附睾蛋白的研究方法及进展

附睾是精子成熟,转运和贮存的场所,哺乳动物精子成熟是通过与附睾管腔微环境相互作用而实现的,精子通过在附睾内转运过程中与附睾蛋白相互作用获得运动和精卵识别结合能力,因而附睾蛋白的研究受到越来越多的关注.本文从不同的实验角度综述了附睾蛋白的研究方法及其进展.     

过氧化物酶6在大鼠附睾中的表达及分布

通过双向电泳结合质谱技术分离鉴定正常成年大鼠附睾头段与尾段管腔液中的蛋白组成,从附睾头段及尾段管腔液的22个差异蛋白点中鉴定出12个蛋白质.其中11个蛋白质在不同种属哺乳动物的附睾组织中已有鉴定报道,而过氧化物酶6(peroxiredoxin 6,Prdx6)为新发现的存在于附睾头段及尾段管腔液中的体液蛋白.采用RT-PCR、Western印迹及免疫组化技术,对该蛋白在大鼠附睾中的表达及分布进行了分析.实验表明,Prdx6与精子的成熟、贮存及保护有一定关系,其具体机制值得进一步深入研究.     

菲菊头蝠冬眠期的精子储存

菲菊头蝠是分布于亚热带和热带的翼手目种类,在中国大陆南部和海南岛有广泛分布。为探讨热带菲菊头蝠是否具有冬眠期(12 月至翌年2 月)储精现象及生殖腺组织结构的变化,对海南岛的菲菊头蝠成年雄蝠和成年雌蝠生殖系统在冬眠期间的变化进行了石蜡切片观察。结果显示:成年雄蝠在冬眠期间附睾内储存大量精子,推测其储存时间超过2 个月;冬眠期间曲细精管横截面积、精子细胞数量和间质细胞数量在冬眠期逐月显著性减少,精原细胞和精母细胞的数量在12 月与翌年1 月间均无显著性变化,而在冬眠末期的2 月显著增多。在雌蝠子宫和卵巢内均未发现精子储存现象,但卵巢内具有初级卵泡和次级卵泡,雄蝠在冬眠期间曲细精管逐月萎缩,但生精上皮精母细胞的数量在冬眠末期明显上升。     

狗施行附睾尾化学注射绝育后寒九附睾内的免疫复合物观察

５只性成熟成年雄性比格犬被施行附睾尾逆向注射１０％精氨酸锌０．５毫升。一般饲料饲养两月后处死作病理检查,发现精子在睾丸曲细精管内的生成及在附睾管内的成熟均受到影响,附睾尾管腔内精子降到了极少程度且多已变性。免疫组织化学反应发现睾丸间质及附睾管上皮间隙内,附睾间质中均有大量免疫复合物的沉积,而其它主要生命器官均未发现异常。结果表明,精氨酸锌附睾尾注射的绝育作用是肯定的,其伴随出现的抗精子抗体及免疫复合物仅对睾丸、附睾及生育功能产生影响,对机体主要生命器官并无损害。     

棕色田鼠睾丸及附睾胚后发育的形态学变化

通过组织学方法,对产后1 d、10 d、25 d、45 d、60 d及70 d的棕色田鼠Lasiopodomys mandarinus睾丸和附睾发育进行了观察,以探讨其精子发生特点.结果 发现,1 d棕色田鼠的生殖细胞主要是生殖母细胞和前精原细胞;10 d出现大量精原细胞,睾丸间质细胞明显;25 d出现精子细胞;45 d有少量精子出现;60 d和70 d具有各级生精细胞,睾丸生精小管和附睾内出现大量成熟精子.睾丸生精小管管径和生精上皮厚度随日龄增加,于60 d达到最大;附睾管腔直径和附睾上皮厚度也于60 d达到最大.这些结果表明,棕色田鼠在生后45 d左右进入青春期,60 d左右达到性成熟,精子的产生及成熟与附睾的发育同步.     

血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞顶体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和/或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

中国石龙子雄性生殖腺的年周期变化

20 0 1年 3月至 2 0 0 2年 2月期间 ,通过每月捕捉浙江丽水中国石龙子 (Eumeceschinensis)雄性成体 ,解剖动物、观测性腺的形态和组织学特征 ,研究雄性生殖周期。睾丸重量和体积、附睾重、输精管重和曲细精管直径有显著的季节变化。睾丸 3月份最重 ,5-9月最轻。睾丸体积和重量的年周期变化规律一致。附睾 3月份最重 ,8-9月份最轻。输精管 4月最重 ,8-10月最轻。生精活动始于 9月下旬 ,翌年 4月最为活跃。 3月下旬曲细精管直径达全年最大值 ,管腔中开始出现呈穗状排列的精子。从基膜到管腔 ,各级生精细胞依次排列。 4月份生精上皮的生精活动最为活跃 ,5月下旬生精活动已近停止 ,7-8月份曲细精管管壁仅由精原细胞 (其间夹有支持细胞 )构成。根据曲细精管生精上皮的年周期变化规律 ,中国石龙子 8月份睾丸生精活动处于第Ⅰ期 ,9月至次年 2月份第Ⅱ期 ,3月上、中旬为Ⅲ期 ,3月底至 4月为Ⅳ期 ,5-6月份为Ⅴ期 ,7月份为Ⅵ期。 4月下旬附睾管腔中有大量的成熟精子 ,7月附睾管腔中已无精子。中国石龙子属于关联型繁殖周期     

血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞项体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和／或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

合欢皮总皂苷对雄性小鼠的抗生育作用研究

目的探讨合欢皮总皂苷对雄性小鼠的抗生育作用。方法将60只健康的雄性性成熟昆明小鼠随机分成3个试验组和1个对照组,各试验组小鼠灌胃540 mg·(kg·d)-1,270 mg·(kg·d)-1和135 mg·(kg·d)-1的合欢皮总皂苷水溶液,对照组自由饮水,灌胃1周后按雌雄比2∶1合笼,继续对雄鼠灌胃至19 d,采集雄性小鼠的血液并测定血液生化指标,剖检灌胃雌性小鼠,统计雌性小鼠的怀孕率,测定雄性小鼠睾丸与附睾的脏器系数及附睾精子活力,并固定睾丸和附睾,研究其组织学变化。结果与对照组相比较,雄性小鼠灌胃合欢皮总皂苷后,导致雌性小鼠的怀孕率降低,睾丸脏器系数和附睾精子活率极显著降低(P<0.01),精子畸形率极显著升高(P<0.01)。光学显微镜下,试验组小鼠睾丸和附睾均有一定程度的损失,睾丸生精细胞排列疏松,间质减少,可见曲细精管内有多核巨噬细胞浸润,各级生精细胞脱落。附睾具正常的管腔结构,但能明显观察到管腔内精子几乎消失。结论合欢皮总皂苷对雄性小鼠具有抗生育作用,主要通过影响精子的生成及破坏生精组织而达到。     

薄荷油对雄性小鼠的抗生育作用研究

目的探讨薄荷油对雄性小鼠的抗生育作用。方法将60只健康的雄性性成熟昆明小鼠随机分成3个试验组和1个对照组,各试验组小鼠灌胃0.135 g/(kg·d),0.27 g/(kg·d)和0.54 g/(kg·d)的薄荷油0.5%羧甲基纤维素钠(sodium carboxymethylcellulose,CMC)混悬液,对照组灌胃0.5%羧甲基纤维素钠溶液,灌胃1周后按雌雄比2∶1合笼,继续对雄鼠灌胃至19 d,采集雄性小鼠的血液并测定血液生化指标,剖检灌胃雌雄小鼠,统计雌性小鼠的怀孕率,测定雄性小鼠睾丸与附睾的脏器系数及附睾精子活力,并固定睾丸和附睾,研究其组织学变化。结果与对照组相比较,雄性小鼠灌胃薄荷油后,导致雌性小鼠的怀孕率降低,睾丸脏器系数和附睾精子活率极显著降低(P<0.01),精子畸形率极显著升高(P<0.01)。光学显微镜下,试验组小鼠睾丸和附睾均有一定程度的损伤,睾丸间质减少,睾丸各级生精细胞排列疏松,曲细精管内有多核巨噬细胞浸润。附睾具正常的管腔结构,但能明显观察到管腔内精子减少。结论薄荷油对雄性小鼠具有抗生育作用,且主要通过影响精子的生成及破坏生精组织而达到,具有实际应用价值,可进一步开发成环境友好型鼠类抗生育药剂。     

Structural characteristics of the patagium of Ptychozoon kuhli (Reptilia: Gekkonidae) in relation to parachuting locomotion

Ptychozoon kuhli is known for its parachuting/gliding capabilities. In this contribution, we document the allometric scaling properties of its patagium, accessory flaps and folds, and its total body surface area and compare them to similar attributes of the agamine lizard Draco volans. Ptychozoon kuhli has passive patagia that lack skeletal support and muscular control. Patagial area in P. kuhli is smaller than that in D. volans for individuals of identical snout-vent length, but the accessory folds and flaps compensate for this shortfall and overall P. kuhli has equivalent total body surface area to D. volans for equally sized individuals. The structure of the patagium of Ptychozoon kuhli was investigated in terms of its scalation patterns and structural integrity, its relationship to the body wall and its mechanism of erection, and the distribution of mechanoreceptive sensilla across its surface. Scalation patterns and the internal architecture of the patagium indicate how its shape and form are maintained as it erects and becomes exposed to air flow. Its cross-sectional shape, together with that of the entire body indicates how it is able to behave as an airfoil. The distribution of sensilla across the patagial surface reflect positioning indicative of the monitoring of scale-to-scale contact on the dorsal surface, and possibly air pressure and flow on the ventral surface.     

Sperm storage in the female reproductive tract of Scotophilus heathii: role of androgen

The aim of the present study was to investigate the mechanism of androgen-mediated, prolonged sperm-storage in the female reproductive tract of the bat, Scotophilus heathii. The bat treated in vivo with flutamide, an androgen antagonist, showed loss of spermatozoa at the storage site, utero-tubal junction. Immunocytochemistry and Western blot analysis revealed the presence of increased expression of Bcl2 in the epithelial cell lining of the utero-tubal junction during the period of sperm-storage. Treatment with testosterone in vitro caused a significant dose-dependent increase in expression of the survival factor Bcl2, whereas treatment with flutamide together with testosterone caused a significant decline in Bcl2 in the utero-tubal junction of S. heathii. Together with the expression of Bcl2, the utero-tubal junction also expresses the death signal, caspase3. Expression of caspase3 decreased during January, but increased in February during the late stage of sperm storage. Androgen stimulated Bcl2 synthesis in the utero-tubal junction via the non-genomic MAP kinase signaling pathway. In conclusion, this study suggests that androgen promotes sperm storage in S. heathii by stimulating the survival factor Bcl2 in the utero-tubal junction. It is further hypothesized that a balance between the survival factor, Bcl2, and the death signal, caspase3, determines the duration of sperm storage in S. heathii.     

肪刺螨属一新种记述及中国已知种检索(中气门目,巨刺螨科)

记述采自海南的小黄蝠Scotophilus temmincki(Horsfield,1824)体表的寄生革螨1新种:吊罗肪刺螨Steatonyssus diaoluoensis sp.nov,.文中给出了中国肪刺螨亚属Steatonyssus 已知种检索表.研究标本保存于贵州大学昆虫研究所标本馆.     

Heritability of semen characteristics in dogs

Retrospective analysis was performed on semen collected from 24 dogs (parents: 14 Labrador retrievers and 10 Golden retrievers) aged between 16 and 28 months of age. The dogs were part of a large breeding programme but lived in the homes of volunteer families. The semen was subjected to a standardised examination procedure including assessment of: percentage normal motility, sperm concentration, total sperm output, percentage of live normal sperm, and total number of live normal sperm. Semen was subsequently collected from one son of each of the parents when the offspring were aged between 16 and 28 mo (offspring: 14 Labrador retrievers and 10 Golden retrievers), and was subjected to the same examination procedures conducted by the same technician. Examination of breeding records demonstrated that each of the 48 dogs achieved at least one pregnancy within a period of 3 months before to 3 months after the semen collection.There was a weak correlation between parents and offspring for each of the 5 semen parameters, although none of these were statistically significant. Narrow sense heritability measures were low for all parameters except for the heritability of high sperm motility (rN2 = 0.57) and the heritability of low total sperm output (rN2 = 0.57).It is plausible that breeding selection procedures may be useful in dog breeding programmes in an attempt to improve semen quality, although any impact upon fertility is yet to be proven.     

Comparison of assessment of pigeon sperm viability by contrast-phase microscope (eosin-nigrosin staining) and flow cytometry (SYBR-14/propidium iodide (PI) staining) [evaluation of pigeon sperm viability

The aim of these experiments was to compare the conventional, microscopic method of evaluating pigeon sperm viability to sperm assessed by flow cytometry. Semen was collected twice a week from two groups of pigeons. In every group were 20 males (Group I: meat-type breed; Group II: fancy pigeon breed). Semen was collected using the lumbosacral and cloacal region massage method. Ejaculates collected from each group were pooled and diluted to 10 × 10⁶ sperm/ml in BPSE solution. Samples were divided into three equal parts and estimated after collection as well as after in vitro storage for 3, 6 and 24 h. The first part was using for semen motility evaluation. The proportion of motile spermatozoa (MOT) and progressive movement (PMOT) of fresh and stored semen were evaluated using the CASA-system. The second part was examined subjectively by microscope (eosin-nigrosin (EN), eosin-nigrosin staining), the third one was assessed using dual fluorescence SYBR-14/propidium iodide (PI) and flow cytometry (FC). There were not any significant differences in sperm viability and motility between the groups at 0, 3, 6, and 24 h post collection. The percentage of viable spermatozoa in fresh semen determined by EN and FC was not different in Groups I and II (I - 88.71 ± 5.42 and 84.01 ± 3.19, respectively; II-90.87 ± 6.01 and 87.38 ± 5.57, respectively). Significantly lower percentages of viable spermatozoa were detected by FC compared to the EN method in both groups after 6 h (P ≤ 0.05) as well as 24 h (P ≤ 0.01) of storage. Moreover, the dual fluorescent SYBR-14/PI staining allowed for the identification a third population of double stained, moribund spermatozoa. High positive correlations in percentage of live spermatozoa were noted between EN and FC methods in both groups of birds. Evaluation of sperm viability by FC is a rapid, accurate, sensitive, and objective method for the assessment of pigeon sperm viability in fresh as well as stored semen.     

Semen characteristics after vasectomy in the ram

The objective of this study was to monitor the changes in semen characteristics in vasectomized rams and to determine if infertility was present 14 days after vasectomy. Experiments were performed using five cross-breed rams, aged between 18 and 30 months. Semen was collected weekly by artificial vagina from 2 months before to 5 months after vasectomy. After sexual rest for 10 days, vasectomy was performed by the cranial midscrotal approach. In all ejaculates the volume, concentration, total sperm number, motility and morphology (normal spermatozoa, loose heads) were determined and sperm viability (SYBR-14/PI) was evaluated in all semen samples collected after vasectomy. In the first ejaculate obtained 14 days post vasectomy all rams showed a significant (P < 0.05) drop in mean volume (from 1.2 to 0.5 mL), total sperm count (from 5176.8 to 51.1 x 10(6)) and morphologically normal sperm (from 84.1 to 15.7%), when compared to the last prevasectomy collection. We could also demonstrate a positive correlation (r = 0.89) between the individual cumulative total number of spermatozoa after vasectomy and the scrotal circumference measured before vasectomy. Sperm motility and viability could never be demonstrated after vasectomy and normal spermatozoa continuously decreased concomitant with an increase in loose heads. On post mortem examination 5 months after surgery, spermatocele formation and multiple sperm granulomas were present in all five rams. Our results show that in the first ejaculate collected by artificial vagina 14 days after vasectomy, no motile and viable spermatozoa could be detected. Despite weekly collections during a 5-month period after sterilization, azoospermia could never be achieved.     

The interactions between spermatozoa and uterine epithelium in the hibernating bat,pipistrellus kuhli natt

In hibernating females of Pipistrellus kuhli, as well as in other bats previously described, the spermatozoa inseminated in autumn are stored in the uterus, both free and attached by their heads to the uterine epithelium, throughout winter. With the electron microscope the apical region of the attached sperm heads appears to be contained in plicae of the endometrial cells. Furthermore, a great number of microvilli protruding from the uterine epithelium participate in the interaction with spermatozoa. In the region of contact between the two cell types, their plasma membranes run parallel with a constant 12-nm gap. The sensitivity of these interactions to EDTA and hyaluronidase suggests an involvement of Ca⁺⁺ and proteoglycans in these bindings, while their insensitivity to trypsin tends to exclude a role of proteins. In addition, evidence of exocytosis of dense granules just at the bottom of the plicae containing the spermatozoon suggests a transfer of material from the uterine epithelium to the intercellular space, thus supporting a physiological role of these interactions.     

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.     

圈养黑熊精液的冷冻保存

将采自23 头成年圈养黑熊的精液,分别用3 种稀释液(Ⅰ:Tris - 乳- 果- 卵;Ⅱ:柠- 葡- 蔗- 卵;Ⅲ:Tris - 柠- 果- 葡- 卵)稀释并在4℃ 下保存,通过检测精液在不同稀释液稀释条件下的保存时间,筛选出最适稀释液用于精液的冷冻保存;从精液解冻后精子的活率、活力、畸形率、顶体完整率4 个指标,分别从3种冷冻保护剂(甘油3%、3.5% 、4% )、两种冷冻方法(两步冷冻法和自动冷冻法)两个方面进行了比较试验。结果表明:精子活力在0.3 以上时,稀释液Ⅲ保存时间为175.42 ± 3.04 h,显著高于稀释液Ⅰ和稀释液Ⅱ(P ﹤0.01),稀释液Ⅱ保存时间也明显高于稀释液Ⅰ (P ﹤0.01);含3.5% 甘油浓度的稀释液解冻后精子活率(41.75 ± 3.46% )、活力(32.63 ±5.27% )和顶体完整率(85.62 ± 4.58% )显著高于其他两组(P ﹤0.01),并且精子畸形率(29.32 ± 8.22% )明显低于其他两组(35.95 ± 8.04% ,36.07 ±7.72% )(P ﹤0.01);采用自动冷冻法冷冻保存圈养黑熊精液,解冻后精子活率、活力和顶体完整率分别为41.75 ±3.46% 、32.63 ± 5.27%和85.62 ±4.58% ,都明显高于两步冷冻法(P ﹤0.01);解冻后畸形率为29.32 ± 8.22% ,明显低于两步冷冻法(P ﹤0.01)。