in response to costs associated with mating with old males in the guppy.

  19. Boundaries of Ethics, Sperm on the Border –The Globalization of Danish Sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    as sperm donation in parliamentary debates, and in the media, often raises ethical questions. This presentation inquires into how the bending of boundaries by “inappropriate parents”, fertility travelers, private sperm banks and fertility clinics have been part in negotiating the changes of the legislation...... in practice, and thus been part of developing a Danish industry of sperm banking. In this presentation, I will show how Danish sperm has become a global commodity even though, or maybe because the technology has been regulated, and continuously has raised and troubled questions of kinship, sexuality...... of the reasons why Danish fertility clinics have enabled women from Denmark and abroad to turn to sperm donation is, that two of the largest sperm banks in Europe are Danish. In contrast to many other countries where sperm is in demand, the Danish sperm banks are able to provide enough sperm for the national...

  20. Assessment of the frequency of sperm annulus defects in a large cohort of patients presenting asthenozoospermia

    OpenAIRE

    Dirami, Thassadite; Rode, Baptiste; Wolf, Jean-Philippe; Gacon, Gérard; Dulioust, Emmanuel; Touré, Aminata

    2015-01-01

    International audience; Background: The annulus is a ring-shaped structure located beneath the plasma membrane that connects the midpiece and the principal piece of mammalian sperm flagellum. It has been suggested that the annulus acts as a morphological organizer, guiding flagellum assembly during spermiogenesis, and as a diffusion barrier, confining proteins to distinct compartments of the flagellum in mature sperm. Previous studies on small cohorts of patients have attempted to correlate a...

  1. Sperm competition promotes diversity of sperm bundles in Ohomopterus ground beetles

    Science.gov (United States)

    Takami, Yasuoki; Sota, Teiji

    2007-07-01

    Diversification of sperm morphology has been investigated in the context of sperm competition, but the adaptive significance of sperm bundles is still unclear. In analyzing 10 taxa of the genus Carabus subgenus Ohomopterus and one related Carabus ground beetles, we found that dimorphic sperm bundles occurred in most species with varied degrees of bimodality, whereas sperm were generally monomorphic. Comparative analyses with phylogenetically independent contrasts revealed that the sizes of large and small sperm bundles evolved more rapidly than, and were not correlated with, the length of sperm, suggesting more intense selection on sperm bundle sizes and their independent responses to different evolutionary forces. The size of large sperm bundles was positively correlated with male genital morphology (pertinent to displacement of rival spermatophores) and postcopulatory guarding duration as well as male body length, suggesting that larger sperm bundles have been favored when the risk of spermatophore displacement is high. Larger sperm bundles may be advantageous because of their ability to migrate more rapidly into the spermatheca. In contrast, no clear association was detected between the small sperm bundle size and mating traits despite its rapid diversification. The present study provides the first record of heteromorphic sperm bundles, the diversity of which may be promoted by sperm competition.

  2. Evaluating the function of calcium antagonist on the Cd-induced stress in sperm of Russian sturgeon, Acipenser gueldenstaedtii.

    Science.gov (United States)

    Li, Zhi-Hua; Li, Ping; Rodina, Marek; Randak, Tomas

    2010-11-15

    In the current study, the sperm of Russian sturgeon (Acipenser gueldenstaedtii) was used to evaluate the roles of Verapamil (VRP), a calcium channel blocker, against cadmium (Cd)-induced stress. Sturgeon sperm were exposed for 2h at 50μg/L VRP, 5.0μg/L Cd, the mixture of 50μg/L VRP+5.0μg/L Cd, 50μg/L Cd and the mixture of 50μg/L VRP+50μg/L Cd. After exposure, the sperm motility parameters (motility and velocity), oxidative stress levels (lipid peroxidation [LPO] and carbonyl protein [CP]) and antioxidant enzyme activities (superoxide dismutase [SOD], glutathione reductase [GR], glutathione peroxidase [GPx]) were measured in sturgeon sperm. Compared to the control, Cd-induced stress was apparent as reflected by depressed motility parameters, induced oxidative stress and inhibited antioxidant enzyme activities at both Cd concentrations. In the presence of VRP, Cd-induced stress was reduced in sturgeon sperm, especially all the measured parameters in the sperm exposed at 5.0μg/L Cd returned to control levels, expect for the sperm motility. The present results indicate that VRP can reduce the Cd-induced stress in sturgeon sperm and suggest that using of sperm in vitro assays may provide a novel and efficient means for evaluating the effects of residual metals in the aquatic environment of sturgeon. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. Evaluating the function of calcium antagonist on the Cd-induced stress in sperm of Russian sturgeon, Acipenser gueldenstaedtii

    Energy Technology Data Exchange (ETDEWEB)

    Li Zhihua, E-mail: zhihuali06@yahoo.com [University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany (Czech Republic); Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou 434000 (China); Li Ping [University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany (Czech Republic); Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou 434000 (China); Rodina, Marek; Randak, Tomas [University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany (Czech Republic)

    2010-11-15

    In the current study, the sperm of Russian sturgeon (Acipenser gueldenstaedtii) was used to evaluate the roles of Verapamil (VRP), a calcium channel blocker, against cadmium (Cd)-induced stress. Sturgeon sperm were exposed for 2 h at 50 {mu}g/L VRP, 5.0 {mu}g/L Cd, the mixture of 50 {mu}g/L VRP + 5.0 {mu}g/L Cd, 50 {mu}g/L Cd and the mixture of 50 {mu}g/L VRP + 50 {mu}g/L Cd. After exposure, the sperm motility parameters (motility and velocity), oxidative stress levels (lipid peroxidation [LPO] and carbonyl protein [CP]) and antioxidant enzyme activities (superoxide dismutase [SOD], glutathione reductase [GR], glutathione peroxidase [GPx]) were measured in sturgeon sperm. Compared to the control, Cd-induced stress was apparent as reflected by depressed motility parameters, induced oxidative stress and inhibited antioxidant enzyme activities at both Cd concentrations. In the presence of VRP, Cd-induced stress was reduced in sturgeon sperm, especially all the measured parameters in the sperm exposed at 5.0 {mu}g/L Cd returned to control levels, expect for the sperm motility. The present results indicate that VRP can reduce the Cd-induced stress in sturgeon sperm and suggest that using of sperm in vitro assays may provide a novel and efficient means for evaluating the effects of residual metals in the aquatic environment of sturgeon.

  4. Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

    Directory of Open Access Journals (Sweden)

    Shiba Kogiku

    2011-08-01

    Full Text Available Abstract Background Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm. Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. Results Large consort males transfer smaller (average total length = 73 μm sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. Conclusions Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external, whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.

  5. Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition.

    Science.gov (United States)

    Seidel, G E; Schenk, J L

    2008-04-01

    In six field trials, doses between 1.0 and 6.0 x 10(6) total sexed, frozen-thawed sperm were inseminated into the uterine body or bilaterally into the uterine horns of heifers and nursing Angus cows 12 or 24h after observed estrus. Except for one comparison in one trial in which uterine body insemination was slightly superior (P0.1) difference between sites of semen deposition. Additionally, except for one small study with limited numbers, there was essentially no difference in pregnancy rates in the range between 1.5 and 6 x 10(6) sexed, frozen-thawed sperm per inseminate. Pregnancy rates with smaller doses of sexed sperm averaged about 75% of controls of 20 x 10(6) total frozen-thawed, unsexed sperm. While 1.0 x 10(6) sexed, frozen-thawed sperm per insemination dose resulted in decreased pregnancy rates compared to larger doses, the lesser fertility with sexed sperm could not be compensated by increasing sperm numbers in the range of 1.5-6 x 10(6) sperm per dose. Pregnancy rates with 2 x 10(6) sexed, frozen-thawed sperm per dose were not markedly less than control pregnancy rates with 20 x 10(6) frozen-thawed unsexed sperm/dose in well-managed herds.

  6. Correlated effects of sperm competition and postmating female mortality.

    Science.gov (United States)

    Civetta, A; Clark, A G

    2000-11-21

    Adaptations in one sex may impair fitness in the opposite sex. Experiments with Drosophila melanogaster have shown that seminal fluid from the male accessory gland triggers a series of postmating responses in the female, including increased egg laying rate and lower remating propensity, but that accessory gland proteins also increase female death rate. Here, we tested the relationships among the longevity of females mated to males from 51 chromosome-extracted D. melanogaster lines, male-mating ability, and sperm-competitive ability. We found significant differences in longevity of females mated to males of different genotypes, and all mated females showed a higher death rate than control virgin females shortly after mating. Both the age-independent mortality parameter (the intercept of the female's survival function) and the slope of the mortality rate curve were significantly correlated with the proportion of progeny sired by the first male to mate relative to tester males (sperm-defense ability, P1). No significant correlation was found between the proportion of progeny sired by the second-mating male relative to tester males (sperm-offense ability, P2) and any mortality parameter. Our results support the hypothesis of a tradeoff between defensive sperm-competitive ability of males and life-history parameters of mated females.

  7. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    Science.gov (United States)

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  8. Control of sperm concentration is necessary for standardization of sperm cryopreservation in aquatic species: evidence from sperm agglutination in oysters.

    Science.gov (United States)

    Dong, Qiaoxiang; Huang, Changjiang; Tiersch, Terrence R

    2007-02-01

    A lack of standardization in sperm cryopreservation of aquatic organisms is one of the main reasons for inconsistency observed among various studies. In particular, there have been few attempts to standardize sperm concentration during procedural optimization. This study was intended to call attention to sperm concentration standardization through research of sperm agglutination in Pacific oysters Crassostrea gigas. Sperm agglutination after thawing is a relatively frequent phenomenon observed for various aquatic species, especially when sub-optimal cryopreservation protocols are used; however, no systematic attempts have been made to explain this phenomenon. The present study evaluated various factors affecting sperm agglutination of thawed samples from diploid and tetraploid Pacific oysters, and is the first detailed report addressing the sperm agglutination phenomenon of thawed samples from any aquatic organism. Agglutination of oyster sperm was classified into six levels with a scale ranging from 0 (homogenous suspension) to 5 (well-developed "noodles"). It was found that agglutination in thawed samples was mainly due to the lack of sufficient cryoprotectant for a specific sperm concentration. Interestingly, high levels of agglutination did not necessarily lead to low fertilization. On the contrary, some sperm cells appeared to gain protection from the formation of peripheral agglutination within 0.5-ml French straws. The exact mechanism of sperm agglutination remains unclear. However, morphological examination of cross sections of the noodles (agglutination level 5) indicated at least two forms of agglutination (formed with and without cryoprotectant) which could be used as a tool to understand the cryopreservation process within the micro-environment of the straw. Furthermore, the fact that the level of sperm agglutination was directly determined by sperm concentration, in addition to the type of cryoprotectant, cryoprotectant concentration, and cooling and

  9. A Method for Preparation, Storage and Activation of Large Populations of Immotile Sea Urchin Sperm

    Science.gov (United States)

    Bracho, Geracimo E.; Fritch, Jennifer J.; Tash, Joseph S.

    1997-01-01

    Reversible protein phosphorylation is associated with initiation and modulation of sperm flagellar motility. Many studies aimed at examining the signal transduction mechanisms underlying the expression of motility have relied on detergent-permeabilized sperm reactivated with exogenous 32 P-ATP. However, the reactivation conditions allow variable levels of motility to be expressed and phosphorylation of many proteins that appear to be unrelated to sperm motility. Thus, identification of the few relevant proteins is difficult. We have developed a method to collect and keep sperm immotile until reactivated for analysis to normal motility levels. Artificial sea water (ASW) buffered with 5 mM 2-[N-morpholino]ethanesulfonic acid at pH 6.0 and containing 50 mM KCI, allows collection and storage of immotile sea urchin sperm for up to 96 h at 4-5 C. Motility under these conditions is essentially zero, but sperm is rapidly reactivated to normal motility by diluting with ASW to standard pH (8.0) and KCI concentration (10 mM).

  10. Fertilization of sea urchin eggs and sperm motility are negatively impacted under low hypergravitational forces significant to space flight

    Science.gov (United States)

    Tash, J. S.; Kim, S.; Schuber, M.; Seibt, D.; Kinsey, W. H.

    2001-01-01

    Sperm and other flagellates swim faster in microgravity (microG) than in 1 G, raising the question of whether fertilization is altered under conditions of space travel. Such alterations have implications for reproduction of plant and animal food and for long-term space habitation by man. We previously demonstrated that microG accelerates protein phosphorylation during initiation of sperm motility but delays the sperm response to the egg chemotactic factor, speract. Thus sperm are sensitive to changes in gravitational force. New experiments using the NiZeMi centrifugal microscope examined whether low hypergravity (hyperG) causes effects opposite to microG on sperm motility, signal transduction, and fertilization. Sperm % motility and straight-line velocity were significantly inhibited by as little as 1.3 G. The phosphorylation states of FP130, an axonemal phosphoprotein, and FP160, a cAMP-dependent salt-extractable flagellar protein, both coupled to motility activation, showed a more rapid decline in hyperG. Most critically, hyperG caused an approximately 50% reduction in both the rate of sperm-egg binding and fertilization. The similar extent of inhibition of both fertilization parameters in hyperG suggests that the primary effect is on sperm rather than eggs. These results not only support our earlier microG data demonstrating that sperm are sensitive to small changes in gravitational forces but more importantly now show that this sensitivity affects the ability of sperm to fertilize eggs. Thus, more detailed studies on the impact of space flight on development should include studies of sperm function and fertilization.

  11. Characterization of Mammalian ADAM2 and Its Absence from Human Sperm.

    Directory of Open Access Journals (Sweden)

    Heejin Choi

    Full Text Available The members of the ADAM (a disintegrin and metalloprotease family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

  12. Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation.

    Science.gov (United States)

    Michailov, Yulia; Ickowicz, Debbi; Breitbart, Haim

    2014-12-15

    Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn(2+), required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn(2+) in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn(2+) activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn(2+) to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn(2+) also occurred in the absence of Ca(2+) in the incubation medium, and required the tmAC, indicating that Zn(2+) activates a GPCR. The AR stimulated by Zn(2+) is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction.

  13. RNA interference of the period gene affects the rhythm of sperm release in moths.

    Science.gov (United States)

    Kotwica, Joanna; Bebas, Piotr; Gvakharia, Barbara O; Giebultowicz, Jadwiga M

    2009-02-01

    The period (per) gene is 1 of the core elements of the circadian clock mechanism in animals from insects to mammals. In clock cells of Drosophila melanogaster, per mRNA and PER protein oscillate in daily cycles. Consistent with the molecular clock model, PER moves to cell nuclei and acts as a repressor of positive clock elements. Homologs of per are known in many insects; however, specific roles of per in generating output rhythms are not known for most species. The aim of this article was to determine whether per is functionally involved in the circadian rhythm of sperm release in the moth, Spodoptera littoralis. In this species, as in other moths, rhythmic release of sperm bundles from the testis into the upper vas deferens occurs only in the evening, and this rhythm continues in the isolated reproductive system. S. littoralis was used to investigate the expression of per mRNA and protein in the 2 types of cells involved in sperm release: the cyst cells surrounding sperm bundles in the testes, and the barrier cells separating testicular follicles from the vas deferens. In cyst cells, PER showed a nuclear rhythm in light/dark (LD) cycles but was constitutively cytoplasmic in constant darkness (DD). In barrier cells, nuclear cycling of PER was observed in both LD and DD. To determine the role of PER in rhythmic sperm release in moths, testes-sperm duct complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment significantly lowered per mRNA and protein in cyst cells and barrier cells and caused a delay of sperm release. These data demonstrate that a molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic sperm release in this species.

  14. Etiology and Evaluation of Sperm Chromatin Anomalies

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee

    2008-01-01

    Full Text Available Evidence suggests that human sperm chromatin anomalies adversely affect reproductive outcomesand infertile men possess substantially amount of sperm with chromatin anomalies than fertilemen.Routine semen analysis evaluates parameters such as sperm motility and morphology, but doesnot examine the nuclear DNA integrity of spermatozoa. It has been suggested that altered nuclearchromatin structure or damaged DNA in spermatozoa could modify the special cellular functionsof human spermatozoa, and thereby affect the fertility potential. Intra-cytoplasmic sperm injection(ICSI bypass the barriers to fertilization for such a sperm, then the effect of chromatin anomalies onthe development remains a concern. Therefore, it is essential to develop and use accurate diagnostictests, which may provide better prognostic capabilities than the standard sperm assessments. Thisreview discusses our current understanding of the structure and organization of sperm DNA,the different procedures for assessment of sperm chromatin anomalies including comet assay,Chromomycin A3 (CMA3, sperm chromatin structure assay (SCSA, acridine orange test (AOT,terminal TdT-mediated dUTP-nick-end labelling (TUNEL assay, aniline blue and sperm chromatindispersion (SCD test and the impact of chromatin anomalies on reproductive outcome.

  15. Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

    Science.gov (United States)

    Serafini, Rosanna; Romano, Juan E; Varner, Dickson D; Di Palo, Rossella; Love, Charles C

    2015-08-01

    The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than inter-assay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (P<0.05). The SCSA measures were inversely correlated with neutral Comet head measures (diameter, area, and intensity) and positively with percentage Ghosts (P<0.05). The % Ghosts and COMP-αt were correlated with some measures of sperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality.

  16. Sperm motility under exposure of hydrogen dioxide

    Directory of Open Access Journals (Sweden)

    V. V. Evdokimov

    2015-01-01

    Full Text Available The paper contains research data on the effect of low concentrations of hydrogen dioxide on human sperm motility and specific enzyme activity of sperms of glyceraldehyde-3-phosphate dehydrogenase. It is shown that incubation of sperms with hydrogen dioxide in a low concentration leads to a change and motility in sperm and activity of sperm enzyme. Intensity of observed effect depended on the concentration of hydrogen dioxide: active mobility increased by 17–19 % and the total mobility – 11 %. Motility changes in sperms were accompanied by increased activity of glyceraldehyde-3-phosphate dehydrogenase by 24 %, in normozoospermia response was higher than in pathozoospermia and also depended on the concentration of hydrogen dioxide. The use of sperm analyzer enabled revealing changes in the diapason of different speeds of the active fraction of sperm, which have been observed in the first 15 min of incubation with hydrogen dioxide. A possible mechanism of action of the detected effect is discussed. Reactive oxygen species easily oxidize enzyme for glyceraldehyde-3-phosphate dehydrogenase of sperms, which leads to a loss of sperm motility, for example, in varicocele. Initially low enzyme activity in varicocele (pathozoospermia may be associated with the suppression of sperm antioxidant defense. Addition of low concentrations of hydrogen dioxide into sperm samples leads to an increase in the concentration of reduced glutathione in a cell. Increase of sperm motility in this case can serve as an indicator of normal operation of the cellular antioxidant defense system. Obtained experimental results provide a background for their introduction into clinical practice in the program of assisted reproductive technologies. 

  17. Sperm motility under exposure of hydrogen dioxide

    Directory of Open Access Journals (Sweden)

    V. V. Evdokimov

    2015-04-01

    Full Text Available The paper contains research data on the effect of low concentrations of hydrogen dioxide on human sperm motility and specific enzyme activity of sperms of glyceraldehyde-3-phosphate dehydrogenase. It is shown that incubation of sperms with hydrogen dioxide in a low concentration leads to a change and motility in sperm and activity of sperm enzyme. Intensity of observed effect depended on the concentration of hydrogen dioxide: active mobility increased by 17–19 % and the total mobility – 11 %. Motility changes in sperms were accompanied by increased activity of glyceraldehyde-3-phosphate dehydrogenase by 24 %, in normozoospermia response was higher than in pathozoospermia and also depended on the concentration of hydrogen dioxide. The use of sperm analyzer enabled revealing changes in the diapason of different speeds of the active fraction of sperm, which have been observed in the first 15 min of incubation with hydrogen dioxide. A possible mechanism of action of the detected effect is discussed. Reactive oxygen species easily oxidize enzyme for glyceraldehyde-3-phosphate dehydrogenase of sperms, which leads to a loss of sperm motility, for example, in varicocele. Initially low enzyme activity in varicocele (pathozoospermia may be associated with the suppression of sperm antioxidant defense. Addition of low concentrations of hydrogen dioxide into sperm samples leads to an increase in the concentration of reduced glutathione in a cell. Increase of sperm motility in this case can serve as an indicator of normal operation of the cellular antioxidant defense system. Obtained experimental results provide a background for their introduction into clinical practice in the program of assisted reproductive technologies. 

  18. Premature sperm activation and defective spermatogenesis caused by loss of spe-46 function in Caenorhabditis elegans.

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    Wei-Siang Liau

    Full Text Available Given limited resources for motility, sperm cell activation must be precisely timed to ensure the greatest likelihood of fertilization. Like those of most species, the sperm of C. elegans become active only after encountering an external signaling molecule. Activation coincides with spermiogenesis, the final step in spermatogenesis, when the spherical spermatid undergoes wholesale reorganization to produce a pseudopod. Here, we describe a gene involved in sperm activation, spe-46. This gene was identified in a suppressor screen of spe-27(it132ts, a sperm-expressed gene whose product functions in the transduction of the spermatid activation signal. While spe-27(it132ts worms are sterile at 25°C, the spe-46(hc197I; spe-27(it132tsIV double mutants regain partial fertility. Single nucleotide polymorphism mapping, whole genome sequencing, and transformation rescue were employed to identify the spe-46 coding sequence. It encodes a protein with seven predicted transmembrane domains but with no other predicted functional domains or homology outside of nematodes. Expression is limited to spermatogenic tissue, and a transcriptional GFP fusion shows expression corresponds with the onset of the pachytene stage of meiosis. The spe-46(hc197 mutation bypasses the need for the activation signal; mutant sperm activate prematurely without an activation signal in males, and mutant males are sterile. In an otherwise wild-type genome, the spe-46(hc197 mutation induces a sperm defective phenotype. In addition to premature activation, spe-46(hc197 sperm exhibit numerous defects including aneuploidy, vacuolization, protruding spikes, and precocious fusion of membranous organelles. Hemizygous worms [spe-46(hc197/mnDf111] are effectively sterile. Thus, spe-46 appears to be involved in the regulation of spermatid activation during spermiogenesis, with the null phenotype being an absence of functional sperm and hypomorphic phenotypes being premature spermatid activation

  19. The voltage-gated sodium channel nav1.8 is expressed in human sperm.

    Directory of Open Access Journals (Sweden)

    Antonio Cejudo-Roman

    Full Text Available The role of Na(+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na(+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM, the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca(2+-containing or Ca(2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na(+, [Na(+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na(+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

  20. Dual function of the selenoprotein PHGPx during sperm maturation.

    Science.gov (United States)

    Ursini, F; Heim, S; Kiess, M; Maiorino, M; Roveri, A; Wissing, J; Flohé, L

    1999-08-27

    The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.

  1. Tektin 3 is required for progressive sperm motility in mice

    Science.gov (United States)

    Roy, Angshumoy; Lin, Yi-Nan; Agno, Julio E.; DeMayo, Francesco J.; Matzuk, Martin M.

    2008-01-01

    Tektins are evolutionarily-conserved flagellar (and ciliary) filamentous proteins present in the axoneme and peri-axonemal structures in diverse metazoan species. We have previously shown that tektin 3 (TEKT3) and tektin 4 (TEKT4) are male germ cell-enriched proteins, and that TEKT4 is essential for coordinated and progressive sperm motility in mice. Here we report that male mice null for TEKT3 produce sperm with reduced motility (47.2% motility) and forward progression, and increased flagellar structural bending defects. Male TEKT3-null mice however maintain normal fertility in two different genetic backgrounds tested, in contrast to TEKT4-null mice. Furthermore, male mice null for both TEKT3 and TEKT4 show subfertility on a mixed B6;129 genetic background, significantly different from either single knockouts, suggesting partial non-redundant roles for these two proteins in sperm physiology. Our results suggest that tektins are potential candidate genes for non-syndromic asthenozoospermia in humans. PMID:18951373

  2. Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane.

    Science.gov (United States)

    Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

    2016-09-13

    Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p membrane of sperm head which could improve sperm plasma membrane integrity.

  3. Impairment on sperm quality and fertility of adult rats after antiandrogen exposure during prepuberty.

    Science.gov (United States)

    Perobelli, Juliana Elaine; Alves, Thaís Regina; de Toledo, Fabíola Choqueta; Fernandez, Carla Dal Bianco; Anselmo-Franci, Janete A; Klinefelter, Gary R; Kempinas, Wilma De Grava

    2012-06-01

    This study evaluated the effects of antiandrogen exposure during the prepubertal period on reproductive development and reproductive competence in adults. Male rats were divided into two groups: flutamide, receiving 25 mg/kg/day of flutamide by oral gavage and control, receiving vehicle daily. Dosing continued from PND 21 to 44, and animals were killed on PND 50 or PND 75-80. The epididymis, prostate, vas deferens and seminal vesicle weights were lower in Flutamide group on PND 50, while on PND 80 only seminal vesicle weight was reduced. Fertility assessed by IUI revealed a decrease in the fertility potential in the flutamide-treated adults. Flutamide accelerated sperm transit time through the epididymis, impairing sperm motility and storage. A quantitative analysis of the cauda sperm membrane proteome revealed a few significant changes in protein expression. Thus, exposure to flutamide during the prepubertal period compromises the function of the epididymis along with epididymal sperm quality at adulthood.

  4. Social imaginaries, sperm and whiteness

    DEFF Research Database (Denmark)

    Andreassen, Rikke

    2017-01-01

    often is associated with nineteenth-century racial science, can surface in contemporary times. Yet the article also reveals how colonial and racial legacies of whiteness tend to be erased from narratives of fertility, as the (neoliberal) focus on the individual as consumer frames reproduction......This article analyses narratives about so-called Viking babies and Viking sperm. Over the last few years an increasing number of British single women and lesbian couples have been creating families by becoming pregnant with Danish donor sperm, termed ‘Viking sperm’. Through analyses of British...... media coverage of these new families, this article explores contemporary constructions of race, especially whiteness, and gender, and shows how imaginaries of Vikings, genes and white superiority circulate in British media and among British mothers. The article illustrates how a racial discourse, which...

  5. Zonadhesin is essential for species specificity of sperm adhesion to the egg zona pellucida.

    Science.gov (United States)

    Tardif, Steve; Wilson, Michael D; Wagner, Rebecca; Hunt, Peter; Gertsenstein, Marina; Nagy, Andras; Lobe, Corrinne; Koop, Ben F; Hardy, Daniel M

    2010-08-06

    Interaction of rapidly evolving molecules imparts species specificity to sperm-egg recognition in marine invertebrates, but it is unclear whether comparable interactions occur during fertilization in any vertebrate species. In mammals, the sperm acrosomal protein zonadhesin is a rapidly evolving molecule with species-specific binding activity for the egg zona pellucida (ZP). Here we show using null mice produced by targeted disruption of Zan that zonadhesin confers species specificity to sperm-ZP adhesion. Sperm capacitation selectively exposed a partial von Willebrand D domain of mouse zonadhesin on the surface of living, motile cells. Antibodies to the exposed domain inhibited adhesion of wild-type spermatozoa to the mouse ZP but did not inhibit adhesion of spermatozoa lacking zonadhesin. Zan(-/-) males were fertile, and their spermatozoa readily fertilized mouse eggs in vitro. Remarkably, however, loss of zonadhesin increased adhesion of mouse spermatozoa to pig, cow, and rabbit ZP but not mouse ZP. We conclude that zonadhesin mediates species-specific ZP adhesion, and Zan(-/-) males are fertile because their spermatozoa retain adhesion capability that is not species-specific. Mammalian sperm-ZP adhesion is therefore molecularly robust, and species-specific egg recognition by a protein in the sperm acrosome is conserved between invertebrates and vertebrates, even though the adhesion molecules themselves are unrelated.

  6. Thermodynamics of the interaction between bovine binder of sperm BSP1 and low-density lipoprotein from hen's egg yolk

    Energy Technology Data Exchange (ETDEWEB)

    Lusignan, Marie-France; Manjunath, Puttaswamy [Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, Universite de Montreal, C.P. 6128, Succ. Centre Ville, Montreal, Quebec, Canada H3C 3J7 (Canada); Lafleur, Michel, E-mail: michel.lafleur@umontreal.ca [Department of Chemistry, Centre for Self-Assembled Chemical Structures (CSACS), Universite de Montreal, C.P. 6128, Succ. Centre Ville, Montreal, Quebec, Canada H3C 3J7 (Canada)

    2011-03-20

    Research highlights: {yields} Isothermal titration calorimetry revealed Binder-of-SPerm BSP1 protein has high affinity for egg yolk low density lipoproteins. {yields} It is estimated that 104 BSP1 molecules could bind one LDL particle. {yields} BSP1 has 2 phosphocholine binding sites and the BSP1/LDL ratio corresponds to 1.6 phosphatidylcholine per bound BSP1. {yields} The strong interaction between LDL particles and BSP1 is proposed to be important for sperm protection by egg yolk extender. - Abstract: Egg yolk is used in extender to protect sperm from cold shock and freezing during preservation. It is the low-density lipoprotein (LDL) fraction of egg yolk that protects sperm. Even though essential for sperm capacitation, the major proteins from bull seminal plasma, the Binder of SPerm (BSP) proteins, are detrimental for sperm preservation because they induce a continual phospholipids and cholesterol efflux from sperm membranes. The BSP proteins were proposed to bind to egg yolk LDL, preventing the sperm membrane damage. We characterized the binding between the BSP proteins and the LDL by isothermal titration calorimetry, providing the thermodynamics and quantitative description of this putative association. The association between BSP1 (major BSP proteins) and LDL is characterized by an affinity constant (K{sub a}) of 3.4 {+-} 0.4 {mu}M{sup -1}. A protein/LDL ratio of 104 {+-} 5 was determined indicating that 104 molecules of BSP1 would bind to one LDL particle. This stoichiometry leads to proposing that the association involves 1.6 {+-} 0.1 phosphatidylcholines (PC) per BSP protein. This finding is satisfactorily consistent with the fact that each BSP1 protein has 2 binding sites for choline group. In conclusion, the formation of a high affinity complex between BSP1 and LDL is proposed to be important for the protection of sperm by egg yolk extender.

  7. Mouse oviduct-specific glycoprotein is an egg-associated ZP3-independent sperm-adhesion ligand.

    Science.gov (United States)

    Lyng, Robert; Shur, Barry D

    2009-11-01

    Mouse sperm-egg binding requires a multiplicity of receptor-ligand interactions, including an oviduct-derived, high molecular weight, wheat germ agglutinin (WGA)-binding glycoprotein that associates with the egg coat at ovulation. Herein, we report the purification and identification of this sperm-binding ligand. WGA-binding, high molecular weight glycoproteins isolated from hormonally primed mouse oviduct lysates competitively inhibit sperm-egg binding in vitro. Within this heterogeneous glycoprotein preparation, a distinct 220 kDa protein selectively binds to sperm surfaces, and was identified by sequence analysis as oviduct-specific glycoprotein (OGP). The sperm-binding activity of OGP was confirmed by the loss of sperm-binding following immunodepletion of OGP from oviduct lysates, and by the ability of both immunoprecipitated OGP and natively purified OGP to competitively inhibit sperm-egg binding. As expected, OGP is expressed by the secretory cells of the fimbriae and infundibulum; however, in contrast to previous reports, OGP is also associated with both the zona pellucida and the perivitelline space of mouse oocytes. Western blot analysis and lectin affinity chromatography demonstrate that whereas the bulk of OGP remains soluble in the ampullar fluid, distinct glycoforms associate with the cumulus matrix, zona pellucida and perivitelline space. The sperm-binding activity of OGP is carbohydrate-dependent and restricted to a relatively minor peanut agglutinin (PNA)-binding glycoform that preferentially associates with the sperm surface, zona pellucida and perivitelline space, relative to other more abundant glycoforms. Finally, pretreatment of two-cell embryos, which do not normally bind sperm, with PNA-binding OGP stimulates sperm binding.

  8. Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid.

    Science.gov (United States)

    Tollner, Theodore L; Vandevoort, Catherine A; Yudin, Ashley I; Treece, Cathy A; Overstreet, James W; Cherr, Gary N

    2009-05-01

    Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.

  9. Localization of CD9 Molecule on Bull Spermatozoa: Its Involvement in the Sperm-Egg Interaction.

    Science.gov (United States)

    Antalíková, J; Jankovičová, J; Simon, M; Cupperová, P; Michalková, K; Horovská, Ľ

    2015-06-01

    Tetraspanin CD9 is one of the egg membrane proteins known to be essential in fertilization process. The presence and localization of CD9 molecule in spermatozoa and its possible function in reproduction are still unclear. In our study, we describe the localization of CD9 on bull spermatozoa. In the immunofluorescence assay, the positive signal has been observed in the high proportion of sperm cells as a fine grains either on the apical part or through the entire anterior region of sperm head. CD9 recognized by monoclonal antibody IVA-50 was detected on freshly ejaculated (83.4 ± 3.7%) and frozen-thawed (84.3 ± 2.3%) sperm. The same reaction pattern was observed on sperm capacitated for 1 h, 2 h, 3 h and 4 h (83.6 ± 2.0%; 84.0 ± 1.5%; 85.7 ± 0.8%; 77.5 ± 10.8%). The presence of CD9 exclusively on plasma membrane of the bovine sperm has been detected by Western blot analysis of the protein fractions after the discontinuous sucrose gradient fractionation of the bull sperm. Moreover, probable role of the sperm CD9 molecule in fertilization process of cattle has been suggested as sperm treatment with anti-CD9 antibody significantly reduced (by 25%, p ≤ 0.001) the number of fertilized oocytes compared to control group in fertilization assay in vitro.

  10. Chicken sperm transcriptome profiling by microarray analysis.

    Science.gov (United States)

    Singh, R P; Shafeeque, C M; Sharma, S K; Singh, R; Mohan, J; Sastry, K V H; Saxena, V K; Azeez, P A

    2016-03-01

    It has been confirmed that mammalian sperm contain thousands of functional RNAs, and some of them have vital roles in fertilization and early embryonic development. Therefore, we attempted to characterize transcriptome of the sperm of fertile chickens using microarray analysis. Spermatozoal RNA was pooled from 10 fertile males and used for RNA preparation. Prior to performing the microarray, RNA quality was assessed using a bioanalyzer, and gDNA and somatic cell RNA contamination was assessed by CD4 and PTPRC gene amplification. The chicken sperm transcriptome was cross-examined by analysing sperm and testes RNA on a 4 × 44K chicken array, and results were verified by RT-PCR. Microarray analysis identified 21,639 predominantly nuclear-encoded transcripts in chicken sperm. The majority (66.55%) of the sperm transcripts were shared with the testes, while surprisingly, 33.45% transcripts were detected (raw signal intensity greater than 50) only in the sperm and not in the testes. The greatest proportion of up-regulated transcripts were responsible for signal transduction (63.20%) followed by embryonic development (56.76%) and cell structure (56.25%). Of the 20 most abundant transcripts, 18 remain uncharacterized, whereas the least abundant genes were mostly associated with the ribosome. These findings lay a foundation for more detailed investigations on sperm RNAs in chickens to identify sperm-based biomarkers for fertility.

  11. Nontransitivity of sperm precedence in Drosophila.

    Science.gov (United States)

    Clark, A G; Dermitzakis, E T; Civetta, A

    2000-06-01

    Sperm competition is an important component of fitness in Drosophila, but we still do not have a clear understanding of the unit of selection that is relevant to sperm competition. Here we demonstrate that sperm competitive ability is not a property of the sperm haplotype, but rather of the diploid male's genotype. Then we test whether the relative sperm competitive ability of males can be ranked on a linear array or whether competitive ability instead depends on particular pairwise contests among males. Sperm precedence of six chromosome-extracted lines was tested against three different visible marker lines (cn bw, bwD, and Cy), and the rank order of the six lines differed markedly among the mutant lines. Population genetic theory has shown that departures from transitivity of sperm precedence may be important to the maintenance of polymorphism for genes that influence sperm competitive ability. The nontransitivity seen in sperm precedence should theoretically increase the opportunity for polymorphism in genes that influence this phenotype.

  12. Microdissection testicular sperm extraction: an update

    Institute of Scientific and Technical Information of China (English)

    Ali A Dabaja; Peter N Schlegel

    2013-01-01

    Patients with non-obstructive azoospermia (NOA) were once considered to be infertile with few treatment options due to the absence of sperm in the ejaculate.In the last two decades,the advent of intracytoplasmic sperm injection (ICSI),and the application of various testicular sperm retrieval techniques,including fine needle aspiration (FNA),conventional testicular sperm extraction (TESE) and microdissection testicular sperm extraction (micro-TESE) have revolutionized treatment in this group of men.Because most men with NOA will have isolated regions of spermatogenesis within the testis,studies have illustrated that sperm can be retrieved in most men with NOA,including Klinefelter's syndrome (KS),prior history of chemotherapy and cryptorchidism.Micro-TESE,when compared with conventional TESE has a higher sperm retrieval rate (SRR) with fewer postoperative complications and negative effects on testicular function.In this article,we will compare the efficacy of the different procedures of sperm extraction,discuss the medical treatment and the role of testosterone optimization in men with NOA and describe the micro-TESE surgical technique.Furthermore,we will update our overall experience to allow counseling on the prognosis of sperm retrieval for the specific subsets of NOA.

  13. Intracellular pH in sperm physiology.

    Science.gov (United States)

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  14. SPERM DNA INTEGRITY IN BUFFALO, BULL AND STALLION

    OpenAIRE

    Serafini, Rosanna

    2015-01-01

    The interest in sperm DNA integrity evaluation and its relationship to subfertility and infertility loaded to development of several sperm DNA assays. The aim of this study was to compare several sperm DNA assays in buffaloes, bulls and stallions, and to identify the relationships between those DNA assays and traditional sperm features. In Italian Mediterranean Buffalo (IMB) bulls traditional sperm features (motility, viability, acrosome integrity and morphology), sperm DNA integrity (neutral...

  15. Current status and potential of morphometric sperm analysis

    OpenAIRE

    Alejandro Maroto-Morales; Olga García-Álvarez; Manuel Ramón; Felipe Martínez-Pastor; M Rocío Fernández-Santos; A Josefa Soler; José Julián Garde

    2016-01-01

    The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cr...

  16. Establishment and optimization of two-dimensional gel electrophoresis for proteomics research of sperm protein of varicocele patients%精索静脉曲张不育患者精子蛋白组双向电泳技术的建立与优化

    Institute of Scientific and Technical Information of China (English)

    何犇; 唐伟; 罗华铭; 郭毅; 李跃华

    2012-01-01

    目的:建立与优化精索静脉曲张不育患者精子蛋白质组双向凝胶电泳技术.方法:采取Percoll密度梯度离心法提取精索静脉曲张不育患者精子,比较不同精子蛋白样品制备方法、不同胶条、不同上样量和不同水化方式对双向凝胶电泳结果的影响.结果:使用clean-up kit法对样品进行预处理,选取pH 5~8的固相化pH梯度胶条,采用250 μg的上样量和主动水化方式能获得背景干净、分辨率高的双向电泳(Two-dimensional electrophoresis,2-DE)图谱.结论:成功建立了精索静脉曲张不育患者精子蛋白双向凝胶电泳平台,所获2-DE图谱的分辨率高、重复性好,为进一步探讨精索静脉曲张导致不育的分子机制奠定了技术基础.%Objective:To establish and optimize a set of two-dimensional gel electrophoresis(2-DE) techniques for proteomic research of sperm protein of varicocele patients. Methods: Spermatozoa in the semen were purified by Percoll gradient technique. The effects of different sample preparation methods,different gels,different amount of protein loads and different rehydration methods to the 2-DE maps were compared. Results:After the sample was treated with clean-up kit,a high resolution and clear 2-DE map for sperm protein of varicocele patients was obtained with pH 5~8 gel, 250 μg protein loads and initiative rehydration method. Conclusion:The 2-DE map of sperm protein of varicocele patients is successfully obtained with good repeatability and high resolution, which paves the way for the further proteomic research of infertility caused by varicocele.

  17. Distinct Roles of Soluble and Transmembrane Adenylyl Cyclases in the Regulation of Flagellar Motility in Ciona Sperm

    Directory of Open Access Journals (Sweden)

    Kogiku Shiba

    2014-07-01

    Full Text Available Adenylyl cyclase (AC is a key enzyme that synthesizes cyclic AMP (cAMP at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC and soluble AC (sAC are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF, as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca2+-dependent chemotactic movement of sperm.

  18. Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumber.

    Science.gov (United States)

    Morita, Masaya; Kitamura, Makoto; Nakajima, Ayako; Sri Susilo, Endang; Takemura, Akihiro; Okuno, Makoto

    2009-04-01

    The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (> 7.8) solution without cAMP, and a phosphorylation assay using (gamma-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+-binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+-sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+-sensitive flagellar protein phosphorylation.

  19. Significance of peristaltic squeezing of sperm bundles in the silkworm, Bombyx mori: elimination of irregular eupyrene sperm nuclei of the triploid

    OpenAIRE

    Sahara, Ken; Kawamura, Naoko; Yamashiki, Naoko; Saitoh, Hiroshi

    2001-01-01

    Silkworm (Lepidoptera) males produce dimorphic sperm: nucleate eupyrene sperm and anucleate apyrene sperm. The eupyrene sperm are ordinary sperm to fertilise the eggs, while the function of apyrene sperm remains uncertain. After meiosis, 256 sperm cells are enclosed by a layer of cyst cells, forming a sperm bundle. We have previously documented that the nucleus of eupyrene sperm anchors to the head cyst cell, which locates at the anterior apex of the bundle, by an acrosome tubule-basal body a...

  20. Toxic effects of Pb(2+) entering sperm through Ca(2+) channels in the freshwater crab Sinopotamon henanense.

    Science.gov (United States)

    Li, Na; Xu, Peng; Jing, Wei-Xin; Hwang, Jiang-Shiou; Wang, Lan

    2017-09-04

    Lead (Pb) is a heavy metal that can damage animal sperm. To study the effects of Pb on calcium homeostasis and calcium channel in the sperm of freshwater crab Sinopotamon henanense, the induction of acrosome reaction (AR) and acrosin activity were investigated when crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L) for 3, 5 and 7 d separately. Fluorescent probe Fluo-3/AM was loaded into the sperm, and [Ca(2+)] in the sperm was measured by fluorescence microscopy and using microplate reader. The calmodulin (CaM) concentration was measured by ELISA method. Verapamil (VRP), a calcium channel blocker, was used to evaluate whether Pb can enter the sperm through calcium channels leading to sperm damage. After sperm were exposed at 50μg/L VRP, 100μg/L Pb, 50μg/L VRP+100μg/L Pb, 1000μg/L Pb and 50μg/L VRP+1000μg/L Pb for 1h in vitro,sperm quality parameters (sperm survival and sperm DNA integrity) and levels of parameters indicating oxidative stress (protein carbonylation [PCO] and malondialdehyde [MDA]) were measured. Our data showed that Pb reduced the induction of acrosome reaction (AR), down-regulated the acrosin activity, decreased the intracellular concentration of Ca(2+) and elevated CaM concentration. Compared to controls, Pb alone induced significant stress, as reflected by decreasing sperm survival and sperm DNA integrity, and increasing PCO and MDA contents. In the presence of VRP, 100μg/L Pb-induced stresses were reduced, all the measured parameters in the sperm exposed at 100μg/L Pb returned to control levels. Our results indicate that Pb enters the sperm of the crab S. henanense through calcium channels, the inhibition of which blocks Pb-induced stresses such as sperm quality decline and oxidative damage. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Protective effect of Rhodiola rosea polysaccharides on cryopreserved boar sperm.

    Science.gov (United States)

    Yang, Shen-Min; Wang, Ting; Wen, Duan-Gai; Hou, Jian-Quan; Li, Hai-Bo

    2016-01-01

    Cryopreservation brings sublethal damage to sperm, resulting in reduced fertile life of sperm. Rhodiola rosea polysaccharides (RPs) have antiviral, antioxidant and antitumor activities. In the present study, the cryoprotective effect of RPs on boar sperm quality parameters after frozen-thawed process was investigated. Boar sperm was cryopreserved in the extender with RPs added at concentrations of 0 (used as control), 2, 4, 6, 8 and 10mg/L and their effects on the quality of frozen-thawed boar sperm were assessed. Addition of RPs significantly improved sperm motility, mitochondrial activity, acrosomal integrity, plasma membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased sperm malonaldehyde level (pboar sperm.

  2. Sperm chromatin structure and male fertility: biological and clinical aspects

    Institute of Scientific and Technical Information of China (English)

    J. Erenpreiss; M. Spano; J. Erenpreisa; M. Bungum; A. Giwercman

    2006-01-01

    Aim: Sperm chromatin/DNA integrity is essential for the accurate transmission of paternal genetic information, and normal sperm chromatin structure is important for sperm fertilizing ability. The routine examination of semen, which includes sperm concentration, motility and morphology, does not identify defects in sperm chromatin structure. The origin of sperm DNA damage and a variety of methods for its assessment are described. Evaluation of sperm DNA damage appears to be a useful tool for assessing male fertility potential both in vivo and in vitro. The possible impact of sperm DNA defects on the offspring is also discussed.

  3. Relationship between Potential Sperm Factors Involved in Oocyte Activation and Sperm DNA Fragmentation with Intra-Cytoplasmic Sperm Injection Clinical Outcomes

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee

    2016-10-01

    Full Text Available Objective: The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP, phospholipase Cζ (PLCζ, and truncated form of the kit receptor (TR-KIT] as candidates of oocyte activation with fertilization rate and early embryonic development. Materials and Methods: In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI candidates and analyzed according to World Health Organization criteria (2010. Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC and the second part was prepared for assessment of sperm morphology (Papanicolaou staining, DNA fragmentation [transferase dUTP nick end labeling (TUNEL], and three Sperm-borne oocyte-activating factor (s (SOAFs-PLCζ, PAWP, and TR-KIT. Results: Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05. There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Conclusion: Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT. These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether

  4. Relationship between Potential Sperm Factors Involved in Oocyte Activation and Sperm DNA Fragmentation with Intra-Cytoplasmic Sperm Injection Clinical Outcomes

    Science.gov (United States)

    Tavalaee, Marziyeh; Kiani-Esfahani, Abbas; Nasr-Esfahani, Mohammad Hossein

    2017-01-01

    Objective The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP), phospholipase Cζ (PLCζ), and truncated form of the kit receptor (TR-KIT)] as candidates of oocyte activation with fertilization rate and early embryonic development. Materials and Methods In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI) candidates and analyzed according to World Health Organization criteria (2010). Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC) and the second part was prepared for assessment of sperm morphology (Papanicolaou staining), DNA fragmentation [transferase dUTP nick end labeling (TUNEL)], and three Sperm-borne oocyte-activating factor (s) (SOAFs)-PLCζ, PAWP, and TR-KIT. Results Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05). There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Conclusion Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT). These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action

  5. Relationship between Potential Sperm Factors Involved in Oocyte Activation and Sperm DNA Fragmentation with Intra-Cytoplasmic Sperm Injection Clinical Outcomes.

    Science.gov (United States)

    Tavalaee, Marziyeh; Kiani-Esfahani, Abbas; Nasr-Esfahani, Mohammad Hossein

    2017-01-01

    The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP), phospholipase Cζ (PLCζ), and truncated form of the kit receptor (TR-KIT)] as candidates of oocyte activation with fertilization rate and early embryonic development. In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI) candidates and analyzed according to World Health Organization criteria (2010). Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC) and the second part was prepared for assessment of sperm morphology (Papanicolaou staining), DNA fragmentation [transferase dUTP nick end labeling (TUNEL)], and three Sperm-borne oocyte-activating factor (s) (SOAFs)-PLCζ, PAWP, and TR-KIT. Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05). There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT). These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should

  6. Intracytoplasmic Sperm Injection Outcomes with Freshly Ejaculated Sperms and Testicular or Epididymal Sperm Extraction in Patients with Idiopathic Cryptozoospermia

    OpenAIRE

    Ketabchi

    2016-01-01

    Background Cryptozoospermia (CO) is a situation in which spermatozoa cannot be observed in a fresh semen sample unless an extended centrifugation and microscopic search are performed. CO patients are suggested to use only intracytoplasmic sperm injection (ICSI) as infertility treatment. But still there is debate about the choice of sperm source in cryptozoospermic men candidate for ICSI. Objectives This study was conducted to eval...

  7. Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation.

    Science.gov (United States)

    Watanabe, Hitomi; Takeo, Toru; Tojo, Hiromasa; Sakoh, Kazuhito; Berger, Thorsten; Nakagata, Naomi; Mak, Tak W; Kondoh, Gen

    2014-05-01

    Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

  8. Cross-talk between free and bound spermatozoa to modulate initial sperm:egg ratios at the site of fertilization in the mammalian oviduct.

    Science.gov (United States)

    Hunter, R H F; Gadea, J

    2014-08-01

    This essay proposes that highly localized communication between free and bound spermatozoa in the caudal portion of the oviduct acts to regulate the numbers detaching from the epithelium and progressing to the site of fertilization close to the time of ovulation. Low initial sperm:egg ratios are essential for monospermic fertilization. Liberation of surface macromolecules and metabolic prompting from activated spermatozoa, together with altered patterns of sperm movement and dynamic differences in intracellular Ca(2+) ion status between neighboring sperm cells, would influence the progressive release of spermatozoa from the reservoir in the oviduct isthmus. Different intensities of preovulatory epithelial binding, reflecting a range of states in the sperm surface membranes and associated proteins, would provide a further explanation for a chronologically staggered periovulatory detachment of spermatozoa. Intimate sperm-sperm interactions within the confines of the oviduct isthmus offer a sensitive means of fine-tuning the vanguard of competent male gametes reaching the isthmo-ampullary junction.

  9. Sperm motility of externally fertilizing fish and amphibians.

    Science.gov (United States)

    Browne, R K; Kaurova, S A; Uteshev, V K; Shishova, N V; McGinnity, D; Figiel, C R; Mansour, N; Agney, D; Wu, M; Gakhova, E N; Dzyuba, B; Cosson, J

    2015-01-01

    We review the phylogeny, sperm competition, morphology, physiology, and fertilization environments of the sperm of externally fertilizing fish and amphibians. Increased sperm competition in both fish and anurans generally increases sperm numbers, sperm length, and energy reserves. The difference between the internal osmolarity and iconicity of sperm cells and those of the aquatic medium control the activation, longevity, and velocity of sperm motility. Hypo-osmolarity of the aquatic medium activates the motility of freshwater fish and amphibian sperm and hyperosmolarity activates the motility of marine fish sperm. The average longevity of the motility of marine fish sperm (~550 seconds) was significantly (P fish sperm (~150 seconds), with the longevities of both marine and freshwater fish being significantly (P fish (140 μm/s) or freshwater fish (135 μm/s) sperm. The longevity of the sperm of giant salamanders (Cryptobranchoidea) of approximately 600 seconds was greater than that of freshwater fish sperm but much lower than anuran sperm. Our research and information from the literature showed that higher osmolarities promote greater longevity in anuran sperm, and some freshwater fish sperm, and that anuran and cryptobranchid sperm maintained membrane integrity long after the cessation of motility, demonstrating a preferential sharing of energy reserves toward the maintenance of membrane integrity. The maintenance of the membrane integrity of anuran sperm in fresh water for up to 6 hours showed an extremely high osmotic tolerance relative to fish sperm. The very high longevity and osmotic tolerance of anuran sperm and high longevity of cryptobranchid sperm, relative to those of freshwater fish, may reflect the complex fertilization history of amphibian sperm in general and anurans reversion from internal to external fertilization. Our findings provide a greater understanding of the reproductive biology of externally fertilizing fish and amphibians, and a

  10. Sperm dynamics in spiders (Araneae: ultrastructural analysis of the sperm activation process in the garden spider Argiope bruennichi (Scopoli, 1772.

    Directory of Open Access Journals (Sweden)

    Oliver Vöcking

    Full Text Available Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present, decapsulated (secretion sheath absent and uncoiled sperm (cell components uncoiled, presumably activated. After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process.

  11. Effects of Cadmium on Rat Sperm Motility Evaluated With Computer Assisted Sperm Analysis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To study effects of cadmium on rat sperm motility evaluated with computer assisted sperm analysis. Methods  Different doses of cadmium chloride (0.2,0.4,0.8mg Cd/kg BW) were administrated ip to adult male Sprague-Dawley rats. Control animals received the same volume of 0.9% NaCl solution. After 7 days, the rats were sacrificed with their testes removed. A part of one testis was used for testicular sperm head counts and daily sperm production observation. The motility of spermatozoa obtained from cauda epididymides using the “diffusion”method was measured by computer assisted sperm analysis(CASA). Results  The sperm head counts and daily sperm production decreased significantly in the high dose group. The motility of spermatozoa in the middle dose group was reduced significantly. No motile sperm was found in the high dose group. The results suggest that germinal epithelium was impaired irreversibly in a short time to produce toxic effects on spermatogenesis at high cadmium doses. Conclusion  Cadmium may reduce sperm motility at a dose far below the dose affecting sperm production at this time point. The motility of sperm is an early and sensitive endpoint for the assessment of cadmium toxicity on male reproduction.

  12. Observation of sperm-head vacuoles and sperm morphology under light microscope.

    Science.gov (United States)

    Park, Yong-Seog; Park, Sol; Ko, Duck Sung; Park, Dong Wook; Seo, Ju Tae; Yang, Kwang Moon

    2014-09-01

    The presence of sperm-head vacuoles has been suspected to be deleterious to the outcomes of assisted reproductive technology (ART). It is difficult to accurately distinguish morphologically abnormal sperm with vacuoles under a light microscope. This study was performed to analyze the result of the observation of sperm-head vacuoles using Papanicolaou staining under a light microscope and whether the male partner's age affects these vacuoles. Sperm morphology with vacuoles was evaluated using Papanicolaou staining and observed under a light microscope (400×) in 980 men. The normal morphology was divided into three categories (group A, 14% of normal morphology). The criteria for the sperm-head vacuoles were those given in the World Health Organization manual. For the analysis of the age factor, the participants were divided into the following groups: 26-30 years, 31-35 years, 36-40 years, 41-45 years, and 46-50 years. The percentage of sperm-head vacuoles increased with normal sperm morphology (group A vs. groups B, C) (p<0.05). In the case of the age factor, a statistically significant difference was not observed across any of the age groups. A majority of the sperm-head vacuoles showed a statistically significant difference among normal morphology groups. Therefore, we should consider the probability of the percentage of sperm-head vacuoles not increasing with age but with abnormal sperm morphology. A further study is required to clarify the effect of the sperm-head vacuoles on ART outcomes.

  13. Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.

    Science.gov (United States)

    Treulen, F; Uribe, P; Boguen, R; Villegas, J V

    2016-02-01

    Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MOMP was associated with ΔΨm dissipation (P DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study

  14. Novel gamete receptors that facilitate sperm adhesion to the egg coat.

    Science.gov (United States)

    Ensslin, Michael A; Lyng, Robert; Raymond, Adam; Copland, Susannah; Shur, Barry D

    2007-01-01

    Mammalian fertilization is initiated by species-specific binding of the sperm to the zona pellucida, or egg coat. Previous studies suggested that sperm adhesion to the egg coat is facilitated, at least in part, through the binding of sperm surface beta1 ,4-galactosyltransferase I (GaIT) to glycoside chains on the egg coat glycoprotein, ZP3. Binding of multiple ZP3 oligosaccharides induces aggregation of GaIT within the sperm membrane, triggering, directly or indirectly, a pertussis toxin sensitive G-protein cascade leading to induction of the acrosome reaction. Consistent with this, spermatozoa bearing targeted deletions in GaIT are unable to bind ZP3 or undergo ZP3-dependent acrosomal exocytosis; however, unexpectedly, GaIT-null sperm are still able to bind to the egg coat. This indicates that sperm-egg binding requires at least two independent binding mechanisms; a GaIT-ZP3-independent event that mediates initial adhesion, followed by a GaIT-ZP3 interaction that facilitates acrosomal exocytosis. Our recent efforts have focused on the identification and characterization of these novel gamete receptors. One recently identified sperm protein that is required for sperm adhesion to the egg coat is SED1. SED1 is a bimotif protein composed of two Notch-like EGF repeats and two discoidin/complement F5/8 domains. SED1 is secreted by the epididymal epithelium and coats spermatozoa as they progress through the epididymis. Spermatozoa null for SED1 fail to bind the egg coat, illustrating its requirement for gamete adhesion. Interestingly, SED1 is also expressed by a variety of other epithelial tissues, where it appears to be required for epithelial morphogenesis and/or maintenance. A second novel gamete receptor has recently been identified on the coat of ovulated oocytes. This ZP3-independent, egg coat component is a high molecular weight, wheat germ agglutinin (WGA)-reactive glycoprotein that is derived from oviduct secretions and appears to participate in initial sperm

  15. Uterine flushings from women treated with levonorgestrel affect sperm functionality in vitro.

    Science.gov (United States)

    Chirinos, Mayel; Durand, Marta; González-González, María Elena; Hernández-Silva, Gabriela; Maldonado-Rosas, Israel; Lopez, Pablo; Larrea, Fernando

    2017-09-08

    Levonorgestrel (LNG), a synthetic 19 nor-testosterone derivative, is widely used for emergency contraception. It is well known that LNG prevents ovulation only when given prior to the surge of serum luteinizing hormone (LH) during the periovulatory phase of the menstrual cycle. This observation suggests that LNG, given its contraceptive efficacy, has additional effects other than those affecting ovulation. In this study, we have evaluated the effects on human sperm functionality of uterine flushings (UF) obtained from women at day LH+1 of a control cycle (CTR-LH+1) and after receiving LNG (LNG-LH+1) two days before the surge of LH. Human sperm from normozoospermic donors were incubated with UF and protein tyrosine phosphorylation, sperm motility, acrosome reaction as well as zona pellucida (ZP) binding capacity were assessed. A significant decrease in total motility and tyrosine phosphorylation accompanied by an increase on spontaneous acrosome reaction was observed when sperm were incubated in the presence of LNG-LH+1. None of these effects were mimicked by purified glycodelin A (GdA). Moreover, the addition of UF obtained during the periovulatory phase from LNG-treated women or the presence of purified GdA significantly decreased sperm-ZP binding. The data were compatible with changes affecting sperm capacitation, motility and interaction with the ZP. These results may offer evidence on additional mechanisms of action of LNG as an emergency contraceptive.

  16. Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function.

    Science.gov (United States)

    Matás, C; Vieira, L; García-Vázquez, F A; Avilés-López, K; López-Úbeda, R; Carvajal, J A; Gadea, J

    2011-08-01

    In this study, different combinations of 2-step, discontinuous gradient centrifugation were used, consisting of three different combinations of isotonic Percoll (45/60, 60/75 and 45/90%) that allowed us to select different sperm subpopulations from fertile and normozoospermic boars. Our objective in this study is to evaluate the effects of centrifugation through three different discontinuous Percoll gradients on sperm function parameters (motility, viability, morphology, acrosome status, chromatin condensation, DNA fragmentation, ROS generation, tyrosine phosphorylation and intracellular calcium concentration) and the sperm penetrating capacity in an IVF system. All the Percoll treatments evaluated increased the percentage of spermatozoa with normal morphology, the proportion of un-damaged DNA, normal chromatin condensation, motion parameters measured by CASA and the percentage of capacitated spermatozoa with tyrosine phosphorylated proteins compared to control group. Finally, the in vitro oocyte penetrating capacity of boar spermatozoa was significantly affected by Percoll centrifugation. All the Percoll treatments increased the penetration rates and mean number of sperm per penetrated oocyte. Despite the efficiency of all three of the sperm treatments tested in selecting spermatozoa with improved sperm parameters and capacity to penetrate oocytes in vitro, the optimum performance of this system was demonstrated after preselecting spermatozoa by centrifugation on a discontinuous 45/90 Percoll gradient. The P45/90 treatment leads to obtain a higher percentage of spermatozoa which develop properly the capacitation process as it was shown measuring tyrosine phosphorylation and intracellular calcium concentration.

  17. Redox regulation of sperm surface thiols modulates adhesion to the fallopian tube epithelium.

    Science.gov (United States)

    Talevi, Riccardo; Zagami, Maria; Castaldo, Marianna; Gualtieri, Roberto

    2007-04-01

    Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.

  18. Fenitrothion Alters Sperm Characteristics in Rats: Ameliorating Effects of Palm Oil Tocotrienol-Rich Fraction

    Science.gov (United States)

    Taib, Izatus Shima; Budin, Siti Balkis; Ghazali, Ahmad Rohi; Jayusman,, Putri Ayu; Mohamed, Jamaludin

    2014-01-01

    Exposure to organophosphate insecticides such as fenitrothion (FNT) in agriculture and public health has been reported to affect sperm quality. Antioxidants may have a potential to reduce spermatotoxic effects induced by organophosphate. The present study was carried out to evaluate the effects of palm oil tocotrienol-rich fraction (TRF) in reducing the detrimental effects occurring in spermatozoa of FNT-treated rats. Adult male Sprague-Dawley rats were divided into four equal groups: a control group and groups of rats treated orally with palm oil TRF (200 mg/kg), FNT (20 mg/kg) and palm oil TRF (200 mg/kg) combined with FNT (20 mg/kg). The sperm characteristics, DNA damage, superoxide dismutase (SOD) activity, and levels of reduced glutathione (GSH), malondialdehyde (MDA), and protein carbonyl (PC) were evaluated. Supplementation with TRF attenuated the detrimental effects of FNT by significantly increasing the sperm counts, motility, and viability and decreased the abnormal sperm morphology. The SOD activity and GSH level were significantly increased, whereas the MDA and PC levels were significantly decreased in the TRF+FNT group compared with the rats receiving FNT alone. TRF significantly decreased the DNA damage in the sperm of FNT-treated rats. A significant correlation between abnormal sperm morphology and DNA damage was found in all groups. TRF showed the potential to reduce the detrimental effects occurring in spermatozoa of FNT-treated rats. PMID:25030881

  19. Predominance of sperm motion in corners

    Science.gov (United States)

    Nosrati, Reza; Graham, Percival J.; Liu, Qiaozhi; Sinton, David

    2016-05-01

    Sperm migration through the female tract is crucial to fertilization, but the role of the complex and confined structure of the fallopian tube in sperm guidance remains unknown. Here, by confocal imaging microchannels head-on, we distinguish corner- vs. wall- vs. bulk-swimming bull sperm in confined geometries. Corner-swimming dominates with local areal concentrations as high as 200-fold that of the bulk. The relative degree of corner-swimming is strongest in small channels, decreases with increasing channel size, and plateaus for channels above 200 μm. Corner-swimming remains predominant across the physiologically-relevant range of viscosity and pH. Together, boundary-following sperm account for over 95% of the sperm distribution in small rectangular channels, which is similar to the percentage of wall swimmers in circular channels of similar size. We also demonstrate that wall-swimming sperm travel closer to walls in smaller channels (~100 μm), where the opposite wall is within the hydrodynamic interaction length-scale. The corner accumulation effect is more than the superposition of the influence of two walls, and over 5-fold stronger than that of a single wall. These findings suggest that folds and corners are dominant in sperm migration in the narrow (sub-mm) lumen of the fallopian tube and microchannel-based sperm selection devices.

  20. Children conceived after intracytoplasmic sperm injection (ICSI)

    DEFF Research Database (Denmark)

    Mau, C; Juul, A; Main, K M;

    2004-01-01

    The aim of the study was to evaluate current medical knowledge about children born after intracytoplasmic sperm injection (ICSI) with respect to congenital malformations, chromosome abnormalities and postnatal growth.......The aim of the study was to evaluate current medical knowledge about children born after intracytoplasmic sperm injection (ICSI) with respect to congenital malformations, chromosome abnormalities and postnatal growth....

  1. Pollen and sperm heteromorphism: convergence across kingdoms?

    Science.gov (United States)

    Till-Bottraud, I; Joly, D; Lachaise, D; Snook, R R

    2005-01-01

    Sperm competition theory predicts that males should produce many, similar sperm. However, in some species of animals and plants, males exhibit a heteromorphism that results in the production of at least two different types of sperm or pollen grains. In animals, sperm heteromorphism typically corresponds to the production of one fertile morph and one (or more) sterile morph(s), whereas in plants two or more pollen morphs (one of which can be either sterile or fertile) are produced in all flowers but sometimes in different anthers. Heteromorphism has arisen independently several times across phyla and at different phylogenetic levels. Here, we compare and contrast sperm and pollen heteromorphism and discuss the evolutionary hypotheses suggested to explain heteromorphism in these taxa. These hypotheses include facilitation, nutritive contribution, blocking, cheap filler, sperm flushing or killing for animals; outcrossing and precise cross-pollen transfer or bet-hedging strategy for plants; cryptic female choice for both. We conclude that heteromorphism in the two phyla is most likely linked to a general evolutionary response to sexual selection, either to increase one male's sperm or pollen success in competition with other males, or mediate male/female interactions. Therefore, although sperm and pollen are not homologous, we suggest that heteromorphism represents an example of convergence across kingdoms.

  2. Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

    Science.gov (United States)

    Aparicio, I M; Bragado, M J; Gil, M C; Garcia-Herreros, M; Gonzalez-Fernandez, L; Tapia, J A; Garcia-Marin, L J

    2007-08-01

    Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

  3. Sperm Whales Suffer the Bends

    Institute of Scientific and Technical Information of China (English)

    Michael; Hopkin; 席芳

    2005-01-01

    据原先推测,鲸鱼对由声纳系统导致的疾病具有免疫力,但是两位美国科学家发现,抹香鲸(Sperm whales)正遭受着骨坏死疾病的侵扰,这是由于声纳系统扰乱了鲸鱼的生活习性所致,这种疾病有可能导致它们的搁浅。鲸鱼亟需得到保护!

  4. Swimming of Xenopus laevis sperm exhibits multiple gears and its duration is extended by egg jelly constituents.

    Science.gov (United States)

    Tholl, Nathan; Naqvi, Sumera; McLaughlin, Ericka; Boyles, Serenity; Bieber, Allan L; Chandler, Douglas E

    2011-06-01

    The motility of Xenopus sperm is initiated by the osmotic shock experienced when these cells are ejaculated into low-salinity pond water. Motility is brief and is required for the sperm to penetrate the jelly layers and fertilize the egg. In this study we demonstrate that extracts of egg jelly contain factors that extend the period of sperm motility as well as providing a chemoattractant activity as previously reported. Both activities are partially dependent on extracellular calcium. Time-lapse and video microscopy show that after activation of motility the number of motile sperm decreases rapidly, with a half-time of about 2 min. Addition of 10% v/v egg jelly extract ("egg water") increased the number of motile sperm 2-fold over controls at 20 s and about 4- to 10-fold over controls at 10 min after initiation of motility. Extension of motility lifetime was not mediated by a nonspecific protein or by allurin, the egg-water protein that has chemoattractant activity. The helical path of Xenopus sperm exhibited tight coupling between rotational and forward velocities in egg jelly, but coupling changed rapidly from moment to moment in low-salinity buffer. Our observations suggest that jelly-derived factors regulate both the longevity and directionality of sperm propulsion.

  5. Alfa-lipoic acid protects testosterone secretion pathway and sperm quality against 4-tert-octylphenol induced reproductive toxicity.

    Science.gov (United States)

    Othman, Azza I; El-Missiry, Mohamed A; Koriem, Khaled M; El-Sayed, Aml A

    2012-07-01

    The protective effect of α-lipoic acid (LA) (50 mg/kg bw) against 4-tert-octylphenol (OP) (50 mg/kg bw) induced reproductive toxicity in male rats was studied. LA was injected 1h prior to OP administration three times a week. OP caused significant increase in oxidative stress in hypothalamus and epididymal sperm, disturbed hormonal levels in serum, decreased sperm quality, increased DNA fragmentation and loss of 35 and 95 kDa proteins in sperm, as well as elevated proliferating index in testis. LA protected against oxidative stress through promoting the levels of glutathione and glutathione-S-transferase in hypothalamus and sperm. In addition, LA prevented the decrease in testosterone, dehydroepiandrosterone sulfate, 3β-hydroxysteroid dehydrogenase, and inhibited the elevations in sex-hormone-binding globulin levels and showed normal sperm quality. LA modulated proliferation of germ cell, protected against DNA fragmentation and maintained membrane protein organization in the sperm. In conclusion, LA normalized oxidative stress and protected testosterone synthesis pathway across hypothalamus-testicular axis and sperm quality indicating its defensive influence against OP-induced oxidative reproductive dysfunction in male rats.

  6. Detection of a mutation in the intron of Sperm-specific glyceraldehyde-3-phosphate dehydrogenase gene in patients with fibrous sheath dysplasia of the sperm flagellum.

    Science.gov (United States)

    Elkina, Y L; Kuravsky, M L; Bragina, E E; Kurilo, L F; Khayat, S S; Sukhomlinova, M Y; Schmalhausen, E V

    2017-03-01

    The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibres of the sperm flagellum. Dysplasia of the fibrous sheath (DFS) is a defect of spermatozoa observed in severe asthenozoospermic patients and characterised by morphologically abnormal flagella with distorted fibrous sheaths. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is a glycolytic enzyme that is tightly associated with the fibrous sheath of the sperm flagellum. The enzymatic activity of GAPDS was investigated in sperm samples of seven patients with DFS and compared to that of normal spermatozoa (n = 10). The difference in GAPDS activity in DFS and normal spermatozoa was statistically significant (0.19 ± 0.11 and 0.75 ± 0.11 μmol NADH per min per mg protein respectively). Immunochemical staining revealed irregular distribution of GAPDS in the flagellum of DFS spermatozoa. Other five samples with typical alterations in the fibrous sheath were assayed for mutations within human GAPDS gene. In all five cases, a replacement of guanine by adenine was revealed in the intron region between the sixth and the seventh exons of GAPDS. It is assumed that the deficiency in GAPDS observed in most DFS sperm samples is ascribable to a disorder in the regulation of GAPDS expression caused by the mutation in the intron region of GAPDS gene.

  7. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

    Science.gov (United States)

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-04-01

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

  8. Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos.

    Science.gov (United States)

    Linhart, Otomar; Rodina, Marek; Flajshans, Martin; Gela, David; Kocour, Martin

    2005-12-01

    The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.

  9. Sperm DNA fragmentation and its role in wildlife conservation.

    Science.gov (United States)

    Gosálvez, Jaime; Holt, William V; Johnston, Stephen D

    2014-01-01

    Until about 20 years ago, sperm assessment in the laboratory was focused on motility, morphology and acrosomal integrity. Then came the gradual realisation that, because the main objective of a spermatozoon is to deliver an intact genetic payload of DNA to the egg, being able to check DNA quality of spermatozoa would be equally important, if not more so. Research over the last two decades has therefore led to the development of several techniques for reliably detecting DNA strand breaks, and the more recent focus has been directed towards understanding the fertility implications of DNA damage. It is now clear that evolutionary history has played an important role in determining the stability of sperm DNA under stressful conditions, and that the nature of the DNA-protein interactions also influence the extent to which fertility is affected by both technical procedures involved in sperm preservation and the basic biology of the species concerned. Here we present an overview of the principles involved in DNA assessment and also provide some cases studies that illustrate the influences of species diversity.

  10. Cellular mechanisms regulating sperm-zona pellucida interaction

    Institute of Scientific and Technical Information of China (English)

    Andrew T Reid; Kate Redgrove; R John Aitken; Brett Nixon

    2011-01-01

    For mammalian spermatozoa to exhibit the ability to bind the zona pellucida(ZP)they must undergo three distinct phases of maturation,namely,spermatogenesis(testis),epididymal maturation(epididymis)and capacitation(female reproductive tract).An impressive array of spermatozoa surface remodeling events accompany these phases of maturation and appear critical for recognition and adhesion of the outer vestments of the oocyte,a structure known as the ZP.It is becoming increasingly apparent that species-specific zona adhesion is not mediated by a single receptor.Instead,compelling evidence now points toward models implicating a multiplicity of receptor-ligand interactions.This notion is in keeping with emerging research that has shown that there is a dynamic aggregation of proteins believed to be important in sperm-ZP recognition to the regions of sperm that mediate this binding event.Such remodeling may in turn facilitate the assembly of a multimeric zona recognition complex(MZRC).Though formation of MZRCs raises questions regarding the nature of the block to polyspermy,formation and assembly of such a structure would no doubt explain the strenuous maturation process that sperm endure on their sojourn to functional maturity.

  11. Low diversity in the mitogenome of sperm whales revealed by next-generation sequencing.

    Science.gov (United States)

    Alexander, Alana; Steel, Debbie; Slikas, Beth; Hoekzema, Kendra; Carraher, Colm; Parks, Matthew; Cronn, Richard; Baker, C Scott

    2013-01-01

    Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20 mitogenomes from 17 sperm whales representative of worldwide diversity using Next Generation Sequencing (NGS) technologies (Illumina GAIIx, Roche 454 GS Junior). Resequencing of three individuals with both NGS platforms and partial Sanger sequencing showed low discrepancy rates (454-Illumina: 0.0071%; Sanger-Illumina: 0.0034%; and Sanger-454: 0.0023%) confirming suitability of both NGS platforms for investigating low mitogenomic diversity. Using the 17 sperm whale mitogenomes in a phylogenetic reconstruction with 41 other species, including 11 new dolphin mitogenomes, we tested two hypotheses for the low CR diversity. First, the hypothesis that CR-specific constraints have reduced diversity solely in the CR was rejected as diversity was low throughout the mitogenome, not just in the CR (overall diversity π = 0.096%; protein-coding 3rd codon = 0.22%; CR = 0.35%), and CR phylogenetic signal was congruent with protein-coding regions. Second, the hypothesis that slow substitution rates reduced diversity throughout the sperm whale mitogenome was rejected as sperm whales had significantly higher rates of CR evolution and no evidence of slow coding region evolution relative to other cetaceans. The estimated time to most recent common ancestor for sperm whale mitogenomes was 72,800 to 137,400 years ago (95% highest probability density interval), consistent with previous hypotheses of a bottleneck or selective sweep as likely causes of low mitogenome diversity.

  12. Human Sperm Immotility Caused by Degeneration in the Epididymis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate whether sperm immotility was caused by degeneration in the epididymisMethods Five patients with totally immotile sperm were selected in this study. Testic ular biopsy was used to obtain testicular sperm to evaluate sperm motility. The com bined hypoosmotic swelling-eosin Y exclusion test was carried out to determine the sperm head and tail membrane integrity for the ejaculated and the testicular sperm.The ultrastructure of ejaculated sperm was examined by transmission electron microscope.Results No motile sperm were found in the ejaculated semen samples from 5 patients,whereas 2% to 11% motile testicular sperm extracted from the testicular biopsy tissues were observed. The percentage of testicular sperm with intact head and tail membranes was higher than that of the ejaculated sperm (P< 0. 01). Ultrastructure of the ejacu lated sperm showed marked degenerative features. Seminal plasma from patients did not influence the motility of normal donor sperm.Conclusion Sperm could undergo degenerative changes during transit through and /or storage in the epididymis, which led to lose sperm motility in these patients. Using motile testicular sperm would benefit the treatment for such cases.

  13. Depolymerization-driven flow and the crawling of nematode sperm

    Science.gov (United States)

    Wolgemuth, Charles

    2008-03-01

    Cell crawling motility is integral in many biological and biomedical processes, such as wound healing, cancer metastasis, and morphogenesis. A complete understanding of the mechanisms by which cells crawl is still lacking, but it is known to entail at least three separate physical processes: (i) cytoskeletal extension at the front of the cell; (ii) adhesion to the substrate at the cell front and release at the rear; and (iii) advance of the cell body. In most cells, the cytoskeletal network is composed of actin. The mechanism by which force is generated to drive translocation of the cell body is still debated. Originally, this force was attributed to an actomyosin system similar to muscle. However, nematode sperm utilize a cytoskeleton composed of a network of Major Sperm Protein (MSP) that forms non-polar filaments for which molecular motors have not been identified. The motility of these cells still exhibits all three fundamental processes required for standard crawling motility. Experiments suggest that depolymerization of the cytoskeletal network is the force-producing mechanism for pulling up the rear. In this talk I will present a mechanical model that describes how depolymerization of the cytoskeleton can drive motility. This model accounts for both cytoskeletal displacements and cytsolic (the fluid component of the cell) flow. The model accurately fits in vitro data using nematode sperm extracts where depolymerization induces contraction of MSP polymer bundles. Application of this model to cell crawling produces testable predictions about how the size and shape of a cell affect crawling speed. Experiments using Caenorhabditis elegans sperm show good agreement with the model predictions. Interestingly, the model requires that cells are anisotropically elastic, being more stiff in the direction of motion than perpendicular to it. A simple physical picture can account for this anisotropy. The model also predicts that cell speed increases with anisotropy and

  14. Prudent sperm use by leaf-cutter ant queens

    DEFF Research Database (Denmark)

    Den Boer, Susanne Petronella A; Baer, Boris; Dreier, Stéphanie Agnès Jeanine

    2009-01-01

    glandular secretions or deteriorating sperm-storage organs. We evaluate our findings with a heuristic model, which suggests that the average queen has sperm for almost 9 years of normal colony development. We discuss the extent to which leaf-cutter ant queens have been able to optimize their sperm...... expenditure and infer that our observed averages of sperm number, sperm robustness and sperm use are consistent with sperm depletion being a significant cause of mortality of mature colonies of Atta leaf-cutter ants.......In many species, females store sperm between copulation and egg fertilization, but the consequences of sperm storage and patterns of sperm use for female life history and reproductive success have not been investigated in great detail. In hymenopteran insect societies (ants, bees, wasps...

  15. Sperm Preservation using Freeze-Drying Method

    Directory of Open Access Journals (Sweden)

    TAKDIR SAILI

    2005-03-01

    Full Text Available Since the discovery of cryopreservation method for bull semen, cryopreservation become an alternative method for maintaining gamet resources of certain animal which is threatened or near extinction. This technology was then applied to the preservation of embryo, oocyte, ovary and testis. The application of intracytoplasmic sperm injection (ICSI for which sperm motility is unnecessary had supported the effort to create simplified method such as freeze-drying for sperm preservation. Due to the benefit of ICSI over the conventional in vitro fertilization (IVF the spermatozoon could be mechanically driven to pass through the zona pellucida and entering the cytoplasm of oocytes prior to fertilization. The freeze-drying method is an alternative method in sperm preservation which ignored the motility of sperm. The sperm resulted from this technique is in drying state, therefore, it might be stored in room temperature or in refrigerator. Many reports have claimed that freeze-dried sperm which is not motile but has an intact DNA was able to fertilize oocytes, even produced offspring in mouse.

  16. Semen analysis and sperm function testing

    Institute of Scientific and Technical Information of China (English)

    Daniel R Franken; Sergio Oehninger

    2012-01-01

    Despite controversy regarding the clinical value of semen analysis,male fertility investigation still relies on a standardized analysis of the semen parameters.This is especially true for infertility clinics in both developing and developed countries.Other optional tests or sophisticated technologies have not been widely applied.The current review addresses important changes in the analysis of semen as described in the new World Health Organization (WHO) manual for semen analysis.The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality.Apart from the above mentioned changes,the initial evaluation and handling methods remain,in most instances,the same as in previous editions.Furthermore,the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology.WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995.1he external quality control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training.This review reports on current sperm functional tests,such as the induced acrosome reaction,and sperm-zona pellucida binding assays,as well as the impact of sperm qualitv in terms of DNA integritv,and the relationship of sperm function tests to sperm morphologv.

  17. Tolerance to environmental desiccation in moss sperm.

    Science.gov (United States)

    Shortlidge, Erin E; Rosenstiel, Todd N; Eppley, Sarah M

    2012-05-01

    • Sexual reproduction in mosses requires that sperm be released freely into the environment before finding and fertilizing a receptive female. After release from the male plant, moss sperm may experience a range of abiotic stresses; however, few data are available examining stress tolerance of moss sperm and whether there is genetic variation for stress tolerance in this important life stage. • Here, we investigated the effects of environmental desiccation and recovery on the sperm cells of three moss species (Bryum argenteum, Campylopus introflexus, and Ceratodon purpureus). • We found that a fraction of sperm cells were tolerant to environmental desiccation for extended periods (d) and that tolerance did not vary among species. We found that this tolerance occurs irrespective of ambient dehydration conditions, and that the addition of sucrose during dry-down improved cell recovery. Although we observed no interspecific variation, significant variation among individuals within species in sperm cell tolerance to environmental desiccation was observed, suggesting selection could potentially act on this basic reproductive trait. • The observation of desiccation-tolerant sperm in multiple moss species has important implications for understanding bryophyte reproduction, suggesting the presence of a significant, uncharacterized complexity in the ecology of moss mating systems. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  18. Measurement and significance of sperm morphology

    Institute of Scientific and Technical Information of China (English)

    Roelof Menkveld; Cas AG Holleboom; Johann PT Rhemrev

    2011-01-01

    The measurement or evaluation and clinical significance of human sperm morphology has always been and still is a controversial aspect of the semen analysis for the determination of a male's fertility potential.In this review the background of the development of the evaluation criteria for sperm morphology will be discussed.Aspects of criticism on the strict criteria definition and use of the criteria for sperm morphology evaluation will be discussed as well as possible reasons for the decline in normal sperm morphology values and how we can compromise for this phenomenon resulting in the very low normal reference value as published in the 2010 WHO manual for the Examination and Processing of Human Semen.One of the possible solutions may be to give more attention to a limited number of abnormal sperm morphology categories and the inclusion of sperm morphology patterns.It is concluded in this review that if done correctly and with care and with strict application of existing guidelines as outlined in the 2010 WHO manual,sperm morphology measurement still has a very important role to play in the clinical evaluation of male fertility potential.

  19. FABP9 Mutations Are Not Detected in Cases of Infertility due to Sperm Morphological Defects in Iranian Men

    Directory of Open Access Journals (Sweden)

    Javad Jamshidi

    2014-01-01

    Full Text Available Background: Fatty acid binding proteins (FABPs are members of the intracellular lipid binding protein (iLBPs family and most of them show tissue specific expression. FABP9/PERF15 (Perforatorial15 is the male germ cell-specific fatty acid-binding protein. It was first identified as the major constituent of the murine sperm perforatorium and perinuclear theca. To date, investigations in mice have demonstrated that this protein has a role in the male reproductive system, especially in spermatogenesis. Also, it has been reported that FABP9 can protect sperm fatty acids from oxidative damage. Recently it was shown that it can affect sperm morphology in mice. Based on these findings, we designed a study to evaluate if mutations of this gene can affect sperm morphology in humans. Materials and Methods: In this case-control study, DNA was extracted from peripheral blood of 100 infertile males with normal sperm count but with a number of morphologically abnormal sperms in their semen that was above normal. Four exons and one intron of the FABP9 gene were amplified by polymerase chain reaction (PCR, re-sequenced and then analyzed for mutation detection. Results: We did not detect any mutation in any area of the four exons, intron 3 and splice sites of FABP9 gene in any of the studied 100 samples. Conclusion: There was no mutation in the exonic regions and the poor sperm morphology. However, we didn’t analyze the promoter, intron 1 and 2 to establish conclusions regarding the association of these genic regions and sperm dysmorphology.

  20. New insights into protamine-like component organization in Mytilus galloprovincialis' sperm chromatin.

    Science.gov (United States)

    Vassalli, Quirino Attilio; Caccavale, Filomena; Avagnano, Stefano; Murolo, Alessandra; Guerriero, Giulia; Fucci, Laura; Ausió, Juan; Piscopo, Marina

    2015-03-01

    We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.

  1. RAB-like 2 has an essential role in male fertility, sperm intra-flagellar transport, and tail assembly.

    Directory of Open Access Journals (Sweden)

    Jennifer C Y Lo

    Full Text Available A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2 as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT proteins and delivers a specific set of effector (cargo proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.

  2. Boundaries of Ethics, Sperm on the Border –The Globalization of Danish Sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    market as well as clinics worldwide. Furthermore, one of the sperm banks market sperm for self-insemination and deliver it to private customers by UPS. Although sperm donation is an old and low technology compared to other reproductive technologies, this development has taken place at the same time...... as sperm donation in parliamentary debates, and in the media, often raises ethical questions. This presentation inquires into how the bending of boundaries by “inappropriate parents”, fertility travelers, private sperm banks and fertility clinics have been part in negotiating the changes of the legislation...... of “ethical” does to a technology and its users such as in the case of the globalization of Danish sperm, as well as I will question how understandings of normality, sexuality, race, age, gender and kinship become part of the negotiations of “the ethical”, or in the doing of ethics in practice...

  3. Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex.

    Science.gov (United States)

    Aydin, Halil; Sultana, Azmiri; Li, Sheng; Thavalingam, Annoj; Lee, Jeffrey E

    2016-06-23

    Fertilization is an essential biological process in sexual reproduction and comprises a series of molecular interactions between the sperm and egg. The fusion of the haploid spermatozoon and oocyte is the culminating event in mammalian fertilization, enabling the creation of a new, genetically distinct diploid organism. The merger of two gametes is achieved through a two-step mechanism in which the sperm protein IZUMO1 on the equatorial segment of the acrosome-reacted sperm recognizes its receptor, JUNO, on the egg surface. This recognition is followed by the fusion of the two plasma membranes. IZUMO1 and JUNO proteins are indispensable for fertilization, as constitutive knockdown of either protein results in mice that are healthy but infertile. Despite their central importance in reproductive medicine, the molecular architectures of these proteins and the details of their functional roles in fertilization are not known. Here we present the crystal structures of human IZUMO1 and JUNO in unbound and bound conformations. The human IZUMO1 structure exhibits a distinct boomerang shape and provides structural insights into the IZUMO family of proteins. Human IZUMO1 forms a high-affinity complex with JUNO and undergoes a major conformational change within its N-terminal domain upon binding to the egg-surface receptor. Our results provide insights into the molecular basis of sperm-egg recognition, cross-species fertilization, and the barrier to polyspermy, thereby promising benefits for the rational development of non-hormonal contraceptives and fertility treatments for humans and other mammals.

  4. In cryptozoospermia or severe oligozoospermia is sperm freezing useful?

    OpenAIRE

    Bessonnat, Julien; Brouillet, Sophie; Sintzel, Sarah; Gillois, Pierre; Bergues, Ulrike; Boutte-Busquet, Caroline; Thomas-Cadi, Claire; Hennebicq, Sylviane

    2014-01-01

    Background Intracytoplasmic Sperm Injection (ICSI) is an Assisted Reproduction Technique (ART) which offers the chance to conceive to patients presenting very low sperm counts (cryptozoospermia/severe oligozoospermia). Sperm freezing before the oocyte pick-up, can prevent from a lack of spermatozoa on the day of the ICSI. It can avoid the cancellation of the ICSI or the use of TESE (Testicular sperm extraction). The objective of this study was to analyse the practice of sperm freezing for the...

  5. Sperm competition and the evolution of gamete morphology in frogs.

    OpenAIRE

    Byrne, Phillip G; Simmons, Leigh W.; Roberts, J. Dale

    2003-01-01

    Despite detailed knowledge of the ultrastructure of spermatozoa, there is a paucity of information on the selective pressures that influence sperm form and function. Theoretical models for both internal and external fertilizers predict that sperm competition could favour the evolution of longer sperm. Empirical tests of the external-fertilization model have been restricted to just one group, the fishes, and these tests have proved equivocal. We investigated how sperm competition affects sperm...

  6. Microscopic analysis of MTT stained boar sperm cells

    OpenAIRE

    B.M. van den Berg

    2015-01-01

    The ability of sperm cells to develop colored formazan by reduction of MTT was used earlier to develop a spectrophotometric assay to determine the viability of sperm cells for several mammalian species. It was the objective of the present study to visualize microscopically the location of the formazan in boar sperm cells. The MTT staining process of boar sperm cells can be divided into a series of morphological events. Incubation of the sperm cells in the presence of MTT resulted after a few ...

  7. Pengaruh Pencucian Sperma dengan Lama Waktu Sentrifugasi yang Berbeda Terhadap Kualitas Sperma Kambing Bligon (Effect of Sperm Washing with Different Centrifugation Duration on Sperm Quality of Bligon Buck

    Directory of Open Access Journals (Sweden)

    Sigit Bintara

    2012-02-01

    spermatozoa. It is concluded that washing Bligon buck sperm using SpermRinse with centrifugation at 1.500 rpm (r = 5.5 cm could increased sperm quality, of which 20 minutes was the best centrifugation time. (Key word: Bligon Buck, Sperm, SpermRinse, Sperm quality

  8. Sperm immobilization by dental focus microorganisms.

    Science.gov (United States)

    Linossier, A; Thumann, A; Bustos-Obregon, E

    1982-01-01

    Focal infections and their ability to produce alterations in different tissues have been in dispute for long time. The purpose of this work was to observe "in vitro" the effect of an Escherichia coli filtrate obtained from open pulpar necrosis on human sperm motility. It was observed that the E. coli filtrate produced a loss in sperm motility. The immobilizating factor was studied and characterized as a heat-stable, resistant to lyophilization and non-dializable substance, which could via blood stream reach the male reproductive system and affect sperm motility.

  9. Sperm Proteomics Reveals Intensified Selection on Mouse Sperm Membrane and Acrosome Genes

    OpenAIRE

    Dorus, Steve; Wasbrough, Elizabeth R.; Busby, Jennifer; Wilkin, Elaine C.; Karr, Timothy L.

    2010-01-01

    Spermatozoa are a focal point for the impact of sexual selection due to sperm competition and sperm–female interactions in a wide range of sexually reproducing organisms. In-depth molecular investigation of the ramifications of these selective regimes has been limited due to a lack of information concerning the molecular composition of sperm. In this study, we utilize three previously published proteomic data sets in conjunction with our whole mouse sperm proteomic analysis to delineate cellu...

  10. Ovarian fluid mediates the temporal decline in sperm viability in a fish with sperm storage.

    Directory of Open Access Journals (Sweden)

    Clelia Gasparini

    Full Text Available A loss of sperm viability and functionality during sperm transfer and storage within the female reproductive tract can have important fitness implications by disrupting fertilization and impairing offspring development and survival. Consequently, mechanisms that mitigate the temporal decline in sperm function are likely to be important targets of selection. In many species, ovarian fluid is known to regulate and maintain sperm quality. In this paper, we use the guppy Poecilia reticulata, a highly polyandrous freshwater fish exhibiting internal fertilization and sperm storage, to determine whether ovarian fluid (OF influences the decline in sperm viability (the proportion of live sperm in the ejaculate over time and whether any observed effects depend on male sexual ornamentation. To address these questions we used a paired experimental design in which ejaculates from individual males were tested in vitro both in presence and absence of OF. Our results revealed that the temporal decline in sperm viability was significantly reduced in the presence of OF compared to a saline control. This finding raises the intriguing possibility that OF may play a role in mediating the decline in sperm quality due to the deleterious effects of sperm ageing, although other possible explanations for this observation are discussed. Interestingly, we also show that the age-related decline in sperm viability was contingent on male sexual ornamentation; males with relatively high levels of iridescence (indicating higher sexual attractiveness exhibited a more pronounced decline in sperm viability over time than their less ornamented counterparts. This latter finding offers possible insights into the functional basis for the previously observed trade-off between these key components of pre- and postcopulatory sexual selection.

  11. Status specific tailoring of sperm behavior in an external fertilizer

    Directory of Open Access Journals (Sweden)

    Torvald Blikra Egeland

    2016-11-01

    Full Text Available Why dominant males experiencing intense sperm competition sometimes show low investments in sperm production is not always obvious. One well-documented example is that of the external fertilizing teleost, the Arctic charr (Salvelinus alpinus, where individuals becoming dominant reduce sperm production and sperm swimming speed in water compared to subordinates. Here we report how ovarian fluid differentially influences sperm velocity of dominant and subordinate male Arctic charr. That is, sperm from dominant males increase their velocity in water diluted ovarian fluid compared to that observed in water, while sperm from subordinates, on the other hand, decrease velocity in ovarian fluid compared to that observed in water. Thus, subordinates, who invest more resources in their sperm and usually show the highest sperm velocity in water, have lower gains from their investment than dominant males when sperm are swimming in ovarian fluid. In sum, our result suggests that ovarian fluid increase sperm velocity more in dominant males than in subordinate males. Although this finding could partly be caused by cryptic female choice exerted by the ovarian fluid for sperm from dominant males, an alternative and more parsimonious explanation is that sperm from dominant males may simply be better designed for swimming in ovarian fluid compared to sperm from subordinate males. Thus, sperm production in the two reproductive roles seems to be adaptively tailored to different external environments.

  12. Evaluation of Sperm Parameters of Infertile Men with Retrograde Ejaculation

    Institute of Scientific and Technical Information of China (English)

    Hong-xing ZHONG; Wei-jie ZHU; Jing LI

    2006-01-01

    Objective To investigate sperm parameters of infertile men with retrograde ejaculation.Methods Twelve infertile men with retrograde ejaculation (group A) were enrolled into this study. Sperm samples were obtained from the postejaculation urine. After sperm recovery and washing procedure, sperm parameters were assessed. Twelve semen samples from normospermic donors were used as the control (group B).Results In all retrograde cases, motile sperm with forward movement were observed in the medium. Motility of group A was significantly lower than that of group B (P<0. 01).In group A, sperm motility ranged from 11% to 56%, sperm with intact both head and tail membranes was 42.2 ± 12.3%, sperm count ranged (13-85)×106/ml, and the sperm survival time was highly shortened. Sperm with normal morphology and intact acrosome were observed in retrograde specimens.Conclusion Sperm parameters recovered from retrograde specimens were highly variable between subjects. The toxicity of urine caused deleterious to sperm functions.Motile sperm could be collected by sperm recovery procedure. Sperm parameters could meet the requirement for the use of assisted reproductive techniques for treating infertile men with retrograde ejaculation.

  13. Tactic-specific differences in seminal fluid influence sperm performance.

    Science.gov (United States)

    Locatello, Lisa; Poli, Federica; Rasotto, Maria B

    2013-03-22

    Seminal fluid often makes up a large part of an ejaculate, yet most empirical and theoretical studies on sperm competition have focused on how sperm characteristics (number and quality) affect fertilization success. However, seminal fluid influences own sperm performance and may potentially influence the outcome of sperm competition, by also affecting that of rivals. As a consequence males may be expected to allocate their investment in both sperm and seminal fluid in relation to the potential level of competition. Grass goby (Zosterisessor ophiocephalus) is an external fertilizer with guard-sneaker mating tactics, where sperm competition risk varies according to the tactic adopted. Here, we experimentally manipulated grass goby ejaculates by separately combining sperm and seminal fluid from territorial and sneaker males. While sperm of sneaker and territorial males did not differ in their performance when they interacted with their own seminal fluid only, sperm of sneakers increased their velocity and fertilization rate in the presence of territorial males' seminal fluid. By contrast, sneaker males' seminal fluid had a detrimental effect on the performance of territorial males' sperm. Sperm velocity was unaffected by the seminal fluid of males employing the same tactic, suggesting that seminal fluid's effect on rival-tactic sperm is not based on a self/non-self recognition mechanism. Our findings show that cross interactions of sperm and seminal fluid may influence the fertilization success of competing ejaculates with males investing in both sperm and seminal fluid in response to sperm competition risk.

  14. High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction.

    Science.gov (United States)

    Azpiazu, Rubén; Amaral, Alexandra; Castillo, Judit; Estanyol, Josep Maria; Guimerà, Marta; Ballescà, Josep Lluís; Balasch, Juan; Oliva, Rafael

    2014-06-01

    Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy? Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins. The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins. This was a case-control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008. Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting. By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values Economia y Competividad; FEDER BFU 2009-07118 and PI13/00699) and

  15. Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis.

    Science.gov (United States)

    Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

    2017-04-18

    Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

  16. Sperm nuclear histone H2B: correlation with sperm DNA denaturation and DNA stainability

    Institute of Scientific and Technical Information of China (English)

    Armand Zini; Xiaoyang Zhang; Maria San Gabriel

    2008-01-01

    Aim: To examine the relationship between sperm DNA damage and sperm nuclear histone (H2B) staining. Methods:We evaluated sperm samples from 14 consecutive asthenoteratozoospermic infertile men and six consecutive fertile controls. Sperm nuclear histone (H2B) staining and sperm chromatin integrity (assessed by sperm chromatin structure assay and expressed using the percentage of (I) DNA fragmentation index [%DFI] and (ii) high DNA stainability [%HDS)]) were evaluated. Results: Histone H2B immunocytochemistry demonstrated two nuclear staining patterns: (I) focal punctate staining; and (ii) diffuse staining. Infertile men had a higher mean percentage of spermatozoa exhibiting diffuse H2B staining than did fertile men (7.7% ± 4.6% vs. 1.6% ± 1.2%, respectively, P < 0.01). We observed significant relationships between the proportion of spermatozoa with diffuse nuclear histone staining and both sperm %DFI (r= 0.63, P < 0.01) and sperm %HDS (r= 0.63, P < 0.01). Conclusion: The data demonstrate that infertile men have a higher proportion of spermatozoa with diffuse histone H2B than do fertile men and suggest that sperm DNA damage might, at least in part, be due to abnormally high histone H2B levels.

  17. Spermatogenesis, the mature sperm, and sperm egg association in Nematospiroides dubius

    Science.gov (United States)

    Clark, W. H., Jr.

    1974-01-01

    Nematode spermatogenesis was investigated using the strongyloid Nematospiroides dubius. The primary spermatocytes, development of spermatids, and changes in the sperm in the female tract are described.

  18. Computer-assisted sperm analysis parameters in young fertile sperm donors and relationship with age.

    Science.gov (United States)

    Fréour, Thomas; Jean, Miguel; Mirallie, Sophie; Barriere, Paul

    2012-04-01

    Sperm parameter values have been shown to decline with age, according to conventional sperm analysis. However, the effect of age on sperm kinematic parameters has been rarely studied, especially in young fertile men. Here, we studied Computer-Assisted Sperm Analysis (CASA) parameters in a large cohort of men with proven fertility, in order to determine if there is a decline with age in this young fertile population. This retrospective analysis of CASA parameters was conducted on all donors included in the sperm donor programme in the Assisted Reproductive Techniques (ART) Centre of the University Hospital of Nantes between 2006 and 2009. Sperm concentration, motility, and kinetic parameters were recorded by a HTM-Ceros system and compared in 3 groups of sperm donors according to their age: donors were analyzed. Values for ALH, VCL, LIN, and STR significantly decreased with age. Sperm concentration, motile sperm proportion, and other kinetic parameters did not differ significantly among the groups. The use of CASA allowed the identification of ALH, VCL, LIN, and STR age-related decrease in young men with proven fertility.

  19. Female sperm use and storage between fertilization events drive sperm competition and male ejaculate allocation.

    Science.gov (United States)

    Requena, Gustavo S; Alonzo, Suzanne H

    2014-12-01

    Sperm competition theory has traditionally focused on how male allocation responds to female promiscuity, when males compete to fertilize a single clutch of eggs. Here, we develop a model to ask how female sperm use and storage across consecutive reproductive events affect male ejaculate allocation and patterns of mating and paternity. In our model, sperm use (a single parameter under female control) is the main determinant of sperm competition, which alters the effect of female promiscuity on male success and, ultimately, male reproductive allocation. Our theory reproduces the general pattern predicted by existing theory that increased sperm competition favors increased allocation to ejaculates. However, our model predicts a negative correlation between male ejaculate allocation and female promiscuity, challenging the generality of a prevailing expectation of sperm competition theory. Early models assumed that the energetic costs of precopulatory competition and the level of sperm competition are both determined by female promiscuity, which leads to an assumed covariation between these two processes. By modeling precopulatory costs and sperm competition independently, our theoretical framework allows us to examine how male allocation should respond independently to variation in sperm competition and energetic trade-offs in mating systems that have been overlooked in the past.

  20. Sperm morphology: consistency of assessment of the same sperm by different observers.

    Science.gov (United States)

    Baker, H W; Clarke, G N

    1987-01-01

    Photographic slides of 36 sperm were shown to a group of 28 observers with different lengths of experience in assessing sperm morphology. Experienced observers were generally consistent (10 or more of the 17 agreeing) in classifying sperm as normal, amorphous, small heads, lacking acrosomes, and having tail defects or cytoplasmic droplets but categorization was more variable for large, tapering and pyriform heads. This study highlights the need for more widespread agreement about definition of sperm shapes and the development of practical objective methods of assessment.

  1. In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster.

    Science.gov (United States)

    Watanabe, Akihiko; Takayama-Watanabe, Eriko

    2014-01-01

    A specific sperm-egg interaction in the oviductal matrix is crucial for internal fertilization of the red-bellied newt, Cynops pyrrhogaster. An understanding of the molecular basis of this interaction is expected to elucidate the evolutionary history of internal fertilization in amphibians. Recently, deep sequencing technology has provided global gene information even in nonmodel animals, allowing us to understand specific features of the molecular mechanisms underlying fertilization in C. pyrrhogaster. In the present study, we screened de novo assembled RNAseq from ovary, testis, and oviduct samples in C. pyrrhogaster and identified the base sequences encoding zona pellucida (ZP) proteins, voltage-dependent Ca(2+) channels, and cysteine-rich secretory proteins (CRISPs), which respectively are sperm receptors for egg envelopes, major mediators of sperm intracellular signaling, and expected extracellular modulators for sperm function in the female reproductive tract. In the ovary, ZP homologues of all six subgroups were found, including a ZP1 homologue that was newly found in amphibians, a ZP4 homologue, and six ZPC homologues. The unique combination of ZP proteins suggests a new mechanism for sperm binding to egg envelopes in the internal fertilization of C. pyrrhogaster. In the testis, CaV1.1, 1.2, and 3.2, which are L- and T-type voltage-dependent Ca(2+) channels, were found as potential mediators for the internal fertilization-specific sperm-egg interaction. We also found CRISP 2 in the oviduct, which is speculated to participate in the sperm-egg interaction. These results indicate that the de novo assembled RNAseq is a powerful tool allowing analysis of the specific sperm-egg interactions in C. pyrrhogaster.

  2. Biochemical characterization of tektins from sperm flagellar doublet microtubules

    OpenAIRE

    1987-01-01

    Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little ...

  3. Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells.

    Science.gov (United States)

    Tsai, Pei-Shiue; De Vries, Klaas J; De Boer-Brouwer, Mieke; Garcia-Gil, Nuria; Van Gestel, Renske A; Colenbrander, Ben; Gadella, Bart M; Van Haeften, Theo

    2007-01-01

    Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.

  4. Rat sperm motility analysis: methodologic considerations

    Science.gov (United States)

    The objective of these studies was to optimize conditions for computer-assisted sperm analysis (CASA) of rat epididymal spermatozoa. Methodologic issues addressed include sample collection technique, sampling region within the epididymis, type of diluent medium used, and sample c...

  5. Comparative evolutionary psychology of sperm competition.

    Science.gov (United States)

    Shackelford, Todd K; Goetz, Aaron T

    2006-05-01

    A comparative evolutionary psychological perspective predicts that species that recurrently faced similar adaptive problems may have evolved similar psychological mechanisms to solve these problems. Sperm competition provides an arena in which to assess the heuristic value of such a comparative evolutionary perspective. The sperm competition that results from female infidelity and polyandry presents a similar class of adaptive problems for individuals across many species. The authors first describe mechanisms of sperm competition in insects and in birds. They suggest that the adaptive problems and evolved solutions in these species provide insight into human anatomy, physiology, psychology, and behavior. The authors then review recent theoretical and empirical arguments for the existence of sperm competition in humans and discuss proposed adaptations in humans that have analogs in insects or birds. The authors conclude by highlighting the heuristic value of a comparative evolutionary psychological approach in this field.

  6. Mammalian Sperm Motility: Observation and Theory

    KAUST Repository

    Gaffney, E.A.

    2011-01-21

    Mammalian spermatozoa motility is a subject of growing importance because of rising human infertility and the possibility of improving animal breeding. We highlight opportunities for fluid and continuum dynamics to provide novel insights concerning the mechanics of these specialized cells, especially during their remarkable journey to the egg. The biological structure of the motile sperm appendage, the flagellum, is described and placed in the context of the mechanics underlying the migration of mammalian sperm through the numerous environments of the female reproductive tract. This process demands certain specific changes to flagellar movement and motility for which further mechanical insight would be valuable, although this requires improved modeling capabilities, particularly to increase our understanding of sperm progression in vivo. We summarize current theoretical studies, highlighting the synergistic combination of imaging and theory in exploring sperm motility, and discuss the challenges for future observational and theoretical studies in understanding the underlying mechanics. © 2011 by Annual Reviews. All rights reserved.

  7. CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline.

    Science.gov (United States)

    Campbell, Anne C; Updike, Dustin L

    2015-05-15

    Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription.

  8. Acrosomo-nuclear syndrome in canine sperm.

    Science.gov (United States)

    Hrudka, F

    1983-01-01

    An acrosomo-nuclear syndrome in sperm of an infertile semicryptorchid dog is described. Based on an EM study of developing and mature sperm the syndrome is defined by simultaneous occurrence of these symptoms: 1) intranuclear inclusions of acrosomal origin, 2) maldifferentiated apical segment of acrosome, 3) intraacrosomal inclusions of Sertoli cell origin, 4) characteristic change of nuclear shape and 5) retained cytoplasmic droplet. The cause of the syndrome and possibility of a transfer of somatic factors are discussed.

  9. Outdoor air pollution and sperm quality.

    Science.gov (United States)

    Lafuente, Rafael; García-Blàquez, Núria; Jacquemin, Bénédicte; Checa, Miguel Angel

    2016-09-15

    Exposure to air pollution has been clearly associated with a range of adverse health effects, including reproductive toxicity, but its effects on male semen quality are still unclear. We performed a systematic review (up to June 2016) to assess the impact of air pollutants on sperm quality. We included 17 semi-ecological, panel, and cohort studies, assessing outdoor air pollutants, such as PM2.5, PM10, NOx, SO2, and O3, and their effects on DNA fragmentation, sperm count, sperm motility, and sperm morphology. Thirteen studies assessed air pollution exposure measured environmentally, and six used biomarkers of air pollution exposure (two did both). We rated the studies using the Newcastle-Ottawa Scale and assessed with the exposure method. Taking into account these factors and the number of studies finding significant results (positive or negative), the evidence supporting an effect of air pollution on DNA fragmentation is weak but suggestive, on sperm motility is limited and probably inexistent, on lower sperm count is inconclusive, and on sperm morphology is very suggestive. Because of the diversity of air pollutants and sperm parameters, and the studies' designs, we were unable to perform a meta-analysis. In summary, most studies concluded that outdoor air pollution affects at least one of the four semen quality parameters included in the review. However, results lack consistency, and furthermore, studies were not comparable. Studies using standardized air pollution and semen measures are required to obtain more reliable conclusions. CRD42015007175. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Sperm DNA oxidative damage and DNA adducts

    Science.gov (United States)

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  11. Formation and Dissociation of Sperm Bundles in Monotremes.

    Science.gov (United States)

    Nixon, Brett; Ecroyd, Heath; Dacheux, Jean-Louis; Dacheux, Francoise; Labas, Valerie; Johnston, Steve D; Jones, Russell C

    2016-10-01

    Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals. © 2016 by the Society for the Study of Reproduction, Inc.

  12. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

    Science.gov (United States)

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2016-09-27

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  13. COMPARISON OF CD46 EXPRESSION ON THE INNER ACROSOMAL MEMBRANE OF SPERMS FROM NORMOSPERMIC AND ASTHENOSPERMIC INDIVIDUALS

    Directory of Open Access Journals (Sweden)

    M NASR ESFAHANI

    2003-03-01

    Full Text Available Introduction: CD46 is a membrane cofactor protein (MCP of complement system wich is present on the membrane of all somatic cells except RBC. It is also present on the inner acrosmal membrane of human sperm. Thus, the aim of this study was to compare the expression of this prote, in on the inner acrosmal membrane of sperms from normospermic and asthenospermic individuals. Method: Semen from 6 normospermic and 17 asthenospermic individuals were examined for CD46 expression. After solublization of sperms in solublizing detergent, the solublized sperm membrane was separated from the rest of cell organelles by centrifugation. Solublized sperm membrane were divide to equal parts and SOS-PAGE gel was canied out in paired on the same gel for each sample. Western blot was carried out on half of the gel and then the nitrocellose papers were stained by a monocolonal Ab and HRP conjugate Ab. The other half were stained by silver stain for identification of MW. Results: After scoring the stained nitrocellose papen in each groups, no statistical significant difference was observed for C046 expression between the two groups. However, a significant Spearmen correlation was observed between CD46 expression and sperm motility (r=0.597, P=0.003. The MW of C046 was between 36 to 45 KD. with a mean of 42 KD. Discussion: This is the first report of a positive Spearmen correlation between sperm CD46 expression and sperm motility which suggest that there might be relation between CD46 expression and sperm motility.

  14. Seasonal changes in reproductive activity, sperm variables and sperm freezability in Blanca Andaluza bucks

    Directory of Open Access Journals (Sweden)

    Lourdes Gallego-Calvo

    2015-12-01

    Full Text Available Interest in the preservation of endangered breeds such as the Blanca Andaluza goat, has increased and some steps should be therefore taken to ensure it. The study was designed to determine the seasonal reproductive pattern of Blanca Andaluza bucks, and whether this affects the quality of their semen and its freezability over the year. Seven bucks were used and their body weight, testicular weight, plasma testosterone concentration and fresh sperm quality determined every week. The collected sperm was cryopreserved and stored; it was then thawed and the same sperm quality variables measured every fortnight. High plasma testosterone concentrations were recorded during the summer and autumn, and low concentrations were recorded during winter and spring (p<0.001. No differences were seen between seasons in terms of the percentage of bucks ejaculating, the percentage of active bucks, or ejaculate volume. However, the sperm concentration, the total number of sperm per ejaculate, and the values for most fresh sperm variables were lower during the winter period (at least p<0.05. After freezing-thawing, the quality of winter-collected sperm was better, in some respects, than that of summer-collected sperm (at least p<0.05. These results reveal that Blanca Andaluza bucks show seasonal reproductive activity in terms of their plasma testosterone concentration, but no clear change in their sexual behaviour between seasons was observed. The values of fresh sperm variables also vary over the year, reaching their lowest during winter. However, after freezing-thawing, winter-collected sperm is of overall better quality than sperm collected during the summer.

  15. Sperm mobility determines the outcome of sperm competition in the domestic fowl.

    Science.gov (United States)

    Birkhead, T R; Martínez, J G; Burke, T; Froman, D P

    1999-01-01

    The aim of this study was to establish whether the mobility of sperm of the domestic fowl, as measured by an in vitro assay, predicted the outcome of sperm competition. Thirteen pairs of New Hampshire roosters, comprising one male categorized as having high-mobility sperm and the other as having average-mobility sperm, were used. Each male provided 25 x 10(6) sperm, which were mixed and artificially inseminated into between four and seven New Hampshire hens, each of which produced 2-11 offspring. The experiment was conducted twice, such that the same pair of males inseminated the same females. Paternity was assigned by using microsatellite markers. There was a clear effect of sperm-mobility phenotype on the outcome of sperm competition: in all 13 pairs the high-mobility male fathered the majority of offspring (75.3% overall; p < 0.0001). The proportion of offspring fathered by the high-mobility male within pairs varied significantly between male pairs (p < 0.0005). This effect was associated with the difference in sperm-mobility scores between males within pairs; there was a significant positive relationship between the proportion of offspring fathered by the high-mobility male and the ratio of mobility scores between males (p < 0.05). In addition, compared with their success predicted from the non-competitive situation, in the competitive situation high-mobility males were disproportionately successful in fertilizing eggs compared with average-mobility males. This may occur because female sperm storage is limited in some way and a greater proportion of high-mobility sperm gain access to the female's sperm storage tubules. There was no evidence that female effects accounted for any of the variation in paternity. PMID:10577160

  16. Seasonal changes in reproductive activity, sperm variables and sperm freezability in Blanca Andaluza bucks

    Energy Technology Data Exchange (ETDEWEB)

    Gallego-Calvo, L.; Gatica, M.C.; Santiago-Moreno, J.; Guzmán, J.L.; Zarazaga, L.

    2015-07-01

    Interest in the preservation of endangered breeds such as the Blanca Andaluza goat, has increased and some steps should be therefore taken to ensure it. The study was designed to determine the seasonal reproductive pattern of Blanca Andaluza bucks, and whether this affects the quality of their semen and its freezability over the year. Seven bucks were used and their body weight, testicular weight, plasma testosterone concentration and fresh sperm quality determined every week. The collected sperm was cryopreserved and stored; it was then thawed and the same sperm quality variables measured every fortnight. High plasma testosterone concentrations were recorded during the summer and autumn, and low concentrations were recorded during winter and spring (p<0.001). No differences were seen between seasons in terms of the percentage of bucks ejaculating, the percentage of active bucks, or ejaculate volume. However, the sperm concentration, the total number of sperm per ejaculate, and the values for most fresh sperm variables were lower during the winter period (at least p<0.05). After freezing-thawing, the quality of winter-collected sperm was better, in some respects, than that of summer-collected sperm (at least p<0.05). These results reveal that Blanca Andaluza bucks show seasonal reproductive activity in terms of their plasma testosterone concentration, but no clear change in their sexual behaviour between seasons was observed. The values of fresh sperm variables also vary over the year, reaching their lowest during winter. However, after freezing-thawing, winter-collected sperm is of overall better quality than sperm collected during the summer. (Author)

  17. Sperm length evolution in the fungus-growing ants

    DEFF Research Database (Denmark)

    Baer, B.; Dijkstra, M. B.; Mueller, U. G.

    2009-01-01

    the evolution of sperm length. Sperm length does not decrease further in multiply mating leaf-cutting ants, despite substantial further increases in colony size. In a combined analysis, sexual dimorphism explained 63.1% of the variance in sperm length between species. As colony size was not a significant...... in phylogenetically independent contrasts. Some of the remaining variation was explained by the relative size of the sperm-storage organ, but only in the multiply mating leaf-cutting ants, suggesting that sperm-storage constraints become important for the evolution of sperm length in this derived group. Mate number...... affected sperm length to a minor extent, and only in interaction with other predictor variables, suggesting that sperm competition has not been a major selective force for sperm length evolution in these ants....

  18. Effect of short abstinence time on sperm motility parameters

    DEFF Research Database (Denmark)

    Alipour, Hiva; Dardmeh, Fereshteh; Van Der Horst, Gerhard

    2015-01-01

    Semen preparation medium have an important role in assisted reproduction techniques and their composition influences sperm binding and motility. Some studies have assessed the influence of pH on sperm kinetics. However, no study to date has assessed the effect of environmental pH on subtle...... differences in the details of the sperm movement (kinematics) of human sperm provided by computerized sperm analysis systems. This study was designed to assess the effect of two different media pH levels on kinematic parameters of the human sperm. Samples were prepared using the 40%/80% Pureception (Sage, USA......) density gradient and resuspended in customized sperm culture media with pH levels of 7.9 and 8.3 (Origio, Denmark). Kinematic parameters of the sperm in both groups were analyzed using the Sperm Class Analyzer (Microptic S.L., Spain) at 0, 6 and 24 hours following the addition of media. Results...

  19. Effect of environmental pH on sperm kinematic characteristics

    DEFF Research Database (Denmark)

    Alipour, Hiva; Dardmeh, Fereshteh; Dissing, Marianne Charlotte

    2015-01-01

    Semen preparation medium have an important role in assisted reproduction techniques and their composition influences sperm binding and motility. Some studies have assessed the influence of pH on sperm kinetics. However, no study to date has assessed the effect of environmental pH on subtle...... differences in the details of the sperm movement (kinematics) of human sperm provided by computerized sperm analysis systems. This study was designed to assess the effect of two different media pH levels on kinematic parameters of the human sperm. Samples were prepared using the 40%/80% Pureception (Sage, USA......) density gradient and resuspended in customized sperm culture media with pH levels of 7.9 and 8.3 (Origio, Denmark). Kinematic parameters of the sperm in both groups were analyzed using the Sperm Class Analyzer (Microptic S.L., Spain) at 0, 6 and 24 hours following the addition of media. Results...

  20. Evidence for the function of hyperactivated motility in sperm.

    Science.gov (United States)

    Suarez, S S; Katz, D F; Owen, D H; Andrew, J B; Powell, R L

    1991-02-01

    After insemination, mammalian sperm undergo a striking change in flagellar beat pattern, termed hyperactivation. In low-viscosity culture medium, nonhyperactivated sperm flagella generate relatively symmetrical, low-amplitude waves, while hyperactivated sperm flagella generate an asymetrical beating pattern that results in nonprogressive movement. Since sperm encounter highly viscous and viscoelastic fluids in the female reproductive tract, the progress of hyperactivated sperm was compared with that of nonhyperactivated and transitional sperm in media of increasing viscosity. Hamster sperm obtained from the caudal epididymis were incubated in a medium that promotes capacitation. After 0, 3, and 4 h of incubation, the majority of the sperm exhibited, respectively, activated, transitional, and hyperactivated motility. At each of these time points, aliquots of sperm were removed from incubation and added to solutions of 0, 5%, 10%, 20%, and 30% Ficoll in medium. Samples containing mostly hyperactivated sperm (4 h) maintained higher swimming and flagellar velocities and were able to generate greater forces in response to increased viscous loading than activated sperm (0 h). Transitional sperm (3 h) showed an intermediate response. The paths of hyperactivated sperm through solutions of 20% and 30% Ficoll were considerably straighter than those made through medium alone. This is the first demonstration that hyperactivation can confer a mechanical advantage upon sperm in the oviduct where they may encounter viscous oviductal fluid and a viscoelastic cumulus matrix.

  1. Effects of ubiquitin-protea some pathway on mouse sperm capacitation, acrosome reaction and in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody against ubiquitinated proteins-UCP1; the polyclonal antibody against ubiquitin anti-Ub, and a special inhibitor against proteasome ALLN. Furthermore, we treated the capacitated sperm or the eggs with these reagents separately and tested whether the normal in vitro fertilization was blocked or not. Results illustrate that UCP1, anti-Ub, and ALLN have little effects on sperm capacitation and acrosome reaction,but they do inhibit fusion of mouse sperm with eggs, which suggests that UPP play an important role in mouse in vitro fertilization.``

  2. Increased receptor for advanced glycation end products in spermatozoa of diabetic men and its association with sperm nuclear DNA fragmentation.

    Science.gov (United States)

    Karimi, J; Goodarzi, M T; Tavilani, H; Khodadadi, I; Amiri, I

    2012-05-01

    Although the majority of patients with diabetes have disorders in sexual function, associations between diabetes mellitus and sperm function at the molecular level are largely unknown. As receptor for advanced glycation end products plays a key role in many diabetic complications, we hypothesised that it may be involved in sperm nuclear DNA fragmentation. RAGE levels were determined using ELISA and western blot analysis in sperm samples from 32 diabetic and 35 nondiabetic men. Sperm DNA fragmentation was assessed using TUNEL assay. Diabetic men had significantly higher mean levels of RAGE protein (P DNA fragmentation (P DNA fragmentation in diabetic men (r = 0.81, P DNA fragmentation in spermatozoa of diabetic men suggests a central role of RAGE in disturbances in sexual function of diabetic men. © 2011 Blackwell Verlag GmbH.

  3. Phenotypic plasticity in sperm traits in scorpionflies (Mecoptera : Panorpidae): Consequences of larval history and seasonality on sperm length and sperm transfer

    NARCIS (Netherlands)

    Vermeulen, Andreas; Engels, Sierk; Engqvist, Leif; Sauer, Klaus Peter

    2009-01-01

    We examined effects of seasonality, larval food availability and larval rearing density on sperm length, sperm transfer rates and body size in the bivoltine scorpionfly Panorpa vulgaris. Males of the first annual generation were larger and had larger sperm. Comparing individuals of two summer genera

  4. Co-Incubation of Human Spermatozoa with Anti-VDAC Antibody Reduced Sperm Motility

    Directory of Open Access Journals (Sweden)

    Bianjiang Liu

    2014-01-01

    Full Text Available Background: Voltage-dependent anion channel (VDAC, a channel protein, exists in the outer mitochondrial membrane of somatic cells and is involved in multiple physiological and pathophysiological processes. Up until now, little has been known about VDAC in male germ cells. In the present study, the relationship between VDAC and human sperm motility was explored. Methods: Highly motile human spermatozoa were incubated in vitro with anti-VDAC antibody. Total sperm motility, straight line velocity (VSL, curvilinear velocity (VCL, and average path velocity (VAP were recorded. Intracellular free calcium concentration ([Ca2+]i, pH value (pHi, and ATP content were determined. Results: Co-incubation with anti-VDAC antibody reduced VSL, VCL, and VAP of spermatozoa. Co-incubation further reduced [Ca2+]i. Anti-VDAC antibody did not significantly alter total sperm motility, pHi and intracellular ATP content. Conclusion: The data suggest that co-incubation with anti-VDAC antibody reduces sperm motility through inhibition of Ca2+ transmembrane flow. In this way, VDAC participates in the modulation of human sperm motility through mediating Ca2+ transmembrane transport and exchange.

  5. Sexual selection and the evolution of egg-sperm interactions in broadcast-spawning invertebrates.

    Science.gov (United States)

    Evans, Jonathan P; Sherman, Craig D H

    2013-08-01

    Many marine invertebrate taxa are broadcast spawners, where multiple individuals release their gametes into the water for external fertilization, often in the presence of gametes from heterospecifics. Consequently, sperm encounter the considerable challenges of locating and fertilizing eggs from conspecific females. To overcome these challenges, many taxa exhibit species-specific attraction of sperm toward eggs through chemical signals released from eggs (sperm chemotaxis) and species-specific gamete recognition proteins (GRPs) that mediate compatibility of gametes at fertilization. In this prospective review, we highlight these selective forces, but also emphasize the role that sexual selection, manifested through sperm competition, cryptic female choice, and evolutionary conflicts of interest between the sexes (sexual conflict), can also play in mediating the action of egg chemoattractants and GRPs, and thus individual reproductive fitness. Furthermore, we explore patterns of selection at the level of gametes (sperm phenotype, gamete plasticity, and egg traits) to identify putative traits targeted by sexual selection in these species. We conclude by emphasizing the excellent, but relatively untapped, potential of broadcast-spawning marine invertebrates as model systems to illuminate several areas of research in post-mating sexual selection.

  6. Association of seminal plasma motility inhibitors/semenogelins with sperm in asthenozoospermia-infertile men.

    Science.gov (United States)

    Terai, K; Yoshida, K; Yoshiike, M; Fujime, M; Iwamoto, T

    2010-01-01

    Seminal plasma motility inhibitors (SPMIs) are proteinase-resistant fragments of semenogelin I and II (Sgs), which are the major proteins of semen coagulum. SPMIs inhibit the motility of spermatozoa, and Sgs are thought to be natural regulators of human sperm function. The mechanism underlying sperm motility regulation and its association with defective motility in infertile men remain unclear. The purpose of this study was to investigate the association between SPMIs and spermatozoa in infertile men with asthenozoospermia. Fifty-four semen samples from 37 asthenozoospermic patients and 17 samples from 9 normal healthy subjects were analyzed. Spermatozoa, washed by Percoll density gradients, were immunostained with anti-SPMI antibody and subjected to flow cytometric analysis. The proportion of spermatozoa labeled with the antibody and the average intensity of fluorescence labeling per spermatozoa were analyzed in relation to the parameters used for semen analysis. A significant negative correlation was found between sperm motility and the proportion (R = -0.68) and intensity (R = -0.38) of labeling. These results suggest that SPMIs remain on the sperm surface after liquefaction. This might account for some disorders of sperm motility observed in infertile men with asthenozoospermia.

  7. Post Testicular Sperm Maturational Changes in the Bull: Important Role of the Epididymosomes and Prostasomes

    Directory of Open Access Journals (Sweden)

    Julieta Caballero

    2011-01-01

    Full Text Available After spermatogenesis, testicular spermatozoa are not able to fertilize an oocyte, they must undergo sequential maturational processes. Part of these essential processes occurs during the transit of the spermatozoa through the male reproductive tract. Since the sperm become silent in terms of translation and transcription at the testicular level, all the maturational changes that take place on them are dependent on the interaction of spermatozoa with epididymal and accessory gland fluids. During the last decades, reproductive biotechnologies applied to bovine species have advanced significantly. The knowledge of the bull reproductive physiology is really important for the improvement of these techniques and the development of new ones. This paper focuses on the importance of the sperm interaction with the male reproductive fluids to acquire the fertilizing ability, with special attention to the role of the membranous vesicles present in those fluids and the recent mechanisms of protein acquisition during sperm maturation.

  8. Na+-permeable channels of human sperm membrane re- assembled into giant liposome

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Previous data showed that a Na+-transmembrane flux was accompanied with acrosome reaction of sperm. However, the electrophysiological recording and characterization of Na+ current in human sperm membrane have not been yet reported. In the present investigation, membrane proteins extracted from human sperms were reassembled into liposome bilayer, and then the liposomes were fused by dehydration-rehydration into giant liposomes with the diameter of more than 10 mm. By patch clamping the giant liposomes two kinds of single channel currents were recorded in a NaCl solution system. Both of them were Na+-carried, TTX-sensitive and strongly rectifying, but with different unit conductance and open probability. Moreover, bursting activity and channel-substates as well as two open time constants were observed in the larger channel.

  9. Canine sperm vitrification with sucrose: effect on sperm function.

    Science.gov (United States)

    Sánchez, R; Risopatrón, J; Schulz, M; Villegas, J; Isachenko, V; Kreinberg, R; Isachenko, E

    2011-08-01

    The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra-rapid cryopreservation in canine sperm was investigated. Swim-up selected spermatozoa of second-fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m) in proportion 1 : 1 v/v with HTF-BSA 1%. From each group, 30-μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF-BSA 1% (42.5 ± 2.3%, P HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF-BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome-reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra-rapid cryopreservation. © 2011 Blackwell Verlag GmbH.

  10. Neonatal outcome and congenital malformations in children born after ICSI with testicular or epididymal sperm

    DEFF Research Database (Denmark)

    Fedder, Jens; Loft, A; Parner, Erik Thorlund;

    2013-01-01

    Does neonatal outcome including congenital malformations in children born after ICSI with epididymal and testicular sperm [testicular sperm extraction (TESE)/percutaneous epididymal sperm aspiration (PESA)/testicular sperm aspiration (TESA) (TPT)] differ from neonatal outcome in children born after...

  11. Sperm chromatin structure assay results in Nigerian men with unexplained infertility

    National Research Council Canada - National Science Library

    Faduola, Paul; Kolade, Charles Oluwabukunmi

    2015-01-01

    .... This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had...

  12. Ocean acidification impacts on sperm mitochondrial membrane potential bring sperm swimming behaviour near its tipping point.

    Science.gov (United States)

    Schlegel, Peter; Binet, Monique T; Havenhand, Jonathan N; Doyle, Christopher J; Williamson, Jane E

    2015-04-01

    Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii. We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in ΔpH -0.3 (35% reduction) and ΔpH -0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification. © 2015. Published by The Company of Biologists Ltd.

  13. SMART USE OF COMPUTER-AIDED SPERM ANALYSIS (CASA) TO CHARACTERIZE SPERM MOTION

    Science.gov (United States)

    Computer-aided sperm analysis (CASA) has evolved over the past fifteen years to provide an objective, practical means of measuring and characterizing the velocity and parttern of sperm motion. CASA instruments use video frame-grabber boards to capture multiple images of spermato...

  14. MONTHLY VARIATION IN SPERM MOTILITY IN COMMON CARP ASSESSED USING COMPUTER-ASSISTED SPERM ANALYSIS (CASA)

    Science.gov (United States)

    Sperm motility variables from the milt of the common carp Cyprinus carpio were assessed using a computer-assisted sperm analysis (CASA) system across several months (March-August 1992) known to encompass the natural spawning period. Two-year-old pond-raised males obtained each mo...

  15. Sperm retrieval outcomes with microdissection testicular sperm extraction (micro-TESE) in men with cryptozoospermia.

    Science.gov (United States)

    Alrabeeah, K; Wachter, A; Phillips, S; Cohen, B; Al-Hathal, N; Zini, A

    2015-05-01

    Several studies support of the use of testicular rather than ejaculated spermatozoa for intracytoplasmic sperm injection (ICSI) in couples with virtual azoospermia or cryptozoospermia, although this approach remains controversial. We sought to evaluate sperm retrieval outcomes with microdissection testicular sperm extraction (micro-TESE) in men with cryptozoospermia. We conducted a retrospective study of 24 consecutive micro-TESEs in men with cryptozoospermia. We also evaluated the outcomes of seven consecutive TESAs (testicular sperm aspiration) in cryptozoospermic men during the same time period (January 2007 and September 2014). Micro-TESE and TESA were performed on the day prior to ICSI. Final assessment of sperm recovery (reported on the day of ICSI) was recorded as (i) successful (available spermatozoa for ICSI) or (ii) unsuccessful (no spermatozoa for ICSI). The decision to perform a unilateral or bilateral micro-TESE was guided by the intra-operative evaluation of sperm recovery from the first testicle. A unilateral procedure was performed in 87.5% (21/24) and 57% (4/7) of the micro-TESE and TESA cohorts, respectively. Sperm recovery was successful in 96% (23/24) of the men who underwent micro-TESE and 43% (3/7) of the men who underwent TESA (p cryptozoospermia and few of these men will require a bilateral procedure. Moreover, sperm retrieval rates are higher with micro-TESE than TESA in this group of men.

  16. MONTHLY VARIATION IN SPERM MOTILITY IN COMMON CARP ASSESSED USING COMPUTER-ASSISTED SPERM ANALYSIS (CASA)

    Science.gov (United States)

    Sperm motility variables from the milt of the common carp Cyprinus carpio were assessed using a computer-assisted sperm analysis (CASA) system across several months (March-August 1992) known to encompass the natural spawning period. Two-year-old pond-raised males obtained each mo...

  17. SMART USE OF COMPUTER-AIDED SPERM ANALYSIS (CASA) TO CHARACTERIZE SPERM MOTION

    Science.gov (United States)

    Computer-aided sperm analysis (CASA) has evolved over the past fifteen years to provide an objective, practical means of measuring and characterizing the velocity and parttern of sperm motion. CASA instruments use video frame-grabber boards to capture multiple images of spermato...

  18. Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Cozzi, J. [Medical School of Grenoble (France)

    1994-09-01

    Sperm microinjection techniques, subzonal sperm insemination (SUZI) and intracytoplasmic sperm injection (ICSI), have achieved a wide spread clinical application for the treatment of male infertility. To date, only one study has focused on sperm karyotypes after microinjection. Martin et al. reported a very high incidence of abnormal human sperm complements after ICSI into hamster oocytes. In the present study, are reported the first human sperm karyotypes after SUZI of hamster oocytes. Spermatozoa from two control donors were treated by calcium ionophore A23187 and injected under the zona of hamster eggs. The microinjected eggs were then cultured for cytogenetic analysis of the pronuclei. Out of 47 analyzed sperm chromosome metaphases, 5 (10.6%) were abnormal, 4 (8.5%) were hypohaploid and 1 (2.1%) had a structural abnormality. The sex ratio was not significantly different from the expected 1:1 ratio. Rates of chromosomal abnormalities in microinjected spermatozoa were similar to those observed in spermatozoa inseminated with zona free eggs, suggesting that SUZI procedure per se does not increase sperm chromosomal abnormalities.

  19. Factors influencing sperm retrieval following testicular sperm extraction in nonobstructive azoospermia patients.

    Science.gov (United States)

    Salehi, Peyman; Derakhshan-Horeh, Marzieh; Nadeali, Zakiye; Hosseinzadeh, Majid; Sadeghi, Erfan; Izadpanahi, Mohammad Hossein; Salehi, Mansour

    2017-03-01

    Azoospermia owing to testicular disorders is the most severe manifestation of male infertility. The main concern for patients with nonobstructive azoospermia (NOA) is the probability of successful sperm retrieval following testicular sperm extraction (TESE). Therefore, the goal of this study was to determine predictive factors correlated with sperm retrieval. We assessed the testicular histopathological patterns, the choice of TESE surgical procedure, hormone levels, and chromosomal abnormalities in patients with NOA (n=170). The histopathology specimens were analyzed based on the histopathological patterns of hypospermatogenesis, maturation arrest, and Sertoli cell-only syndrome. The mean rate of sperm retrieval was 48.8%. The rate of sperm retrieval was significantly higher in the hypospermatogenesis group than in the other groups (p<0.001). There was a positive correlation between micro-TESE (vs. conventional TESE) and the sperm retrieval rate (odds ratio, 8.077; p<0.01). A logistic regression model demonstrated that high levels of follicle-stimulating hormone (FSH) and small testicular volume were significantly associated with lower chances of successful sperm retrieval. Some parameters, including testicular histopathology patterns, FSH levels, testicular volume, and method of TESE surgery, may be able to predict the chances of obtaining spermatozoa in patients with NOA. However, despite the efficiency of some predictive models, the hope of retrieving any functioning spermatozoa may be sufficient to disregard predictive factors of the success of intracytoplasmic sperm injection in these patients.

  20. Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success.

    Science.gov (United States)

    Gage, Matthew J G; Macfarlane, Christopher P; Yeates, Sarah; Ward, Richard G; Searle, Jeremy B; Parker, Geoffrey A

    2004-01-06

    Sperm competition occurs when sperm from more than one male compete for fertilizations. This form of post-copulatory sexual selection is recognized as a significant and widespread force in the evolution of male reproductive biology and as a key determinant of differential male reproductive success. Despite its importance, however, detailed mechanisms of sperm competition at the gamete level remain poorly understood. Here, we use natural variation in spermatozoal traits among wild Atlantic salmon (Salmo salar), a species naturally adapted to sperm competition, to examine how the relative influences of sperm (i) number, (ii) velocity, (iii) longevity, and (iv) total length determine sperm competition success. Atlantic salmon fertilize externally, and we were therefore able to conduct controlled in vitro fertilization competitions while concurrently measuring spermatozoal traits within the aqueous micro-environment to which salmon gametes are naturally adapted. Microsatellite DNA fingerprinting revealed that a male's relative sperm velocity was the primary determinant of sperm competition success. There was no significant relationship between fertilization success and either relative sperm number or total length; sperm longevity showed an inverse relationship with competition success. These relationships were consistent for two experimental repeats of the in vitro fertilization competitions. Our results therefore show, under the natural microenvironment for salmon gametes, that relative sperm velocity is a key spermatozoal component for sperm competition success. Atlantic salmon sperm can be considered to enter a competition analogous to a race in which the fastest sperm have the highest probability of success.

  1. Improved risk assessment by screening sperm parameters.

    Science.gov (United States)

    Plassmann, S; Urwyler, H

    2001-02-28

    The question of whether a 4 or 9 week premating treatment period is more suitable in studies for effects on fertility and early embryonic development, and the extent to which the screening of sperm parameters may contribute to the detection of effects, has been under discussion since the ICH guideline changed in 1994/1995. This study presents a comparison between 4 and 9 weeks treatment with known male reproductive toxicants with regard to sperm motility, count, morphology, abnormal movements and testicular and epididymal histopathology. Mating outcome was examined after 4 weeks treatment. Three compounds with different targets and mechanisms of action were chosen: two testicular toxicants, Pyridoxine and Adriamycin and the epididymal toxicant, alpha-Chlorohydrine. Sperm motility was reduced in males treated with Pyridoxine (markedly) and alpha-Chlorohydrine (slightly) after 4 weeks treatment and in males treated with Adriamycin after 9 weeks treatment. With Pyridoxine and Adriamycin, sperm count was significantly increased after 4 weeks. Histopathological examination after 4 weeks showed characteristic changes leading to marked testicular tubular atrophy at 8/9 weeks, which was confirmed by a significantly reduced sperm count at 8/9 weeks. With alpha-Chlorohydrine, sperm count was not affected and the results of the histopathological examination were equivocal. Changes in sperm morphology were observed after 4/9 weeks of treatment with Pyridoxine. Mating outcome after 4 weeks was markedly affected with both Pyridoxine and alpha-Chlorohydrine, but no effect was observed with Adriamycin. The results of this study indicate that the two testicular toxicants would have been detected as male reproductive toxicants in a 4-week general toxicity study with routine testicular histopathology and examination of sperm parameters, without the need for mating trials. For the epididymal toxicant, alpha-Chlorohydrine, there was slightly reduced sperm motility after 4 weeks

  2. Structure and evolution of the sea star egg receptor for sperm bindin.

    Science.gov (United States)

    Hart, Michael W

    2013-04-01

    Selection on coevolving sperm- and egg-recognition molecules is a potent engine of population divergence leading to reproductive isolation and speciation. The study of receptor-ligand pairs can reveal co-evolution of male- and female-expressed genes or differences between their evolution in response to selective factors such as sperm competition and sexual conflict. Phylogeographical studies of these patterns have been limited by targeted gene methods that favour short protein-coding sequences amplifiable by PCR. Here, I use high-throughput transcriptomic methods to characterize the structure and divergence of full-length coding sequences for the gene encoding the protein component of a large complex egg surface glycopeptide receptor for the sperm acrosomal protein bindin from the sea star Patiria miniata. I used a simple but effective method for resolving nucleotide polymorphisms into haplotypes for phylogeny-based analyses of selection. The protein domain organization of sea star egg bindin receptor (EBR1) was similar to sea urchins and included a pair of protein-recognition domains plus a series of tandem repeat domains of two types. Two populations separated by a well-characterized phylogeographical break included lineages of EBR1 alleles under positive selection at several codons (similar to selection on sperm bindin in the same populations). However, these populations shared the same alleles that were under selection for amino acid differences at multiple codons (unlike the pattern of selection for population divergence in sperm bindin). The significance of positively selected EBR1 domains and alleles could be tested in functional analyses of fertilization rates associated with EBR1 (and bindin) polymorphisms.

  3. Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

    Science.gov (United States)

    Das, Sujoy; Saha, Sudipta; Majumder, Gopal Chandra; Dungdung, Sandhya Rekha

    2010-01-01

    Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility. PMID:20706623

  4. Purification and characterization of a sperm motility inhibiting factor from caprine epididymal plasma.

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    Sujoy Das

    Full Text Available Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II has been purified from caprine epididymal plasma (EP by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 microg/ml (12.5 nM level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due t