Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immun-ofluo...Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immun-ofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary sperma-tocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion: GCNF may play important roles in spermatogenesis, capacitation and fertilization.展开更多
Although the cytoplasm of spermatids is removed at the end of spermiogenesis, a tiny portion is usually retained in the sperm flagellum, which is termed the cytoplasmic droplet （CD） in mammals. CDs are believed to p...Although the cytoplasm of spermatids is removed at the end of spermiogenesis, a tiny portion is usually retained in the sperm flagellum, which is termed the cytoplasmic droplet （CD） in mammals. CDs are believed to play a role in sperm volume adaptation. However, we have noticed that epididymal spermatozoa that display initial （flagellation in situ） and progressive motility mostly possess CDs, whereas spermatozoa without CDs are rarely motile, suggesting that CDs have a role in motility development during sperm epididymal maturation. In the present study, we analyzed the relationship between the presence or absence of CDs, motility development and positional changes of CDs during sperm epididymal maturation in mice and monkeys. We also examined CDs on spermatozoa of three knockout mouse lines with late spermiogenic defects. Our data suggest that the CD is a normal organelle transiently present exclusively on epididymal spermatozoa, and normal CD morphology and location are associated with normal motility development during epididymal maturation of spermatozoa. Abnormal CD formation, e.g., a complete lack of CDs or ectopic CDs, is indicative of defective spermiogenesis. If CDs are essential for sperm motility development, then CDs may represent an ideal drug target for the development of non-hormonal male contraceptives.展开更多
Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endot...Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1ng/mL, 10ng/mL, 1000ng/mL, 10 000ng/ mL, and 50 000ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0. 5ng/mL, 1 ng/mL, and 10 ng/ mL ) , the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0. 5ng/mL-1000ng/mL endotoxin in media with 2mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0. 05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000ng/mL, and 1 000ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0. 01). In media containing 0. 5 ng/mL and 1 ng/ mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10ng/mL, the development of the embryos was arrested. Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.展开更多
It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare...It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling （TUNEL） assay, the comet assay, the sperm chromatin structure assay （SCSA） and the sperm chromatin dispersion （SCD） test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay.展开更多
Spermatozoa emerging from the testis undergo a maturation process in the epididymis during which they change morphologically, biochemically and physiologically to gain motility and the ability to fertilize ova. We exa...Spermatozoa emerging from the testis undergo a maturation process in the epididymis during which they change morphologically, biochemically and physiologically to gain motility and the ability to fertilize ova. We examined mouse epididymal sperm with immunostaining and transmission electron microscopy （EM） and identified a previously unknown structure on the apical hook. The structure has a coiled configuration around 11 nm in thickness and is present at the tip of each corner of the triangular-rod shaped perforatorium. Surveying sperm isolated from various regions of the epididymis indicated that mouse sperm acquire the hook rim （HR） structure during its passage through the proximal two-thirds of the caput epididymidis. The structure withstands vigorous sonication and harsh chemical treatments and remains intact after the acrosome reaction. Its location and sturdiness suggest a function in protecting the apical hook from mechanical wear during fertilization. Our EM images of epididymal sperm also revealed additional novel structures as well as lateral asymmetry of the sperm head, indicating that mouse sperm head has a structure more complex than previously recognized.展开更多
Objective To investigate effects of aging on spermatogenesis in testis, sperm maturation in epididymis, and fertility in mice. Methods Testicular specimens, caput epididymal sperm and cauda epididymal sperm were ob...Objective To investigate effects of aging on spermatogenesis in testis, sperm maturation in epididymis, and fertility in mice. Methods Testicular specimens, caput epididymal sperm and cauda epididymal sperm were obtained from Kuming mice （18-month aged group, n =15; 6-month young group as control, n=15）. The testicular histological examinations and quantitative evaluations on spermatogenesis were performed. Sperm parameters including sperm density, sperm viability, sperm motility, and normal morphological rate were assessed. The fertilization rate and embryo development were measured by in vitro fertilization and embryo culture. Results The histological changes of testes in aged mice were mainly seminiferous tubule atrophy and hypospermatogenesis. In aged testes, a significant decline was .found in the numbers of round spermatids and elongated spermatids per Sertoli cell （P 〈0.01）. Sperm density, sperm motility and normal morphological rate in caput epididymis and cauda epididymis in aged mice significantly decreased （P〈0.05）. The fertilization rate and embryo development of aged group were lower than those in the control（P〈0. 01）. Conclusions Spermatogenesis and sperm functions could be maintained in the aging male. However, aging affects spermatogenesis and sperm maturation, which leads to lower the quality of sperm, including sperm fertilizing capacity. The development of embryo from aging sperm would have more abnormalities.展开更多
50只体重为18～22 g的雄性昆明种小鼠随机分为5组（每组10只）,即溴氰菊酯（DM）1.8、3.6 mg/kg组,氯氰菊酯（CP）4.0、8.0 mg/kg组及色拉油对照组,连续1个月经口灌胃染毒后处死,测定主要脏器的脏器系数,检测小鼠精子运动参数、精子畸...50只体重为18～22 g的雄性昆明种小鼠随机分为5组（每组10只）,即溴氰菊酯（DM）1.8、3.6 mg/kg组,氯氰菊酯（CP）4.0、8.0 mg/kg组及色拉油对照组,连续1个月经口灌胃染毒后处死,测定主要脏器的脏器系数,检测小鼠精子运动参数、精子畸形发生率。结果显示：（1）DM两个剂量组小鼠脾脏的脏器系数与对照组比较明显增高（P〈0.05）;CP 8.0 mg/kg组小鼠脑的脏器系数与对照组比较却明显降低（P〈0.05）。（2）DM 3.6 mg/kg组平均直线运动速度（velocity of rectilinear motion,VSL）、平均路径速度（velocity of average pathway,VAP）、侧摆幅度（amplitude of oscillation,ALH）、运动的直线性（linearity of mo-tion,LIN）、运动的摆动性（wobble of motion,WOB）与对照组相比明显降低（P〈0.05）;CP 8.0 mg/kg组平均曲线运动速度（ve-locity of curve motion,VCL）、VSL、VAP与对照组相比明显降低（P〈0.05）;其余各剂量组精子运动参数与对照组相比,均无统计学意义。（3）各剂量组精子畸形率的增加与对照组相比无统计学意义。提示本实验条件下,Ⅱ型拟除虫菊酯对雄性小鼠脾脏、脑脏器系数和小鼠精子活力及运动方式有一定的影响。展开更多
文摘Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immun-ofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary sperma-tocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion: GCNF may play important roles in spermatogenesis, capacitation and fertilization.
文摘Although the cytoplasm of spermatids is removed at the end of spermiogenesis, a tiny portion is usually retained in the sperm flagellum, which is termed the cytoplasmic droplet （CD） in mammals. CDs are believed to play a role in sperm volume adaptation. However, we have noticed that epididymal spermatozoa that display initial （flagellation in situ） and progressive motility mostly possess CDs, whereas spermatozoa without CDs are rarely motile, suggesting that CDs have a role in motility development during sperm epididymal maturation. In the present study, we analyzed the relationship between the presence or absence of CDs, motility development and positional changes of CDs during sperm epididymal maturation in mice and monkeys. We also examined CDs on spermatozoa of three knockout mouse lines with late spermiogenic defects. Our data suggest that the CD is a normal organelle transiently present exclusively on epididymal spermatozoa, and normal CD morphology and location are associated with normal motility development during epididymal maturation of spermatozoa. Abnormal CD formation, e.g., a complete lack of CDs or ectopic CDs, is indicative of defective spermiogenesis. If CDs are essential for sperm motility development, then CDs may represent an ideal drug target for the development of non-hormonal male contraceptives.
基金This study was supported by the Science&Technology Commission of Guangdong Province,P.R.China
文摘Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1ng/mL, 10ng/mL, 1000ng/mL, 10 000ng/ mL, and 50 000ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0. 5ng/mL, 1 ng/mL, and 10 ng/ mL ) , the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0. 5ng/mL-1000ng/mL endotoxin in media with 2mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0. 05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000ng/mL, and 1 000ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0. 01). In media containing 0. 5 ng/mL and 1 ng/ mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10ng/mL, the development of the embryos was arrested. Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.
文摘It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling （TUNEL） assay, the comet assay, the sperm chromatin structure assay （SCSA） and the sperm chromatin dispersion （SCD） test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay.
文摘Spermatozoa emerging from the testis undergo a maturation process in the epididymis during which they change morphologically, biochemically and physiologically to gain motility and the ability to fertilize ova. We examined mouse epididymal sperm with immunostaining and transmission electron microscopy （EM） and identified a previously unknown structure on the apical hook. The structure has a coiled configuration around 11 nm in thickness and is present at the tip of each corner of the triangular-rod shaped perforatorium. Surveying sperm isolated from various regions of the epididymis indicated that mouse sperm acquire the hook rim （HR） structure during its passage through the proximal two-thirds of the caput epididymidis. The structure withstands vigorous sonication and harsh chemical treatments and remains intact after the acrosome reaction. Its location and sturdiness suggest a function in protecting the apical hook from mechanical wear during fertilization. Our EM images of epididymal sperm also revealed additional novel structures as well as lateral asymmetry of the sperm head, indicating that mouse sperm head has a structure more complex than previously recognized.
基金This study was supported by Nature Science Foundation of Guangdong Province, China (No.010399).
文摘Objective To investigate effects of aging on spermatogenesis in testis, sperm maturation in epididymis, and fertility in mice. Methods Testicular specimens, caput epididymal sperm and cauda epididymal sperm were obtained from Kuming mice （18-month aged group, n =15; 6-month young group as control, n=15）. The testicular histological examinations and quantitative evaluations on spermatogenesis were performed. Sperm parameters including sperm density, sperm viability, sperm motility, and normal morphological rate were assessed. The fertilization rate and embryo development were measured by in vitro fertilization and embryo culture. Results The histological changes of testes in aged mice were mainly seminiferous tubule atrophy and hypospermatogenesis. In aged testes, a significant decline was .found in the numbers of round spermatids and elongated spermatids per Sertoli cell （P 〈0.01）. Sperm density, sperm motility and normal morphological rate in caput epididymis and cauda epididymis in aged mice significantly decreased （P〈0.05）. The fertilization rate and embryo development of aged group were lower than those in the control（P〈0. 01）. Conclusions Spermatogenesis and sperm functions could be maintained in the aging male. However, aging affects spermatogenesis and sperm maturation, which leads to lower the quality of sperm, including sperm fertilizing capacity. The development of embryo from aging sperm would have more abnormalities.
文摘50只体重为18～22 g的雄性昆明种小鼠随机分为5组（每组10只）,即溴氰菊酯（DM）1.8、3.6 mg/kg组,氯氰菊酯（CP）4.0、8.0 mg/kg组及色拉油对照组,连续1个月经口灌胃染毒后处死,测定主要脏器的脏器系数,检测小鼠精子运动参数、精子畸形发生率。结果显示：（1）DM两个剂量组小鼠脾脏的脏器系数与对照组比较明显增高（P〈0.05）;CP 8.0 mg/kg组小鼠脑的脏器系数与对照组比较却明显降低（P〈0.05）。（2）DM 3.6 mg/kg组平均直线运动速度（velocity of rectilinear motion,VSL）、平均路径速度（velocity of average pathway,VAP）、侧摆幅度（amplitude of oscillation,ALH）、运动的直线性（linearity of mo-tion,LIN）、运动的摆动性（wobble of motion,WOB）与对照组相比明显降低（P〈0.05）;CP 8.0 mg/kg组平均曲线运动速度（ve-locity of curve motion,VCL）、VSL、VAP与对照组相比明显降低（P〈0.05）;其余各剂量组精子运动参数与对照组相比,均无统计学意义。（3）各剂量组精子畸形率的增加与对照组相比无统计学意义。提示本实验条件下,Ⅱ型拟除虫菊酯对雄性小鼠脾脏、脑脏器系数和小鼠精子活力及运动方式有一定的影响。
