Objective To explore the effects of cryovial vector on the cryosurvival of human spermatozoa and optimize the protocol of cryopreservation of human semen. Methods Semen samples were obtained from 20 healthy volunteer donors, and each one was aliquoted equally and transferred into the experimental cryotube wrapped with cotton (the experiment group) and the non-treated cryotube (the control group) respectively. All control and post-thaw samples were assessed by a computer-assisted semen analyzer(CASA) with a phase contrast microscope for determination of sperm motility parameters. Some statistical methods were used for sperm motility parameter comparison between the control and the experimental group. Results The recovery rate of sperm motility (66.71±6.51)% in the experimental group wassignificantly higher than that of the control group (59.67 ±7.74)%, (P<0.01). Conckution The cryotube wrapped with cotton showed some protective effects on cryosurvival of sperm. Therefore, it might be critical to design a special storage instrument for sperm cryopreservation.

目的探讨精液冷冻管承载工具对人类精液活动力及复苏率的影响,优化精子冷冻保存的方案.方法取本库20例健康自愿供精的精液,同一例供精者的精液分成两份:实验组冷冻管用棉花包裹,对照组不做任何处理.通过相差显微镜镜检,利用计算机辅助精液分析(CASA)系统分析两组冷冻前后人类活动精子的运动参数,并作相关的统计学分析及比较.结果实验组冷冻复苏率为(66.71±6.51)%,对照组冷冻复苏率为(59.67±7.74)%(平均提高了7.04%)(P<0.01).结论冷冻管包裹棉花的冷冻前处理对精子有一定的保护作用,因而冷冻管承载工具的专门设计对精液冷冻有实质性意义.

OBJECTIVE To analyze the distribution characteristics of the main semen parameters of healthy semen donors and normal fertile men in Shanghai, compare the semen quality between the two groups, and investigate the normal reference values of the semen parameters of the fertile population in Shanghai. METHODS We obtained semen samples from 100 healthy donors and 41 fertile men, performed semen analyses according to the WHO (2010) guidelines, and determined the semen volume, sperm concentration, sperm progressive motility, total sperm count and total progressively motile sperm count. We analyzed the distribution of the semen parameters of the normal fertile men, and obtained the lower limits of their normal reference values. RESULTS There were no statistically significant differences in the main semen parameters between the healthy donors and normal fertile men (P < 0.05). The lower reference limits for the semen parameters of normal fertile men in Shanghai (P < 0.05) were as follows: sperm concentration > or = 27.3 x 10(6)/ml, sperm progressive motility > or = 8.1%, semen volume > or = 0.82 ml, total sperm count > or = 44.73 x 10(6) per ejaculate, and total progressively motile sperm count > or = 24.68 x 10(6) per ejaculate. CONCLUSION For the evaluation of male fecundity, total sperm count and total progressively motile sperm count may be two better predictors than others.

Objective To investigate the effects of vapor nitrogen freezing method and direct liquid nitrogen freezing method on the motility,morphology and ultrastructure of post-thaw human sperm.Methods Twenty-five semen samples from semen donors were selected,and glycerol-yolk-sodium citrate(GYC) was added as cryoprotectant with the proportion of 1∶ 1.Each semen sample was equally distributed to two cryovials,and were cryopreserved with vapor nitrogen freezing method and direct liquid nitrogen freezing method respectively.The total motility rate,progressive motility rate and motility parameters of post-thaw sperm were examined by IVOS sperm analyzer.The morphology of post-thaw sperm was observed with papanicolaou staining,and the rate of post-thaw sperm with normal morphology was calculated.Besides,the ultrastructure of post-thaw sperm was observed by transmission electron microscope.Results It cost 468 s to drop in temperature from 28 ℃ to-185 ℃ by vapor nitrogen freezing method,and an hour later the temperature went to-196 ℃.However,it only cost 90 s to drop in temperature from 30℃ to-196 ℃ by direct liquid nitrogen freezing method.The total motility rates,progressive motility rates,motility parameters and rates of post-thaw sperm with normal morphology in two cryopreservation methods were significantly lower than those before cryopreservation(P0.05).The revival rate,total motility rate and progressive motility rate in vapor nitrogen freezing method were significantly higher than those in direct liquid nitrogen freezing method(P0.05).There was no significant difference in motility parameters and rates of post-thaw sperm with normal morphology between two cryopreservation methods(P0.05).Transmission electron microscopy indicated that no obvious damage was found on the ultrastructure of serosa and acrosome membrane at the head of post-thaw sperm in two different cryopreservation methods by using GYC as cryprotectant Conclusion GYC has a protective effect on the ultrastructure of sperm.Vapor nitrogen freezing method may yield better post-thaw results than direct liquid nitrogen freezing method in cryopreservation of human sperm.

OBJECTIVE:To study the effect of direct fumigation on the post-thaw recovery rate of cryopreserved spermatozoa, and to search for a best method for human sperm cryopreservation. METHODS:We collected semen samples from 100 donors conforming to the normal reference values in WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed), divided them into two groups, and subjected them to cryopreservation by programmable freezing (Group A) and direct fumigation (Group B), respectively. We detected the progressive motility of pre-freezing and post-thaw sperm with a computer-assisted semen analyzer, and compared the effects of the two methods on the functional integrity of sperm membrane and the rate of abnormal sperm using the percentage of hypo-osmotic swelling sperm and modified Papanicolaou staining. RESULTS:Statistically significant differences were found in post-thaw sperm progressive motility between the Groups A and B ([34.0 +/- 18.4]% vs [43.0 +/- 19.5]%, P<0.05), both remarkably decreased as compared with pre-freezing ([57.0 +/- 16.7]%, P<0.05). Such differences were also found in the post-thaw recovery rate of progressively motile sperm between the two groups ([52.2 +/- 20.6]% vs [67.1 +/- 20.0]%, P<0.05). The post-thaw percentage of hypo-osmotic swelling sperm was obviously decreased in both Groups A and B ([67.1 +/- 11.1]% and [70.6 +/- 10.0]%) in comparison with pre-freezing ([84.5 +/- 7.5]%, P<0.05), with significant differences between A and B (P<0.05). However, the rate of sperm abnormality was evidently increased in Groups A and B ([85.0 +/- 8.7% and [85.7 +/- 9.1]%), significantly higher than pre-freezing ([77.8 +/- 9.6]%, P<0.05), but with no significant differences between A and B (P>0.05). CONCLUSION:Direct fumigation is superior to programmable freezing for its easier operation, wider application, and higher sperm recovery rate.

AIM To study the expression pattern of the retinoic acid metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development at both mRNA and protein levels. METHODS Real-time polymerase chain reaction and Western blot analysis were performed to determine the relative quantity of RALDH2 and CYP26b1 at both mRNA and protein levels at postnatal day 1, 5, 10, 20, and in adult mice (70 days testes). Testicular localization of RALDH2 and CYP26b1 during mouse postnatal development was examined using immunohistochemistry assay. RESULTS Aldh1a2 transcripts and its protein RALDH2 began to increase at postnatal day 10, and remained at a high level through postnatal day 20 to adulthood. Cyp26b1 transcripts and CYP26b1 protein did not change significantly during mouse postnatal testis development. RALDH2 was undetectable in the postnatal 1, 5 and 10 day testes using immunohistochemistry assay. At postnatal day 20 it was detected in pachytene spermatocytes. Robust expression of RALDH2 was restricted in round spermatids in the adult mouse testis. In the developing and adult testis, CYP26b1 protein was confined to the peritubular myoepithelial cells. CONCLUSION Our results indicate that following birth, the level of retinoic acid in the seminiferous tubules might begin to increase at postnatal day 10, and maintain a high level through postnatal day 20 to adulthood.

OBJECTIVE To investigate the correlation of exogenous estrogens with the expression of FasL in Sertoli cells and the blood-testis barrier during the differentiation and maturation period of Sertoli cells, and to discuss the related factors that influence the blood-testis barrier of pubertal rats. METHODS Super-physiological doses of exogenous estrogenic compounds (diethylstilbestrol and estradiol) were administered to pubertal Sprague-Dawley rats in vitro and in vivo, the FasL expression in the Sertoli cells of the rats detected by immunohistochemistry and Western blot, and the changes in the blood-testis barrier observed with the electron microscope. RESULTS After the exposure to exogenous estrogens, the FasL expression was markedly up-regulated in the immature Sertoli cells (P < 0.05) as well as in the Sertoli cell membrane and the blood-testis barrier of the epithelium. The tracer lanthanum passed through the blood-testis barrier and reached the whole layer of the epithelium at 18 days. CONCLUSION Super-physiological dose of exogenous estrogens can change the expression and distribution of FasL in immature Sertoli cells and affect the structure of the blood-testis barrier.

应用金霉素(CTC)荧光检测、精子穿卵试验和顶体酶β-D-半乳糖苷酶活力测定等方法检测冻存对大鼠精子顶体的影响.CTC荧光反应显示,与未冻存精子比较,冻存后精子顶体反应的类型发生改变,AR型(发生了顶体反应)精子比例明显下降,冻存前为68.6%,冻存后为13.4%,但是获能精子的比例未发生明显变化(92.6%对90.8%).与未冻存的精子相比,冻存组精子的穿卵试验的受精指数下降(23.4±7.02对10.2±3.95),而且冻存后精子顶体酶--β-D-半乳糖苷酶活力明显下降(9.39±1.98对4.50±1.40).结果表明冻存后精子的顶体功能受到较明显的破坏.实验结果为今后针对性地改进大鼠精子的冻存方法,改善大鼠精子的冻存保护剂提供实验和机制上的依据.

Objective: To study the relationship among the motility,the membrane integrity,acrosomal reaction and the mitochondrial function of cryopreserved spermatozoa from rat.Methods:(1)The membrane integrity of frozen spermatozoa was determined with FDA fluorescence staining. (2)The structure of rat sperm mitochondrial sheath was checked with rhodamine fluorescence staining.(3)The effect of cryopreservation on the acrosomal reaction of spermatozoa was analyzed with chlortetracycline (CTC) staining. (4)The motibility of both the unfrozen and frozen spermatozoa was determined with computer aided sperm assay.Results: After being cryopreserved,the cytoplasmic membrane of rat spermatozoa was intact,but the rate of acrosomal reaction reduced significantly,and the structure of mitochondrial sheath damaged. The motibility of the frozen spermatozoa decreased significantly.Conclusion: Cryopreservation impaired the function of acrosome and the mitochondria of rat spermatozoa,while there was no impairment on the cytoplasmic membrane. The relationship among the mitochondrial function,acrosomal reaction and the motility of rat spermatozoa need to be further studied,which will be useful to improve the cryopreservation protocol and cryoprotectant components for rat spermatozoa.

目的冻存未受精的成熟卵母细胞(MII期)比冻存同一品系的处于卵裂期的胚胎更困难.因为卵母细胞的某些内部结构对低温很敏感.本研究的目的是希望能够更有效地观察冻存对卵母细胞的影响.方法我们运用激光扫描共聚焦显微术(CLSM)的光学切片和三维图像重建技术,研究冻存对自发性高血压大鼠(SHR)卵母细胞的结构,尤其是对纺锤体、染色体的影响,以及冻存对体外受精的影响.结果我们不仅观察到有的卵母细胞冻存后仍维持着完整的桶状纺锤体以及紧密排列的染色体,而且能观察到卵母细胞内均匀分布着细胞质微管星体.冻存也造成对一些卵母细胞纺锤体和细胞质微管星体的破坏.结论 CLSM能够清晰有效地观察冻存对未受精卵纺锤体、染色体以及胞质微管星体的影响,我们推想冻存后卵母细胞的体外受精率下降与这些结构的受损有关.

目的;研究雌激素对大鼠附睾上皮细胞非基因组作用及其机制.方法:青春期大鼠附睾上皮细胞,FLUO-3/AM标记,激光共聚焦显微镜技术观察.结果;17β-雌二醇能快速诱导附睾上皮细胞内Ca2+[Ca2+]升高,并具有剂量依赖性(10-2～10-9M);无Ca2+液及2mM EGTA孵育附睾上皮细胞不影响雌二醇诱导效应;Ca2+通道阻滞剂nifedipine、verapamil也不影响雌二醇诱导效应;而细胞内Ca2+储器释放抑制剂neomycin能完全抑制雌二醇诱导效应.雌激素核内受体阻滞剂RU-486、mRNA转录抑制剂放线菌素D、蛋白质合成抑制剂放线菌酮等对雌二醇的效应均无作用.结论:雌激素以非基因组方式调控青春期大鼠附睾上皮细胞功能.

本工作报道了血管内照射(650nm,5mW,cw)对家兔尾核、下丘脑DA和NE的影响,发现DA在尾核和下丘脑中均有下降现象,但均P＞0.05.在大鼠光照"扶突"穴中亦发现DA在脑干和尾核中,亦有下降倾向,NE有脑干和尾核中则有上升倾向.5-HT则均下降,但亦均未达显著性差异,(P＞0.05).另发现在人体血管内照射时,血液中NO.含量随照射次数增加而递减倾向.这些变化作者认为与光照的光化学反应引发的神经体液性调控有关.在本工作中还发现光照后神经未梢中的线粒体有肿胀现象.

为探讨低强度激光产生生物效应的机制,采用半导体激光器(波长650 nm,功率10mW,连续可调),对雄性SD大鼠和自发性高血压大鼠进行颈部"扶突"穴穴位照射,用高效液相色谱电化学检测方法测脑干和尾核中五羟色胺、去甲肾上腺素和多巴胺等单胺类神经递质含量,用TBA荧光法测定血浆中丙二醛(MDA),同时用氢清除法组织血流仪监测大鼠海马区的脑血流量.结果提示,光照后,海马脑血流量下降,脑干和尾核中单胺类神经递质含量明显下降,而血浆中MDA含量略有上升.揭示激光照射"扶突"穴会引起单胺类神经递质下降.