小鼠睾丸、附睾和前列腺组织细胞外囊泡蛋白质组分的鉴定及功能分析

【目的】对睾丸、附睾和前列腺组织细胞外囊泡(extracellular vesicles,EVs)蛋白质组分进行鉴定,以明确其在精子发生、成熟及功能维持方面所发挥的作用。【方法】用总外泌体分离试剂盒 (total exosome isolation,TEI)分别富集小鼠睾丸、附睾和前列腺组织的EVs,用免疫印迹法对EVs进行鉴定。用透射电镜观察EVs的形态,动态光散射粒径分析仪(dynamic light scattering,DLS)分析其直径分布。用液相色谱串联质谱法(LC-MS/MS)分析EVs的蛋白质组分,运用DAVID 6.8在线分析工具从细胞组分、分子功能、生物学过程三方面对筛选出的蛋白质进行基因本体(gene ontology,GO)富集分析。【结果】用TEI法分离纯化的EVs均可检测到分化抗原63(cluster differentiation antigen 63,CD63)和肿瘤易感基因101(tumor susceptibility gene 101,TSG101)标记蛋白,表明EVs分离成功。透射电镜下观察EVs呈凹杯状,睾丸组织来源的EVs以直径30~140 nm的居多,附睾组织EVs直径在105 nm处有峰值,前列腺组织EVs直径在50和90 nm处有峰值。用LC-MS/MS分析3种EVs的蛋白组分,睾丸组织EVs筛选到1 148种蛋白,附睾组织EVs筛选到902种蛋白,前列腺组织EVs筛选到651种蛋白。睾丸组织EVs特有蛋白有667种,主要参与RNA加工及剪切、精子发生、DNA损伤应答等生物学过程;附睾组织EVs特有蛋白有311种,主要参与转运、代谢和免疫作用等生物学过程;前列腺组织EVs特有蛋白有142种,主要参与代谢活动等。三者共有蛋白277种,主要发挥蛋白质结合、RNA结合等分子功能,参与蛋白质翻译和运输等生物学过程。【结论】睾丸、附睾和前列腺组织EVs分别在精子发生、精子的睾丸后成熟及功能维持中发挥一定的作用。     

Evaluation of gonadal macroscopic and microscopic morphometry reveals precocious puberty in Bísaro pig

Bísaro pig (BP), like many other local breeds reared in extensive non-industrial systems, faces many constraints to comply with the EU welfare regulation, particularly regarding the restrictions to surgical castration. In order to adapt an immunocastration protocol to overtake this issue, a puberty timeline is needed. Using gonadal morphometry data from 91 young male BP, this study intended to characterize testicular development, describing the prominent cell types and structures, to ultimately assess the age at puberty in male BP through a mixed prediction model. As expected, the relations between several macro and microscopic parameters and their relation with age were as described within the literature. Post-pubertal animals have larger and heavier gonads, lower Sertoli cell density/tubule, higher Leydig cell density and larger seminiferous tubules. Meiosis was firstly seen in 44-day-old animals, elongated spermatids in 70-day-old animals. Complete spermatogenesis was firstly identified in a 90-day-old animal. Spermatozoa were present in the epididymis of 23 animals, aged from 70 to 240 days old, and in the vas deferens of 14 animals (105 to 240 days old). The prediction model inferred that male Bísaro pigs reach puberty between 14 and 17 weeks (3 and 4 months old) and become sexually capable from 15 to 19 weeks (3.5 and 4.4 months old). These parameters confirm the sexual precocity of this breed.     

绵羊附睾尾部精液低温保存效果的研究

由成年绵羊离体睾丸上获得附睾尾部精液,进行3种浓度稀释液(葡萄糖、柠檬酸三钠、卵黄含量分别为:Ⅰ组1 5g,0 7g,10ml;Ⅱ组0 4g,1 4g,10ml;Ⅲ组1 5g,0 7g,4ml)低温保存(4℃)效果的观察,并对低温保存前精液的处理方法进行了比较。结果表明:Ⅲ组绵羊精液低温保存总存活时间和存活指数均好于Ⅰ、Ⅱ两组(P<0 01;P<0 05);Ⅲ组保存前经上游处理的精液在总存活时间、存活指数和体外受精效果均明显高于未经上游处理的精液。     

GPx6在大白猪附睾细胞中的表达及其与繁殖性能的相关性

本试验旨在研究谷胱甘肽过氧化物酶6（glutathione peroxidase 6,GPx6）在大白公猪附睾细胞中的表达和定位,并探究其与公猪繁殖性能的相关性。选取15月龄的公猪和母猪各3头,取睾丸、附睾、前列腺、尿道球腺、精囊腺、卵巢和输卵管,提取蛋白,通过蛋白免疫印迹和免疫组化（IHC）检测组织和细胞中GPx6蛋白的表达和定位;根据评定大白公猪受精能力的数学模型,按照公猪配种胎次≥20胎、3次配种公猪为同一头的标准,筛选并采集符合要求的20头公猪精液,同时,统计相对应的1 279头母猪的生产成绩,计算得到公猪繁殖性能指标（包括窝产总仔数、窝产活仔数、分娩率和繁殖力）。提取精子蛋白和精清蛋白,通过BCA和ELISA检测精子和精清中GPx6蛋白的含量。使用SPSS软件的独立样本t检验及单因素方差分析（one-way ANOVA）,进行差异显著性分析,用双变量Pearson进行相关性分析,P<0.05表示差异或相关显著。结果表明,GPx6蛋白在附睾中高表达,IHC结果显示,GPx6蛋白在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达,在肌样细胞中不表达;精清蛋白中GPx6的含量是精子蛋白的7倍,精液中GPx6蛋白的含量与窝产活仔数、窝产总仔数呈负相关关系。结果提示,GPx6在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达,且其在精液中的含量会影响公猪的繁殖性能,这为GPx6对公猪受精能力影响的研究奠定了理论和试验基础。     

冬眠中华鳖雌、雄生殖道的精子储存

应用光镜和电镜技术,系统观察冬眠中华鳖(Trionyx sinensis)雌、雄生殖道的精子储存情况,显示与精子储存相关的形态结构和细胞特征。结果表明,中华鳖在冬眠期生精活动处于相对静止状态,仅有1～3层精原细胞排列在睾丸曲细精管基膜附近,其余生精细胞排列松散而紊乱,生精上皮中未见有各级细胞规律性排列,不能形成完整的管壁。附睾管腔增大,腔中储存有大量精子。附睾上皮及附睾管腔中见有PAS反应阳性物质。附睾上皮主要由主细胞、亮细胞和基细胞组成。超微结构显示,附睾上皮细胞中含有大量分泌颗粒,内质网膨大。储存在附睾中的精子结构完整,精子中段由35～40个同心圆状线粒体构成线粒体鞘,中段胞质中含有大量糖原颗粒。雌性输卵管分布着数量不等的精子,尤其狭部最多。狭部黏膜皱襞高度融合,形成大量纵行于输卵管的储精细管。在细管中含有大量精子,精子头部嵌入上皮细胞纤毛中。蛋白部和子宫部也有少量精子存在,但在这些部位不能形成明显的储精细管。PAS反应显示,输卵管狭部储精细管上皮分泌有糖蛋白类物质。储精细管上皮下固有层中分布有大量的管状腺,通过腺导管与输卵管管腔相通。雌、雄中华鳖隔离4个月(12月初至来年3月底)后,在雌性输卵管峡部的储精细管中仍可观察到大量结构完整的精子,表明中华鳖精子在输卵管中至少可以储存120 d。以上结果显示,在冬眠期,雄性附睾和雌性输卵管中有大量精子储存,储存的精子能够渡过漫长的冬季用于来年春天交配或受精,这一特殊的生殖策略对其成功繁殖具有重要意义。本研究还对中华鳖的繁殖特性进行了讨论。     

小鼠生后不同发育阶段附睾组织中Crb3的表达

应用HE染色法和免疫荧光组织化学技术分别对小鼠生后不同发育阶段附睾组织结构及Crb3在不同发育时期附睾组织中的定位表达进行了研究。结果显示,附睾在4周龄时上皮为2层～3层的假复层纤毛柱状细胞,顶端纤毛细长;6周龄上皮细胞层数减少至1层～2层;8周龄附睾管上皮厚度增至最大,上皮细胞呈单层高柱状;12周龄后附睾管上皮开始变薄,呈单层柱状。免疫荧光染色结果显示,Crb3蛋白主要分布在附睾上皮柱状细胞的胞膜,在精子尾部有微弱的表达,这提示了Crb3不仅与附睾上皮细胞的极性建立和维持有关,而且对精子活力和血-附睾屏障的形成也可能具有潜在的影响。     

Dose-dependent Response to an Intratesticular Injection of Calcium Chloride for Induction of Chemosterilization in Adult Albino Rats

This study concerned the minimum and optimum effective doses of calcium chloride needed for induction of chemosterilization in male albino rats, 30 days after a single intratesticular injection of calcium chloride (CaCl₂.2H₂O) solution at 2.5, 5, 10 or 20 mg per 100 g body weight per testis. There was a significant diminution in the relative wet weight of the sex organs (p<0.01), epididymal sperm count (p<0.001), plasma concentration of testosterone (p<0.01), testicular activities of D,3-hydroxy steroid dehydrogenase (D,3-HSD), 17-hydroxy steroid dehydrogenase (17-HSD) (p<0.01), glutathione S-transferase (GST) (p<0.01), superoxide dismutase (SOD) (p<0.01), and peroxidase (p<0.01), significant elevations in testicular content of malondialdehyde (MDA) and conjugated dienes (p<0.01), along with derangement of seminiferous tubular architecture and degeneration of the Leydig cells in the testis and elevations in the concentrations in the plasma of LH and FSH (p<0.01), commencing at a dose of 5 mg, with the greatest effects at a dose of 20 mg. No significant alterations in these factors occurred at the dose of 2.5 mg in comparison to the control that received only the vehicle. There was no significant alteration in the plasma concentrations of prolactin (p>0.05), corticosterone (p>0.05) or fasting blood glucose or in the rectal temperature (p>0.05) at any of the doses relative to the control group, suggesting that this chemosterilizing procedure did not exert any chronic stress on the experimental animals. From these observations, it may be suggested that 5 mg should be considered as the minimum dose, and 10 mg or 20 mg as the optimum dose, whereas 2.5 mg was ineffective for induction of chemosterilization. There would seem to be little point in using more than 20 mg of calcium chloride for this purpose. Intratesticular injection of calcium chloride at an effective dose may be considered as an alternative to surgical castration.     

哺乳动物附睾特异GPx5功能及其调节机制的研究进展

精子在附睾中的成熟过程是哺乳动物雄配子获得受精能力的关键。谷胱甘肽过氧化物酶-5（glutathione peroxidase-5,GPx5）作为附睾特异性表达的抗氧化酶具有强抗氧化作用,可调节附睾微环境中的活性氧浓度,保护精子免受脂质过氧化损伤,以维持精子DNA完整性。GPx5还可能对精子活力和顶体反应产生一定影响。GPx5基因的染色体定位及其外显子数存在一定的种间差异,其在不同物种、部位和发育期的表达具有特异性,受雄激素、PEA3因子和ETV4家族等调节。作者就哺乳动物附睾特异GPx5基因的结构与定位、表达特性、功能及其调节机制的研究进展进行综述,以期为进一步研究精子抗氧化机制和提高雄性动物繁殖力提供理论依据。     

Immunohistochemical study of galectin-3 in mature and immature bull testis and epididymis

Galectin-3, a member of the β-galactoside-binding protein family, has been implicated in mammalian sperm maturation. We examined galectin-3 expression in the testis and epididymis of sexually mature and immature bulls. Western blot analysis showed varying levels of galectin-3 in the bull testis and epididymis, and galectin-3 immunoreactivity was higher in the mature testis and epididymis than in immature organs. Galectin-3 was primarily localized in interstitial cells of the immature bull testis and in the peritubular myoid and interstitial cells of the mature testis. In the immature epididymis head, galectin-3 was primarily in the principal and basal cells of the epithelium. In the mature epididymis head, moderate levels of galectin-3 were detected in the sperm, while low levels were found in the stereocilia, epithelium and connective tissue. In the immature epididymis body, moderate protein levels were detected in the principal cells, while lower levels were found in the basal cells. The mature epididymis body showed moderate levels of galectin-3 immunostaining in the stereocilia and epithelium, but low levels in the connective tissue. In the immature epididymis tail, only low levels of galectin-3 staining were found in the epithelium, whereas the mature epididymis tail showed high levels of galectin-3 in the principal cells, moderate levels in the basal cells and low levels in connective tissue. These findings suggest that galectin-3 expression plays a role in the maturation and activation of sperm in bulls.     

GPX5在成年绵羊附睾中的表达与蛋白定位

【目的】研究谷胱甘肽过氧化物酶-5（glutathione peroxidase type-5, GPX5）在成年绵羊附睾的表达特征以及GPX5蛋白在附睾中的定位,为绵羊精子在附睾中抗氧化机制的研究提供理论依据。【方法】采取年龄相近的成年蒙古绵羊的附睾、睾丸和输精管。每一只公羊的附睾分别按照附睾头、附睾体和附睾尾进行分割。样品保存于-80 ℃冰箱,采用实时荧光定量PCR和Western blotting的方法,对成年绵羊的附睾、睾丸和输精管的GPX5 表达量进行分析。将新鲜的组织样品各部分切取适合大小浸泡于4%多聚甲醛溶液中24h,按照石蜡切片的方法制做组织切片。用GPX5特异性抗体孵育组织切片,利用DAB显色试剂盒对阳性信号进行标记。【结果】实时荧光定量PCR结果显示,成年绵羊附睾头GPX5基因的表达量极显著高于附睾体和附睾尾（P＜0.01）,而在输精管和睾丸几乎不表达。Western blotting的结果显示,GPX5蛋白在成年绵羊附睾头高表达,在附睾体和附睾尾有微量蛋白存在,睾丸和输精管中未发现GPX5蛋白。这表明GPX5主要在成年绵羊附睾头表达;经过免疫组化染色分析,GPX5蛋白主要位于附睾头上皮细胞质以及静纤毛,附睾体和附睾尾中的GPX5蛋白主要集中在静纤毛。在附睾头、附睾体和附睾尾中的精子上以及附睾腔都可以观察到GPX5蛋白,表明GPX5蛋白从附睾上皮分泌到附睾腔中,随着精子在附睾中的运输与精子结合。【结论】在成年绵羊附睾中,GPX5主要由附睾头上皮细胞合成和分泌,在附睾管腔中与精子结合,为精子功能的正常发挥提供保护。