Sample records for acrosin

  1. Acrosin structure-based design, synthesis and biological activities of 7-azaindol derivatives as new acrosin inhibitors

    Institute of Scientific and Technical Information of China (English)

    Jun Hang Jiang; Xue Fei Liu; Can Hui Zhen; You Jun Zhou; Ju Zhu; Jia Guo Lv; Chun Quan Sheng


    A series of 7-azaindol derivatives were designed based on the homologous 3D model of human acrosin. These compounds were synthesized and evaluated for their human acrosin inhibitory activities in vitro. Compounds 7a, 7i, 7j, 7k and 7n showed highly inhibitory activity against human acrosin. The three-dimensional structure-activity relationship was investigated through a CoMFA model, which provided valuable information to further study of potential human acrosin inhibitors.

  2. Acrosin inhibitor detection along the boar epididymis. (United States)

    Maňásková-Postlerová, Pavla; Cozlová, Nina; Dorosh, Andriy; Šulc, Miroslav; Guyonnet, Benoit; Jonáková, Věra


    Epididymal sperm maturation represents a key step in the reproduction process. Spermatozoa are exposed to epididymal fluid components representing the natural environment essential for their post-testicular maturation. Changes in sperm membrane proteins are influenced by proteolytic, glycosylation and deglycosylation enzymes present in the epididymal fluid. Accordingly, the occurrence of inhibitors of these enzymes in the epididymis is very important for the regulation of sperm membrane protein processing. In the present study, we monitored acrosin inhibitor distribution in boar epididymal fluid and in spermatozoa from different segments of the organ. Using specific polyclonal antibody we registered increasing signal of the acrosin inhibitor (AI) from caput to cauda epididymis. Mass spectroscopy examination of the immunoprecipitated acrosin inhibitor (12 kDa) unequivocally identified sperm-associated acrosin inhibitor (SAAI) in the epididymal tissue. Lectin staining showed N-glycosylation in AI from boar epididymis. Protein detection of AI was supported by the results of semi-quantitative RT-PCR showing the presence of mRNA specifically coding for SAAI and similarly increasing throughout the epididymal duct, from its proximal to distal part. Additionally, the immunofluorescence technique showed the AI localization in the secretory tissue of caput, corpus and cauda epididymis, and in the acrosome region and midpiece of the sperm.

  3. Determination of sperm acrosin activity for evaluation of male fertility

    Institute of Scientific and Technical Information of China (English)

    Yun-HeCUI; Rui-LanZHAO; QiangWANG; Zi-YingZHANG


    Aim: To investigate a simple method for assaying acrosin activity for the evaluation of male fertility. Methods: The acrosin activity of 7.5 × 106 sperm without seminal plasma and acrosin activity inhibitors was assayed using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and detergent (Triton X-100) as substrate. Results: The acrosin activity of 60 normal fertile men (35±10μIU/106 sperm ) was higher than that of 168 infertile men ( 16±8μIU/106 sperm) (P < 0.01 ). It was indicated that there was a significant positive correlation between the acrosin activity and the sperm motility ( r≥0. 6534, P < 0.01 ) and a significant negative correlation between the sperm malformed rate and the WBC number ( r≤-0. 5426, P < 0.01 ). The temperature and time of incubation and the sperm concentration could influence the assay results. Conclusion: Acrosin activity is an important index for the evaluation of male fertility. The approach developed by the authors is a simple method for the determination of acrosin activity.

  4. Discovery of novel human acrosin inhibitors by virtual screening (United States)

    Liu, Xuefei; Dong, Guoqiang; Zhang, Jue; Qi, Jingjing; Zheng, Canhui; Zhou, Youjun; Zhu, Ju; Sheng, Chunquan; Lü, Jiaguo


    Human acrosin is an attractive target for the discovery of male contraceptive drugs. For the first time, structure-based drug design was applied to discover structurally diverse human acrosin inhibitors. A parallel virtual screening strategy in combination with pharmacophore-based and docking-based techniques was used to screen the SPECS database. From 16 compounds selected by virtual screening, a total of 10 compounds were found to be human acrosin inhibitors. Compound 2 was found to be the most potent hit (IC50 = 14 μM) and its binding mode was investigated by molecular dynamics simulations. The hit interacted with human acrosin mainly through hydrophobic and hydrogen-bonding interactions, which provided a good starting structure for further optimization studies.

  5. Acrosin activity is a good predictor of boar sperm freezability. (United States)

    Pinart, Elisabeth; Yeste, Marc; Bonet, Sergi


    The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze-thawing. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze-thawing procedures took place, i.e., in the refrigeration step at 15 °C.

  6. Proteolysis of specific porcine zona pellucida glycoproteins by boar acrosin. (United States)

    Dunbar, B S; Dudkiewicz, A B; Bundman, D S


    The morphologic and biochemical effects on the structure and constituent glycoproteins of the zona pellucida (ZP) by a specific sperm enzyme, acrosin, and a nonsperm enzyme, trypsin, have been evaluated. Intact porcine ZP matricies, exposed to either acrosin or trypsin, were analyzed microscopically. Changes in specific glycoproteins were monitored by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and the silver-based color stain, GELCODE. Although these enzymes did not alter the macroscopic properties of the ZP matrix, the 2D-PAGE ZP protein patterns were markedly altered. The high molecular weight glycoprotein families (II and III) were sensitive to proteolytic digestion, whereas the major glycoprotein family (I) of the porcine zona was only partially proteolyzed by acrosin and trypsin. Furthermore, it was demonstrated that acrosin had unique substrate specificity compared to that of trypsin, since the ZP peptide patterns were found to be different. These studies are the first to demonstrate which integral glycoproteins of the native porcine ZP matrix are specifically proteolyzed by acrosin from the homologous species and that this proteolysis occurs without the dissolution of the native porcine matrix.

  7. Roles of tumor necrosis factor alpha on sperm acrosin activity and acrosome reaction

    Institute of Scientific and Technical Information of China (English)

    Shu-LingBian; Guo-YiLiu; Hai-XiaWen; Shu-ZhenWang; JiangNi; WeiZhang; HuiSi


    Aim: To study the roles of tumor necrosis factor alpha (TNF-a)on the sperm acrosin activity and acrosome reaction. Methods:The sperm acrosin activity was tested by the method of BAEE/ADH Unity and the acrosome reaction by the Triple-stain technique. Results: TNF-a decreased the sperm acrosin activityand acrosome reaction (P<0.01, P<0.01, respectively);

  8. Assessment of released acrosin activity as a measurement of the sperm acrosome reaction

    Institute of Scientific and Technical Information of China (English)

    Rui-Zhi Liu; Wan-Li Na; Hong-Guo Zhang; Zhi-Yong Lin; Bai-Oong Xue; Zong-Oe Xu


    Aim: To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction (AR). Methods: Human semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization (WHO) criteria. Calcium ionophore A23187 and progesterone (P4) were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA). The percentage of sperm undergoing AR (AR%) was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis. Results: The AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimula-tion with A23187 (P < 0.001). There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining (r = 0.916, P < 0.001). Conclusion: Spectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR.

  9. Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo). (United States)

    Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A


    This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After

  10. Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity. (United States)

    Glogowski, J; Jankowski, J; Faruga, A; Ottobre, J S; Ciereszko, A


    We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.

  11. Effect of dialysis on the proacrosin/acrosin system and motility of turkey (Meleagris gallopavo) spermatozoa during liquid storage. (United States)

    Słowińska, M; Dietrich, G J; Liszewska, E; Kozłowski, K; Jankowski, J; Ciereszko, A


    1. The effect of dialysis on the proacrosin/acrosin system and motility of turkey spermatozoa were examined after 24 and 48 h of liquid storage at 4°C. 2. Fifteen pools of semen diluted in extender were dialysed against Clemson Turkey Semen Diluent (dialysed semen) or stored in aerobic conditions (undialysed semen). Semen quality was assessed by measuring spermatozoa motility, amidase activity of spermatozoa suspension, spermatozoa extract and seminal plasma and anti-trypsin activity of seminal plasma. 3. Extracted amidase activity of dialysed semen was lower than undialysed by 28%. Higher values for speed parameters of spermatozoa were found in dialysed semen in comparison to undialysed, for example, 81.6 µm/s versus 75.0 µm/s for straight-line velocity (VSL), 114.7 µm/s versus 110.3 µm/s for curvilinear velocity (VCL) and 86.6 µm/s versus 79.8 µm/s for average path velocity (VAP). 4. It was concluded that dialysis caused lower amidase activity of spermatozoa and increased speed parameters of progressively motile turkey spermatozoa during storage. Lower extracted amidase activity of dialysed semen reflected better membrane integrity of dialysed semen and suggests that the proacrosin/acrosin system of dialysed spermatozoa is less susceptible to activation compared to undialysed semen.

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

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


    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.

  13. Synthesis and Crystal Structure of a Novel Ethyl 5-(4-(2-Phenylacetamido)phenyl)-lH-pyrazole- 3-carboxylate as an Acrosin Inhibitor%Synthesis and Crystal Structure of a Novel Ethyl 5-(4-(2-Phenylacetamido)phenyl)-lH-pyrazole- 3-carboxylate as an Acrosin Inhibitor

    Institute of Scientific and Technical Information of China (English)

    QI Jing-Jing; ZHOU You-Jun; LIU Xue-Fei; DING Li-Li; ZHENG Can-Hui; SHENG Chun-Quan; LV Jia-Guo; ZHU Ju


    The title compound (ethyl5-(4-(2-phenylacetamido)phenyl)-lH-pyrazole-3-carboxylate, C20H19N3O3) was synthesized by the reaction of Claisen condensation, cyclization, reduction and acylation. The structure was characterized by X-ray diffraction, MS, NMR and IR. It belongs to the monoclinic system, space group C2/c with a = 22.723(9), b = 9.324(4), c = 18.890(8) A, β = 114.259(6)°, V = 3649(3) A^3, Dc = 1.272 Mg·m^3, Z = 8, Mr = 349.38, p = 0.087 mm^-1, F(000) = 1472, the final R = 0.0615 and wR = 0.1643. The biological test shows that the title compound has a moderate acrosin inhibition activity.

  14. Study on relationship between sperm morphology with seminal plasma zinc and sperm acrosin activity in male infertile patients%男性不育患者精子形态与精浆锌和精子顶体酶活性关系的研究

    Institute of Scientific and Technical Information of China (English)

    邴晏如; 莫和国; 黄健云; 李璐琳; 欧水连; 黎泳仪; 陆小琴; 蔡锦梅


    Objective To investigate the relationship of sperm morphology with seminal plasma zinc and sperm acrosin activity in male infertile patients.Methods 455 patients with male infertility were divided into 5 groups and 8 groups according to age and sperm morphology respectively.The sperm morphology,seminal plasma zinc and sperm acrosin activity were detected by the Diff-Quik stain,modified 5-Br-PAPS and modified Kennedy methods respectively.Results The percentages of morphologically normal sperm and seminal plasma zinc concentration in the infertile men groups were not different in various age groups(P >0.05 ).The sperm acrosin activity in the age group of 36-50 years was remarkably decreased (P 0.05).Seminal plasma zinc concentration in the infertile men groups were not different in the 8 groups (P >0.05 ).Sperm acrosin activity was decreased with the percentage decrease of morphologically normal sperm (r =0.93,P <0.01).Conclusion The percentage of morphologically normal sperm is not affectd by the age and the seminal plasma zinc,which is positively correlated with the sperm acrosin activity.%目的:研究男性不育患者精子形态与精浆锌和精子顶体酶活性关系。方法455例男性不育患者分别根据年龄和精子正常形态百分率分成5组和8组,采用 Diff-Quik 染色法、改良 PRA 法和改良 Kennedy 法分别进行精子形态、精浆锌和精子顶体酶活性的检测。结果各年龄组的正常形态精子百分率和精浆锌浓度差异无统计学意义(P >0.05),精子顶体酶活性在36~50岁年龄段显著降低(P <0.05),其他年龄段精子顶体酶活性差异无统计学意义(P >0.05)。在精子正常形态百分率不同的8组中,年龄差异无统计学意义,精浆锌总量差异无统计学意义,精子顶体酶活性随着精子正常形态百分率降低而降低(r =0.93,P <0.01)。结论精子正常形态百分率不受年龄和精浆锌的影响,与精

  15. Association between calcium ionophore A23187 induced acrosome reaction rate, total acrosin activity and semen analysis, the outcome of in vitro fertilization%钙离子载体A23187诱发的精子顶体反应率、顶体酶总活性与精液参数及体外受精结局的关系研究

    Institute of Scientific and Technical Information of China (English)

    郭小桥; 余波澜; 周华; 司沙沙; 曹定娅; 刘见桥; 孙筱放


    Objective To investigate the association between the calcium ionophore A23187 induced sperm acrosome reaction rate, total acrosin activity and semen parameters and the IVF outcome, and to compare the predictive value for the male fertility and the outcome of IVF between calcium ionophore A23187 induced sperm acrosome reaction rate and sperm total acrosoin activity. Methods Sperm acrosome reaction was induced by calcium ionophore A23187,while sperm acrosin activity was determined by the modified Kennedy method. In this study, 150 cycles of IVF-ET were included, which were divided into Normal semen Group (85 cycles), Teratospermia Group (44 cycles), Asthenozoospermia Group (6 cycles). Compare the association between the calcium ionophore A23187 induced sperm acrosome reaction rate, total acrosin activity and normal morphology sperm percentage, concentration, activity in the 150 cycles and subgroups. Analyze the difference of the sperm acrosome reaction rate and total acrosin activity in Pregnancy Group and Non-pregnancy Group. Use the Receiver Operating Characteristic curve to make out the threshold of the sperm acrosome reaction rate and total acrosin activity for predicting the IVF pregnancy outcomes. Results In this 150 samples, there are no statistically significant differences between sperm acrosome reaction rate and sperm concentration, progressive velocity rate, the percentage of normal sperm morphology, fertility rate, and their related coefficient are respectively as follow:0.120, 0.018, 0.084, 0.054(P>0.05). But the negative correlation is statistically significant different between sperm acrosome reaction rate and teratozoospermia index(r=-0.183, P=0.025). There are significant correlations between sperm acrosin activity and sperm concentration(r=0.172, P0.05) and fertility rate(r=0.039, P>0.05). There are no statistically significant differences between sperm acrosome rate and fertility rate in Normal semen Group(r=-0.039, P>0.05), Teratospermia Group

  16. The Expression and Tyrosine Phosphorylation of Sperm Protein 32 Regulate the Activation of the Boar Proacrosin/Acrosin System%精子蛋白32的表达及酪氨酸磷酸化调控猪精子顶体蛋白的活化

    Institute of Scientific and Technical Information of China (English)

    孙培亮; 崔明勋; 姜园园; 曹立朋; 金一


    为了探究猪精子蛋白32(Sperm protein,sp32)表达及酪氨酸磷酸化调控猪精子顶体蛋白活化的关系,本研究对不同处理(鲜精、冷冻-解冻、获能、顶体反应)的猪精子顶体膜蛋白进行分离,通过考马斯亮蓝染色、SDS-PAGE电泳和Western blot检测和分析.结果表明,猪精子经过获能处理、冷冻-解冻处理以及顶体反应处理后,前顶体蛋白(Proacrosin)和顶体蛋白(Acrosin)在转化过程中,sp32表达有所差异,获能和顶体反应处理的sp32的表达量略高于冷冻-解冻处理组,而显著高于鲜精处理组.与其他处理组相比,冷冻-解冻精子处理组sp32酪氨酸磷酸化水平产生显著的差异.但在SDS-PAGE电泳中,猪鲜精处理组在蛋白质分子质量38~170 ku区间的蛋白表达条带较其他对比组明显,这表明猪精子在受精前所必需经历的获能和顶体反应过程中,顶体膜蛋白伴随着大分子的蛋白质修饰和降解.结论:sp32作为一种前顶体蛋白结合蛋白,在前顶体蛋白活化过程中它的表达水平及酪氨酸磷酸化水平上调.

  17. 精子顶体酶活性与体外受精受精率的相关性研究%Study on the relationship between sperm acrosin activity and insemination during in-vitro fertilization

    Institute of Scientific and Technical Information of China (English)



    目的 探讨精子顶体酶活性的变化与体外受精(IVF)受精率的关系.方法 46例不育夫妇男方精液,测定其密度、活力、前向运动精子及精子顶体酶活性,探讨与IVF受精率的相关性.结果精子顶体酶活性与精子的密度无相关性,与精子的活率和前向运动精子有正相关,精子顶体酶活性与IVF受精率有直线正相关性.结论 精子顶体酶活性与IVF受精率有相关性,通过测定精子顶体酶活性可能预测IVF受精率.

  18. Study on the relationship of sperm acrosin activity with fertilization rate during in-vitro fertilization%优选前后精子顶体酶活性与IVF受精率相关性的研究

    Institute of Scientific and Technical Information of China (English)

    段红艳; 章晓梅; 李永刚; 高梦莹; 邓莲; 冯怀英


    目的 探讨优选处理前和处理后精子顶体酶活性的变化,及与体外受精(IVF)受精率的相关性.方法 采用分光光度比色法,对接受IVF治疗的53例不育夫妇男方精液,分别测定优选处理前和处理后精子顶体酶活性,分析其与IVF受精率的相关性.结果 优选处理后精子顶体酶活性与优选前比较有显著性差异(P<0.05);达到常规IVF标准并选择常规IVF治疗者,优选后的精子顶体酶活性与IVF受精率有相关性,精子顶体酶活性降低与IVF受精率降低有关.结论 精子顶体酶活性与IVF受精率有相关性,并且通过精子顶体酶活性可以预测IVF受精率.

  19. 猪精子顶体素水平与精液质量参数和体外受精间的联系%Sperm acrosin levels and its relationship with other quality parameters and in vitro fertilization rate in boar semen

    Institute of Scientific and Technical Information of China (English)

    太男; 金一; 单世梁; 韩明铭


    本研究目的是评估不同处理对精子顶体素活性的影响以及顶体素活性、精液参数和体外受精间的联系.鲜精、稀释精液(1:1)和液相精液(17℃)的精子顶体素活性分别是5.27,4.28和4.41 mIU/106.冻精或液相精液保存4 d后顶体素活性显著下降(P<0.05).公猪间液相精液顶体素活性和低渗膨胀精液百分数有显著的差异(P<0.05).冷、热应激对精子顶体素活性没有影响.鲜精和冻精的顶体素活性与低渗膨胀精子百分数和顶体完整率呈正相关.不同种猪液相精液精子顶体素活性与体外受精率和随后的囊胚发育率相关性不大.因此建议顶体素活性与其它精子功能参数相结合才能精确预测精子受精力.

  20. Association between phthalate metabolites and biomarkers of reproductive function in 1066 Chinese men of reproductive age. (United States)

    Pan, Yitao; Jing, Jun; Dong, Fengshou; Yao, Qi; Zhang, Wei; Zhang, Hongxia; Yao, Bing; Dai, Jiayin


    Phthalates are suspected endocrine disrupting chemicals that impair male reproductive function in animal and epidemiological studies. We investigated associations between urinary phthalate metabolites and acrosin activity, along with that between insulin like-factor 3 (INSL3), a Leydig cell function marker, in Chinese adult men and assessed the association between the metabolites and male reproductive function. Serum levels of INSL3 and other hormones, semen parameters, and urinary concentrations of 14 phthalate metabolites in 1066 men were measured. The unadjusted concentrations of phthalates were included as independent variables and urinary creatinine as a separate covariate. INSL3 was negatively associated with mono-2-ethylhexyl phthalate (MEHP) and %MEHP [percentage of MEHP to all di(2-ethylhexyl) phthalate (DEHP) metabolites]. Acrosin activity was negatively associated with mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), MEHP and %MEHP. MBP and MiBP were also negatively associated with total testosterone (T), free androgen index (FAI), free testosterone (FT), luteinizing hormone (LH) and sperm morphology and positively associated with DNA fragmentation index (DFI). A negative association between %MEHP and sperm motility was observed. Several other metabolites were also associated with reproductive function. This is the first report on the inverse associations of phthalate metabolites with acrosin activity and INSL3. Phthalates may cause multiple adverse results on reproductive function at environmental levels.

  1. Effect of You Gui Wan on mouse sperm fertilising ability in vivo and in vitro. (United States)

    Jiang, X-H; Yie, S-M; Zhen, X; Den, Y-L; Liang, X; Hu, X; Li, L-M; Li, Q-J; Cao, S; Lu, H


    This study is to explore whether YGW has an impact on sperm fertilising ability in mice. Twenty male mice were randomly divided into two groups. In vivo experiments, one group of animals were orally administrated with YGW decoction and another group administered with saline for 14 days. Afterwards, the animals were mated with their female partners. Percentages of retrieved zygotes were then compared. In vitro experiments, in vitro fertilisation (IVF) assay, sperm acrosome reaction and acrosin activity were used to compare sperm fertilising ability between the two groups. The YGW-treated group had a significantly higher percentage of zygotes than the saline controls (P = 0.005). The IVF rates induced by spermatozoa from the herb-treated mice were also significantly higher than those from the control animals (P = 0.015). The sperm acrosin activity of the herb-treated group was significantly higher than that of the saline-treated group (P = 0.048), although there was no significant difference in testicular weight, sperm count and sperm motility. These data suggest that YGW decoction has a significant effect on normal sperm fertilising ability both in vivo and in vitro, which may be due to, at least in part, increments in the sperm acrosin activity.

  2. Inhibition of Hamster Sperm Acrosomal Enzyme by Gossypol is Closely Associated with the Decrease in Fertilization Capacity

    Institute of Scientific and Technical Information of China (English)

    YuanYu-ying; ShiQi-xian


    To elucidate the mechanism of sterility induced by gossypol,we studied the relationship between the activities of acrosomal enzymes and their fertilizing capacity in the hamster. The results showed that the ability of spermatozoa to penetrate into bovine cervical mucus,hyperactivated motility(HAM)and fertility in vivo were significantly inhibited when spermatozoa were exposed to gossypol (2.5μg-60μg/mL)for 15 min in vitro.Also,following administration of gossypol (12.5mg/kg/day)for 6 weeks,sperm motility,HAM and rate of fertilization in vitro by the hamster cauda epididymal spermatozoa were significantly decreased and the extracts of testis delayed dispersion of the cumulus oophorus cells,suggesting that hyaluronidase and other acrosomal enzymes might be inhibited by gossypol.In addition, acrosin and arylsulfatase activities were also markedly inhibited.These data show that the inhibition of acrosin and arylsulfatase activities is the main cause of gossypol-induced infertility.The inhibition was dependent upon gossypol dose and the duration of administration.Thus, the assay of acrosin and arylsulfatase activities may provide a useful tool for monitoring sterility induced by gossypol.

  3. Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein. (United States)

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


    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

  4. Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm-zona pellucida receptors. (United States)

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


    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.

  5. Isolation and characterization of an ovoinhibitor, a multidomain Kazal-like inhibitor from Turkey (Meleagris gallopavo) seminal plasma. (United States)

    Słowińska, Mariola; Liszewska, Ewa; Nynca, Joanna; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Szubstarski, Jarosław; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej


    Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

  6. In Vitro Effect of Cell Phone Radiation on Motility, DNA Fragmentation and Clusterin Gene Expression in Human Sperm

    Directory of Open Access Journals (Sweden)

    Adel Zalata


    Full Text Available Background: Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU gene expression. Materials and Methods: In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26, ii. asthenozoospermia (A, n=32, iii. asthenoteratozoospermia (AT, n=31 and iv. oligoasthenoteratozoospermia (OAT, n=35. The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where pAT>A>N groups, respectively (p<0.05. Conclusion: Cell phone emissions have a negative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases.

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


    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.

  8. Effect of estrogens on boar sperm capacitation in vitro

    Directory of Open Access Journals (Sweden)

    Ded Lukas


    Full Text Available Abstract Background Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1, 17beta-estradiol (E2 and estriol (E3 as well as the synthetical one, 17alpha-ethynylestradiol (EE2 on boar sperm capacitation in vitro. Methods Boar sperm were capacitated in vitro in presence of estrogens. Capacitation progress in control and experimental samples was analyzed by flow cytometry with the anti-acrosin monoclonal antibody (ACR.2 at selected times of incubation. Sperm samples were analyzed at 120 min of capacitation by CTC (chlortetracycline assay, immunocytochemistry and flow cytometry with anti-acrosin ACR.2 antibody. Furthermore, sperm samples and capacitating media were analyzed by immunocytochemistry, ELISA with the ACR.2 antibody, and the acrosin activity assay after induced acrosomal reaction (AR. Results Estrogens stimulate sperm capacitation of boar sperm collected from different individuals. The stimulatory effect depends on capacitation time and is highly influenced by differences in the response to estrogens such as E2 by individual animals. Individual estrogens have relatively same effect on capacitation progress. In the boar samples with high estrogen responsiveness, estrogens stimulate the capacitation progress in a concentration-dependent manner. Furthermore, estrogens significantly increase the number of acrosome-reacted sperm after zona

  9. Protective effect of resveratrol on spermatozoa function in male infertility induced by excess weight and obesity. (United States)

    Cui, Xiangrong; Jing, Xuan; Wu, Xueqing; Yan, Meiqin


    Male infertility is a complex, multifactorial and polygenic disease that contributes to ~50% cases of infertility. Previous studies have demonstrated that excess weight and obesity factors serve an important role in the development of male infertility. An increasing number of studies have reported that resveratrol may regulate the response of cells to specific stimuli that induce cell injury, as well as decrease germ cell apoptosis in mice or rats. In the present study, the semen quality and serum sex hormone levels were evaluated in 324 men, which included 73 underweight, 82 normal weight, 95 overweight and 74 obese men. All patients were referred to The Reproductive Medicine Center of Shanxi Women and Infants Hospital (Taiyuan, China) between January 2013 and January 2015. The aim of the present study was to investigate the effects of resveratrol treatment on the motility, plasma zinc concentration and acrosin activity of sperm from obese males. The sperm concentration, normal sperm morphology, semen volumes, DNA fragmentation rates and testosterone levels in men from the overweight and obese groups were markedly decreased when compared with men in the normal weight group. In addition, the progressive motility, seminal plasma zinc concentration and spermatozoa acrosin activity were notably decreased in the obese group compared with the normal weight group. However, estradiol levels were significantly increased in the overweight, obese and underweight groups compared with the normal weight group. Notably, semen samples from obese males with astenospermia treated with 0‑100 µmol/l resveratrol for 30 min demonstrated varying degrees of improvement in sperm motility. When these semen samples were treated with 30 µmol/l resveratrol, sperm motility improved when compared to other doses of resveratrol. Therefore, 30 µmol/l resveratrol was selected for further experiments. Upon treatment of semen samples with resveratrol (30 µmol/l) for 30 min, the seminal

  10. Protective effect of resveratrol on spermatozoa function in male infertility induced by excess weight and obesity (United States)

    Cui, Xiangrong; Jing, Xuan; Wu, Xueqing; Yan, Meiqin


    Male infertility is a complex, multifactorial and polygenic disease that contributes to ~50% cases of infertility. Previous studies have demonstrated that excess weight and obesity factors serve an important role in the development of male infertility. An increasing number of studies have reported that resveratrol may regulate the response of cells to specific stimuli that induce cell injury, as well as decrease germ cell apoptosis in mice or rats. In the present study, the semen quality and serum sex hormone levels were evaluated in 324 men, which included 73 underweight, 82 normal weight, 95 overweight and 74 obese men. All patients were referred to The Reproductive Medicine Center of Shanxi Women and Infants Hospital (Taiyuan, China) between January 2013 and January 2015. The aim of the present study was to investigate the effects of resveratrol treatment on the motility, plasma zinc concentration and acrosin activity of sperm from obese males. The sperm concentration, normal sperm morphology, semen volumes, DNA fragmentation rates and testosterone levels in men from the overweight and obese groups were markedly decreased when compared with men in the normal weight group. In addition, the progressive motility, seminal plasma zinc concentration and spermatozoa acrosin activity were notably decreased in the obese group compared with the normal weight group. However, estradiol levels were significantly increased in the overweight, obese and underweight groups compared with the normal weight group. Notably, semen samples from obese males with astenospermia treated with 0–100 µmol/l resveratrol for 30 min demonstrated varying degrees of improvement in sperm motility. When these semen samples were treated with 30 µmol/l resveratrol, sperm motility improved when compared to other doses of resveratrol. Therefore, 30 µmol/l resveratrol was selected for further experiments. Upon treatment of semen samples with resveratrol (30 µmol/l) for 30 min, the seminal plasma

  11. Advancement in biochemical assays in andrology

    Institute of Scientific and Technical Information of China (English)

    Wolf-BernhardSchill; RaftHenkel


    Determination of maikers of sperm function, accessory sex gland secretion and silent male genital tract inflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemical tests into the analysis of male factor has the advantage that standardized assays with a coefficient of variafion characteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability .Biochemical parameters may be used in clinical practice to evaluate the sperm fertitizing capacity (acrosin, aniline blue,ROS), to characterize male accessory sex gland secretinns (fructose, a-glucosidase, PSA), and to identify men with silent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS). (As/an J Androl 1999 Jun; 1: 45-51)

  12. Sperm immobilization activity of Allium sativum L. and other plant extracts

    Institute of Scientific and Technical Information of China (English)

    KausikiChakrabarti; SulagnaPal; AsokK.Bhattacharyya


    Aim: To identify possible spermicidal agents through screening a number of edible medicinal plants with antimicrobial activity. Methods: Initial screening was made on the basis of ram cauda epididymal sperm immobiliza-tion immediately after addition of extracts. The most potent extract was selected and was evaluated on both ram and human spermatozoa. To unravel its mode of action several sperm functional tests were carried out, namely viability of cells, hypo-osmotic swelling test for membrane integrity and assays of membrane-bound enzyme 5'-nucleotidase and acrosomal marker enzyme acrosin. Results: The crude aqueous extract of the bulb ofAllium sativum L. Showed the most promising results by instant immobilization of the ram epididymal sperm at 0.25 g/mL and human ejaculated sperm at 0.5 g/mL. Sperm immobilizing effects were irreversible and the factor of the extract responsible for immobilization was thermostable up to 90℃. On boiling at 100℃ for 10 minutes, this activity was markedly reduced. Moreover, this extract was able to cause aggregation of ram sperms into small clusters after 30 minutes of incubation at 37℃. However this property was not found in human spermatozoa. More than 50 % reduction in sperm viability and hypo-osmotic swelling occurred in treated sperm as compared with the controls, indicating the possibility of plasma membrane disintegration which was further supported by the significant reduction in the activity of membrane bound 5''-nucleotidase and acrosomal acrosin. Conclusion: The crude aqueous extract of A. Sativum bulb possesses spermicidal activity in vitro. (Asian J Androl 2003 Jun, 5:131-135 )

  13. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma. (United States)

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej


    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  14. Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo). (United States)

    Słowińska, Mariola; Olczak, Mariusz; Wojtczak, Mariola; Glogowski, Jan; Jankowski, Jan; Watorek, Wiesław; Amarowicz, Ryszard; Ciereszko, Andrzej


    The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

  15. Red wine consumption may affect sperm biology: the effects of different concentrations of the phytoestrogen myricetin on human male gamete function. (United States)

    Aquila, Saveria; Santoro, Marta; De Amicis, Francesca; Guido, Carmela; Bonofiglio, Daniela; Lanzino, Marilena; Cesario, Maria Grazia; Perrotta, Ida; Sisci, Diego; Morelli, Catia


    Myricetin is a natural flavonoid, particularly enriched in red wines, whose occurrence is widespread among plants. Despite extensive research, the beneficial effects of Myricetin on human health are still controversial. Here, we tested the estrogen-like effect of the phytoestrogen Myricetin on human ejaculated sperm biology. To this aim, human normozoospermic samples were exposed to increasing concentrations (10 nM, 100 nM, and 1 µM) of Myricetin. Motility, viability, capacitation-associated biochemical changes (i.e., cholesterol efflux and tyrosine phosphorylation), acrosin activity, as well as glucose utilization and fatty-acid oxidation (i.e., glucose and lipid metabolism) were all significantly increased by low doses of Myricetin. Importantly, both estrogen receptors α and β (ERs) and phosphatidylinositol-3-OH kinase (PI3K)/AKT signaling are activated in the presence of Myricetin since these were both abrogated by specific inhibitors of each pathway. Our results show how Myricetin, through ERs and PI3K/AKT signalings, potentiates sperm function. This effect is dose-dependent at low concentrations of Myricetin (up to 100 nM), whereas higher amounts do not seem to improve any further sperm motility, viability, or other tested features, and, in some cases, they reduced or even abrogated the efficacy exerted by lower doses. Further studies are needed to elucidate if high levels of Myricetin, which could be attained even with moderate wine consumption, could synergize with endogenous estrogens in the female reproductive tract, interfering with the physiological sperm fertilization process.

  16. Endocrinological, biophysical, and biochemical parameters of semen collected via masturbation versus sexual intercourse. (United States)

    Sofikitis, N V; Miyagawa, I


    In clinical programs of assisted reproduction involving infertile males, it is essential to obtain semen of maximum quality. To evaluate ways of achieving this objective, and to assess the fertilizing capacity of the sperm, six semen samples were collected from each of 38 infertile men via masturbation. Six more samples were then collected from each man at sexual intercourse using a semen collection device (SCD). We confirmed that the volume of seminal plasma, total sperm count, sperm motility, and percentage of morphologically normal spermatozoa were significantly higher in samples collected at intercourse than masturbation, as reported previously. In addition, the markers of the secretory function of the prostate and the outcome of sperm function tests (hypoosmotic swelling test, acrosin assay, and sperm penetration assay) were significantly higher for the samples collected at intercourse. There were no significant differences in markers of the secretory function of the seminal vesicles and epididymis between the samples. The improved spermatozoal parameters in the samples collected at intercourse may reflect a higher prostatic secretory function at that time. There were no significant differences in the serum concentrations of gonadotropins, or in the serum or seminal plasma concentrations of testosterone, before or after masturbation or sexual intercourse. Therefore, the differences in prostatic secretory function and semen parameters may not be attributed to differences in hormonal levels. Semen collection during intercourse using an SCD appears to be the method of choice for selecting semen samples for artificial insemination.

  17. Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells

    Directory of Open Access Journals (Sweden)

    Charles A. Easley, IV


    Full Text Available Human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been shown to differentiate into primordial germ cells (PGCs but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm. These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

  18. Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). (United States)

    Huleihel, Mahmoud; Nourashrafeddin, Seyedmehdi; Plant, Tony M


    In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.

  19. A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): purification, primary structure, and inhibitory properties. (United States)

    Kubiak, Agnieszka; Jakimowicz, Piotr; Polanowski, Antoni


    Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K(a)=1.1 x 10(9) M(-1) and 2.5 x 10(9) M(-1), respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K(a)=4.5 x 10(8) M(-1) and 1.2 x 10(10) M(-1)). Weak interaction with human plasmin (K(a)=1.2 x 10(7) M(-1)) was also revealed.

  20. Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition. (United States)

    Deng, Shou-Long; Chen, Su-Ren; Wang, Zhi-Peng; Zhang, Yan; Tang, Ji-Xin; Li, Jian; Wang, Xiu-Xia; Cheng, Jin-Mei; Jin, Cheng; Li, Xiao-Yu; Zhang, Bao-Lu; Yu, Kun; Lian, Zheng-Xing; Liu, Guo-Shi; Liu, Yi-Xun


    Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10(-7) M) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl-2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro-generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage.

  1. Sperm quality and fertility of boar seminal doses after 2 days of storage: does the type of extender really matter? (United States)

    Pinart, Elisabeth; Yeste, Marc; Prieto-Martínez, Noelia; Reixach, Josep; Bonet, Sergi


    The present approach was designed to evaluate the extender effects on sperm quality and fertility of short-term refrigerated seminal doses from Landrace boars lodged in husbandry-controlled conditions. For this purpose, we analyzed the sperm quality of seminal doses diluted in short-term (Beltsville Thawing Solution) and extra-long-term (Duragen) extenders from Days 0 to 2 of storage at 17 °C during an 8-month period. Pregnancy rates and litter size were evaluated from double inseminations within an interval of 12 hours (36 and 48 hours of refrigeration) of multiparous females using seminal doses diluted in each extender type. Sperm quality was assessed from the analyses of sperm motility and kinetics, sperm viability, expressed as plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, and acrosin activity. Results indicated significant differences between the extenders in the sperm quality of seminal doses. Therefore, the seminal doses diluted in Duragen had higher percentages of progressive motile spermatozoa and membrane-intact spermatozoa than those diluted in Beltsville Thawing Solution throughout all the experimental months. Nevertheless, despite these differences in preserving the sperm quality, pregnancy rates (>90%) and litter sizes (>10 piglets born per litter) were similar between the extenders. Our results had great relevance from a practical point of view because they reported lack of an extender effect on the reproductive performance of seminal doses during short-tem storage.

  2. Effects of Sperm Acrosomal Integrity and Protamine Deficiency on In Vitro Fertilization and Pregnancy Rate

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee


    Full Text Available Background: The objective of this study was to evaluate the relationship between protaminedeficiency, and acrosomal integrity with fertilization and pregnancy rate in patients undergone in vitrofertilization (IVF.Material &Methods: Semen samples from 70 infertile couples undergoing IVF at Isfahan Fertility andInfertility center were assessed in this study. Semen analysis was carried out according to WHO criteria.Protamine deficiency, Sperm morphology and acrosin activity were assessed by Chromomycin A3(CMA3, Papanicolaou staining and Gelatinolysis tests, respectively. Coefficients of correlation andstudent t-test were carried out using the Statistical Package for the Social Studies (SPSS 11.5 and Pvaluelower than 0.05 was considered as significant.Results: Fertilization rate, percentage of halo formation, mean halo diameter and abnormalmorphology show a significant correlation with percentage of CMA3 positivity. CMA3 positivity,percentage of halo, mean halo and sperm morphology showed a significant correlation with fertilizationrate. Among the aforementioned parameters percentage of halo had the highest correlation. In thepresent study patients were divided into two groups according to pregnancy status. None of the studiedparameters were significantly different between pregnant and non-pregnant patients. However,percentage of halo formation showed a slightly significant difference (r=0.306; P=0.058.Conclusion: The results of this study revealed that, even though sperm morphology, sperm protaminecontent and acrosome formation are events related to spermiogenesis, sperm acrosomal integrityassessed by percentage of halo formation has more profound effect on fertilization rate and pregnancyoutcome during IVF procedure.

  3. Sperm Proteases that May Be Involved in the Initiation of Sperm Motility in the Newt, Cynops pyrrhogaster

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    Misato Yokoe


    Full Text Available A protease of sperm in the newt Cynops pyrrhogaster that is released after the acrosome reaction (AR is proposed to lyse the sheet structure on the outer surface of egg jelly and release sperm motility-initiating substance (SMIS. Here, we found that protease activity in the sperm head was potent to widely digest substrates beneath the sperm. The protease activity measured by fluorescein thiocarbamoyl-casein digestion was detected in the supernatant of the sperm after the AR and the activity was inhibited by 4-(2-aminoethyl benzenesulfonyl fluoride (AEBSF, an inhibitor for serine or cysteine protease, suggesting the release of serine and/or cysteine proteases by AR. In an in silico analysis of the testes, acrosins and 20S proteasome were identified as possible candidates of the acrosomal proteases. We also detected another AEBSF-sensitive protease activity on the sperm surface. Fluorescence staining with AlexaFluor 488-labeled AEBSF revealed a cysteine protease in the principal piece; it is localized in the joint region between the axial rod and undulating membrane, which includes an axoneme and produces powerful undulation of the membrane for forward sperm motility. These results indicate that AEBSF-sensitive proteases in the acrosome and principal piece may participate in the initiation of sperm motility on the surface of egg jelly.

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

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


    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.

  5. A Catalog of Proteins Expressed in the AG Secreted Fluid during the Mature Phase of the Chinese Mitten Crabs (Eriocheir sinensis). (United States)

    He, Lin; Li, Qing; Liu, Lihua; Wang, Yuanli; Xie, Jing; Yang, Hongdan; Wang, Qun


    The accessory gland (AG) is an important component of the male reproductive system of arthropods, its secretions enhance fertility, some AG proteins bind to the spermatozoa and affect its function and properties. Here we report the first comprehensive catalog of the AG secreted fluid during the mature phase of the Chinese mitten crab (Eriocheir sinensis). AG proteins were separated by one-dimensional gel electrophoresis and analyzed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Altogether, the mass spectra of 1173 peptides were detected (1067 without decoy and contaminants) which allowed for the identification of 486 different proteins annotated upon the NCBI database ( and our transcritptome dataset. The mass spectrometry proteomics data have been deposited at the ProteomeXchange with identifier PXD000700. An extensive description of the AG proteome will help provide the basis for a better understanding of a number of reproductive mechanisms, including potentially spermatophore breakdown, dynamic functional and morphological changes in sperm cells and sperm acrosin enzyme vitality. Thus, the comprehensive catalog of proteins presented here can serve as a valuable reference for future studies of sperm maturation and regulatory mechanisms involved in crustacean reproduction.

  6. Shengjing prescription improves semen parameters of oligoasthenozoospermia patients:Efficacy and mechanism%生精片对男性少弱精子症患者精液参数的影响及作用机制研究

    Institute of Scientific and Technical Information of China (English)

    孙振高; 连方; 姜鲲鹏; 张建伟; 马凤梅; 张宁; 孙金龙; 杨武文


    目的:观察生精片治疗男性少弱精子症患者的临床有效性,并对其作用机制进行探讨. 方法:选取少弱精子症患者120例,采取随机对照研究的方法,治疗组口服生精片,对照组服用维生素E,服用12周后,观察治疗前后两组精子浓度、精子活力及正常形态精子百分率,血清FSH、T、LH水平,DNA碎片指数,低渗肿胀精子百分率,精浆弹性蛋白酶、α-葡糖苷酶、精子顶体酶、果糖及精浆锌等指标变化. 结果:生精片能显著改善精子浓度、活力和形态(P<0.01),血清FSH水平明显降低,T明显升高(P<0.01),DNA碎片指数明显降低,低渗肿胀精子百分率显著升高,弹性蛋白酶明显降低,α-葡糖苷酶、精子顶体酶、果糖、精浆锌等水平明显升高. 结论:生精片通过多层次、多环节、多途径改善少弱精子症患者的精液参数,具有良好的临床疗效.%Objective: To investigate the clinical efficacy of Shengjing prescription for oligoasthenozoospermia and its action mechanism. Methods: We equally assigned 120 patients with oligoasthenozoospermia to receive Shengjing prescription (treatment group) and vitamin E (control group) , respectively, for 12 weeks. Before and after the treatment, were obtained sperm concentration, sperm motility, the percentage of morphologically normal sperm, the levels of serum follicle - stimulating hormone (FSH ) , testosterone ( T) and luteinizing hormone ( LH ) , sperm DNA fragmentation index (DFI), the percentage of hypotonic swelling sperm, and the levels of seminal plasma elastase, α-glucosidase, fructose, zinc and acrosin. Results: Compared with vitamin E, Shengjing prescription significantly improved sperm concentration, motility and morphology (P<0.01) , decreased the serum FSH level, elevated the serum T level (P <0.01) , reduced DFI and seminal plasma elastase, and increased the percentage of hypotonic swelling sperm as well as the levels of seminal plasma a

  7. The human acrosome reaction

    Institute of Scientific and Technical Information of China (English)

    H.W.G.Baker; D.Y.Liu; C.Garrett; M.Martic


    We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, spermzona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization (IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one woman affected. Low or zero fertilization in standard IVFwas usually caused by sperm abnormalities. Poor sperm-zona pellucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morphology. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion of acrosome intact sperm in the insemination medium was related to the IVF rate. Inducing the acrosome reaction with a calcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented spermzona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding. Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normal sperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrated the zona. In contrast, fertilization and pregnancy rates were high with intracytoplasmic sperm injection. We call thiscondition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signal transduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler tests and treatments for the patients and also provide new leads for contraceptive development.

  8. In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues. (United States)

    Sato, Takuya; Katagiri, Kumiko; Kojima, Kazuaki; Komeya, Mitsuru; Yao, Masahiro; Ogawa, Takehiko


    Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

  9. Bone morphogenetic protein 4 (BMP4) induces buffalo (Bubalus bubalis) embryonic stem cell differentiation into germ cells. (United States)

    Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Singh, Manoj Kumar; Manik, Radhey Sham; Singla, Suresh Kumar; Palta, Prabhat; Chauhan, Manmohan Singh


    The aim of the present study was to investigate the effect of Bone morphogenetic protein 4 (BMP4) stimulation on differentiation of buffalo embryonic stem (ES) cells into germ lineage cells. ES cells were subjected to in vitro differentiation in floating and adherent cultures, under different BMP4 concentrations (20, 50 and 100 ngml(-1)) for different culture intervals (4, 8 and 14 days). qPCR analysis revealed that BMP4 at a concentration of 50-100 ngml(-1) for a culture period of 14 days led to maximum induction of germ lineage genes like DAZL, VASA, PLZF (PGC-specific); SYCP3, MLH1, TNP1/2 and PRM2 (Meiotic genes); BOULE and TEKT1 (Spermatocyte markers); GDF9, ZP2 and 3 (Oocyte markers). The expression levels of all the genes were significantly higher under BMP4 differentiation as compared to BMP4 + NOGGIN and spontaneously differentiated cultures. Immunocytochemical analysis of embryoid bodies (EBs) and monolayer adherent cultures revealed expression of PGC- (c-KIT, DAZL and VASA); Meiotic- (SYCP3, MLH1 and PROTAMINE1); Spermatocyte- (ACROSIN and HAPRIN); and Oocyte- markers (GDF9 and ZP4). Western blotting was positive for VASA, GDF9 and ZP4. Oocyte-like structures (OLS) obtained in monolayer differentiated cultures harbored a big nucleus and a ZP4 coat. They showed embryonic development and progressed through 2-cell, 4-cell, 8-cell and blastocyst-like structures. Global DNA methylation analysis showed significantly (p < 0.05) decreased levels of 5-methyl-2-deoxycytidine in EBs obtained in optimum differentiation medium. The expression of meiotic markers coupled with expression of spermatocyte and oocyte markers is an indication of post-meiotic progression into spermatogenesis and oogenesis, respectively.

  10. MnSOD expression inhibited by electromagnetic pulse radiation in the rat testis. (United States)

    Zeng, LiHua; Ji, XiTuan; Zhang, YanJun; Miao, Xia; Zou, ChangXu; Lang, HaiYang; Zhang, Jie; Li, YuRong; Wang, XiaoWu; Qi, HongXing; Ren, DongQin; Guo, GuoZhen


    Male Sprague Dawley rats were exposed to EMP irradiation of 100 kV/m peak-to-peak e-field intensity and different numbers of pulses. Rat sperm samples were prepared for analysis of sperm qualities; Testes were assessed by transmission electron microscopy and serum hormone concentrations were examined by radioimmunoassay; Enzymatic activities of Total-superoxide dismutase(T-SOD) and manganese-superoxide dismutase (MnSOD), the mRNA levels of MnSOD and cuprozinc-superoxide dismutase (CuZnSOD), and the density of malondialdehyde (MDA) were also determined. EMP irradiation did not affect spermatozoon morphology, micronucleus formation rate, sperm number or viability, but the acrosin reaction rate decreased at 24 h and 48 h and recovered by 72 h after irradiation as compared to the controls. The ultrastructure of rat testis displayed more serious damage at 24 h than at other time points (6 h, 12 h, 48 h). Serum levels of luteotrophic hormone (LH) and testosterone (T) were elevated in irradiated rats as compared to controls. After irradiation, enzymatic activities of T-SOD and MnSOD were reduced by 24 h, consistent with the changes observed in MnSOD mRNA expression; MDA content increased at 6 h in turn. These studies have quantified the morphological damage and dysfunction in the rat reproductive system induced by EMP. The mechanism of EMP induced damage may be associated with the inhibition of MnSOD expression.

  11. Follicle cell trypsin-like protease HrOvochymase: Its cDNA cloning, localization, and involvement in the late stage of oogenesis in the ascidian Halocynthia roretzi. (United States)

    Mino, Masako; Sawada, Hitoshi


    We previously reported that the sperm trypsin-like protease HrAcrosin and its precursor HrProacrosin participate in fertilization of the ascidian Halocynthia roretzi. The HrProacrosin gene is annotated in the H. roretzi genome database as Harore.CG.MTP2014.S89.g15383; our previously reported sequence of HrProacrosin gene appeared to include four nucleotides inserted near the 3'-end of HrProacrosin, resulting in a frame-shift mutation and a premature termination codon. The gene architecture of HrProacrosin and Harore.CG.MTP2014.S89.g15383 resembles that of Xenopus laevis ovochymase-1/OVCH1 and ovochymase-2/OVCH2, which encode egg extracellular polyproteases. Considering these new observations, we evaluated the cDNA cloning, expression, localization, and function of Harore.CG.MTP2014.S89.g15383, herein designated as HrOvochymase/HrOVCH. We found that HrOVCH cDNA consists of a single open reading frame of 1,575 amino acids, containing a signal peptide, three trypsin-like protease domains, and six CUB domains. HrOVCH was transcribed by the testis and ovary, but the majority of protein exists in ovarian follicle cells surrounding eggs. An anti-HrOVCH antibody inhibited elevation of the vitelline coat at a late stage of oogenesis, during the period when self-sterility is acquired. As trypsin inhibitors are reported to block the acquisition of self-sterility during oogenesis, whereas trypsin induces the acquisition of self-sterility and elevation of the vitelline coat in defolliculated ovarian eggs, we propose that HrOVCH may play a role in the acquisition of self-sterility by late-stage H. roretzi oocytes.

  12. Upregulation of RHOXF2 and ODF4 Expression in Breast Cancer Tissues

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    Golnesa Kazemi-Oula


    Full Text Available Objective: During the past decade, the importance of biomarker discovery has been highlighted in many aspects of cancer research. Biomarkers may have a role in early detection of cancer, prognosis and survival evaluation as well as drug response. Cancer-testis antigens (CTAs have gained attention as cancer biomarkers because of their expression in a wide variety of tumors and restricted expression in testis. The aim of this study was to find putative biomarkers for breast cancer. Materials and Methods: In this applied-descriptive study, the expression of 4 CTAs, namely acrosin binding protein (ACRBP, outer dense fiber 4 (ODF4, Rhox homeobox family member 2 (RHOXF2 and spermatogenesis associated 19 (SPATA19 were analyzed at the transcript level in two breast cancer lines (MCF-7 and MDA-MB-231, 40 invasive ductal carcinoma samples and their adjacent normal tissues as well as 10 fibroadenoma samples by means of quantitative real-time reverse transcription polymerase chain reaction (RT-PCR. Results: All four genes were expressed in both cell lines. Expression of ODF4 and RHOXF2 was detected in 62.5% and 60% of breast cancer tissues but in 22.5 and 17.5% of normal tissues examined respectively. The expression of both RHOXF2 and ODF4 was upregulated in cancerous tissues compared with their normal adjacent tissues by 3.31- and 2.96-fold respectively. The expression of both genes was correlated with HER2/neu overexpression. RHOXF2 expression but not ODF4 was correlated with higher stages of tumors. However, no significant association was seen between expression patterns and estrogen and progesterone receptors status. Conclusion: ODF4 and RHOXF2 are proposed as putative breast cancer biomarkers at the transcript level. However, their expression at protein level should be evaluated in future studies.

  13. Differentiation of murine male germ cells to spermatozoa in a soft agar culture system

    Institute of Scientific and Technical Information of China (English)

    Mahmoud Abu Elhija; Eitan Lunenfeld; Stefan Schlatt; Mahmoud Huleihel


    Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility.In the present study,we developed in vitro culture conditions using three-dimensional agar culture system (SACS),which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis,including spermatozoa generation.Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated,and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS).The lower layer of the SACS contained only RPMI medium supplemented with FCS.Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size.Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa,Dazl,OCT-4,C-Kit,GFR- α-1,CD9 and α-6-integrin),meiotic cells (LDH,Crem-1 and Boule) and post-meiotic cells (Protamine-1,Acrosin and SP-10).Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS.The spermatozoa showed normal morphology and contained acrosomes.Thus,our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

  14. 精子质量与短时受精后受精结局关系的研究%The relationship between the sperm quality and fertilization outcome after short-time insemination

    Institute of Scientific and Technical Information of China (English)

    郭海彬; 殷宝莉; 张翠莲; 李杭生; 夏松; 张天; 贾楠; 杨锦建


    Objective To evaluate the predictive value of the sperm quality to fertilization outcomes after short-time insemination.Methods A total of 558 cycles of short-time insemination in the Reproductive Medical Center of Henan Provincial People's Hospital during January 2009 to June 2010 excluding patients aged > 38 years and M Ⅱ oocyte number < 3 were analyzed retrospectively.According to whether undergo rescue intracytoplasmic sperm injection( Re-ICSI),all cycles were divided into in vitro fertilization (IVF)group (472 cycles) and rescue intracytoplasmic sperm injection (Re-ICSI) group (86 cycles).Both IVFgroup and Re-ICSI group were subdivided into primary infertility and secondary infertility according to previous history of pregnancy.269 primary infertility cycles and 203 secondary infertility cycles were characterized in IVF group; and 64 primary infertility cycles and 22 secondary infertility cycles were characterized in Re-ICSI group.x2 test was applied for comparison of embryo plant rate,clinical pregnancy rate,early miscarriage rate between IVF and Re-ICSI groups,while Fisher test was used for comparison of live birth rate.and Mann-Whitney U test was utilized for comparison of duration of infertility,forward moving sperm counts,abnormal sperm rate,sperm acrosin activity between IVF and Re-ICSI groups.Results The embryo plant rate,clinical pregnancy rate,early miscarriage rate,live birth rate of IVF group were:29.4%,44.9%,13.4%,37.0% respectively; the above indicators in Re-ICSI group were:25.7%,34.6%,10.7%,29.6% respectively,the differences of the indicators between the two groups had no statistical sigmficance (x2 =0.869,2.963,0.010,P =0.351,0.085,0.922,0.098).Median of duration of infertility,forward moving sperm counts,abnormal sperm rate,sperm acrosin activity of primary infertility cycles in IVF group were:4.00(3.00 -6.00) years,58.37(33.64 - 102.27) × 106,81.09% (79.41% -88.69% ),76.30 (48.50 - 92.46 ) μIU/106 sperm

  15. Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells. (United States)

    Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S


    Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P embryonic development and progressed through two-cell, four-cell, eight-cell, morula, and blastocyst-like structures, indicative of their developmental competence

  16. Effect of the working environment on the male reproductive system%铅接触对男性生殖系统影响的研究

    Institute of Scientific and Technical Information of China (English)



      目的探讨化学因素中的重金属因素对男性生殖功能的影响.方法选取本市某家具厂中200名与铅长期接触超过1年的男性工人作为本次研究的观察组,另外选取本市不接触铅和其他有害物质的男性200名作为对照组.采用问卷调查和测定被试精液当中计算精子数、顶体酶活性、雄激素、促卵泡素、促间质细胞激素的含量.结果观察组中生殖症状率为28.2%,对照组为13.6%,性欲减退观察组为23.3%,对照组为10.1%,配偶流产率观察组为2.0%,对照组为8.0%,配偶早产率观察组为18.0%,对照组为9.0%,差异均有统计学意义(P 0.05);精液检查结果显示,两组患者的精子数的差异无统计学意义(P >0.05).而在顶体酶活性、雄激素、促卵泡素、促间质细胞激素方面差异均有统计学意义(P 0.05).The results of semen examination showed that the sperm count of the two groups were not significantly different (P >0.05). And the differences of acrosin activity, T, FSH and LH were statistically significant (P <0.05). Conclusion The exposure of lead has an effect on the male reproductive system in some way. It has a great effect on the activity of acrosome enzyme, and can weaken the sexuality and erectile function.In order to increase the health of the reproductive system of male workers, we should prompt protection measures and improved working conditions of lead workers, if necessary can suspend the work of a male who was exposure in lead.

  17. 壳聚糖凝胶对精子活力的影响及机制研究%The study on the effect of chitosan gel on sperm motility and the mechanism of this effect

    Institute of Scientific and Technical Information of China (English)

    陈彦瑾; 丁钰; 刘莹


    Objective To explore the effect of chitosan gel on sperm motility and the mechanism of this effect. Methods A total of 200 semen samples were randomly divided into two groups,control group( n = 100)without any treatment,chitosan gel was added to chitosan gel group(n = 100). Percentage of sperm motility of grade A(fast forward motion)and grade B(slow or sluggish),the number of normal,head de-fect,body defect and tail defect sperms,the activity of sperm acrosin enzyme,and the levels of seminal acid phosphatase,neutral α - glucosid and seminal zinc were determined and compared. Results The activity of sperm in chitosan gel group was lower than that in control group,there was statistical difference in the percentage of grade A sperms( P ﹤ 0. 05),but no difference in the percentage of grade B sperms( P ﹥ 0. 05)be-tween two groups. The number of deformity sperm in the chitosan gel group was significantly higher than that in the control group. There were sig-nificant differences in the number of normal,head defect and tail defect sperms( P ﹤ 0. 05),but not in the number of middle defect sperms( P﹥ 0. 05). The activity of sperm acrosin enzyme in chitosan gel group was lower than that in control group,but not statistically significant( P ﹥0. 05). The levels of seminal acid phosphatase,neutral α - glucosid and seminal zinc in chitosan gel group were lower than those in control group, with statistical significance(all P ﹤ 0. 05). Conclusion Chitosan gel could significantly reduce the sperm motility and biochemical parameters of seminal plasma,and make sperm deformity. Therefore,chitosan has significant killing sperm effect in vitro and,which was regarded as a new type of contraceptive drug for further research.%目的:研究壳聚糖凝胶对精子活力的影响及其影响机制。方法将200例精液常规在正常范围的精液标本随机分为两组,每组各100例,对照组无任何处理,壳聚糖凝胶组加入壳聚糖凝胶。分别测定

  18. The potential effect of smoking on semen quality through TNF-αand IL-6%肥胖对男性精液质量影响与血清TN F-α及I L-6相关性

    Institute of Scientific and Technical Information of China (English)

    武学清; 王振强; 李晓芳; 崔向荣; 井宣; 毕星宇; 张栋栋; 贾洪响; 李艳宏; 战晓宇; 李艳


    目的:探讨血清中肿瘤坏死因子α(TNF-α)及白细胞介素6(IL-6)水平的改变与肥胖的相关性及其对精液常规参数的影响。方法选择2015年1月—2015年5月于山西省妇幼保健院生殖医学中心就诊的男性患者324例为研究对象,将324例研究对象分为低体质量组、正常组、超重组及肥胖组,对各组患者行精液常规参数、精子形态、精浆锌含量、精子顶体酶活性及血清TNF-α及IL-6水平检测。同时就血清TNF-α及IL-6水平与精液常规参数之间进行相关性分析。结果低体质量组、超重组及肥胖组与正常组相比,前向运动精子百分率均显著下降,差异有统计学意义。肥胖组与正常组相比,头部缺陷精子显著增多,差异有统计学意义。而低体质量组及超重组与正常组相比精子形态差异无统计学意义。肥胖组与正常体质量组相比,精浆锌含量及精子顶体酶活性显著降低,差异有统计学意义。低体质量组及超重组与正常体质量组相比差异无统计学意义。超重与肥胖组患者血清中TNF-α及IL-6含量与正常体质量组相比均显著升高,差异有统计学意义。低体质量组与正常体组相比,血清中TNF-α及IL-6含量均差异无统计学意义。血清TNF-α及IL-6水平与男性精液密度及前向运动精子间存在相关性,而血清TNF-α及IL-6水平与患者精液量间未见相关性。结论男性肥胖患者体内不仅TNF-α及IL-6水平升高,而且与男性精液密度及前向运动精子率下降存在一定的相关性。%Objective Toinvestigatethepotentialeffectofover-weightandobesityonsemenqualitymediated TNF-αand IL-6. Methods According to the body mass index values we divided 324 male cases into low-weight group, normal weight group,over-weight group and obese group.Then we determined the semen parameters,sperm morphology, sperm DNA integrity, spermatozoa acrosin activity, seminal

  19. The Influence of Sperm DNA Damage on Test Tube Baby Help Pregnant Outcome%精子 DNA 损伤对试管婴儿助孕结局的影响

    Institute of Scientific and Technical Information of China (English)



    Objective:Detection of DNA damage of sperm,the assessment of the sperm quality and then explore the in-fluence of sperm DNA damage on test tube baby help pregnant outcome.Methods:We collect 68 patients who turn to help for test tube baby assisted reproductive technology from January 201 1 to December 2014 in our hospital.The 68 patients were in accordance with the relevant provisions which the ministry of health on the revision of 《human assisted reproductive technology and human sperm bank relevant technical standards,basic standards and ethical principles》,for men,there are not serve oligozoospermia or obstructive azoospermia.According to the DNA damage of sperm into sperm DNA damage group(sperm DNA damage rate ≥ 30%)and normal group (sperm DNA damage rate < 30%). Through a variety of means testing,get the acrosin activity of the 68 cases of male sperm as well as the sperm concen-tration,sperm activity,invitro fertilization rate,the percentage of normal sperm morphology,good quality embryo rate and live birth rate,deformity rate,pregnancy outcome and other indexes of results.Then observe and compare the a-bove indexes between the two groups.Results:The test results of injury group were less than the normal group.The a-bortion rate of the normal group compared to the abortion of the injury group does not have statistical significance. Conclusion:The integrity of the sperm will affect the fertilization ability,so the detection outcome of the sperm DNA damage rate of test tube baby help pregnancy success rate has certain influence.%目的::检测精子 DNA 损伤情况,评估精子的质量,进而探究精子 DNA 损伤对试管婴儿助孕结局的影响。方法:以2011年1月—2014年12月在我院进行试管婴儿辅助生殖技术的其中68例患者为例,均符合《卫生部关于修订人类辅助生殖技术与人类精子库相关技术规范、基本标准和伦理原则的通知》的相关规定,男性均不存在严重少精症