精子发生中miRNAs调控功能研究进展

<正>源源不断的精子发生过程是雄性繁殖机能的基础。精原干细胞(SSCs)持续不断地自我更新和分化又是精子发生的基础。精子发生起始于精原干细胞的分化,历经漫长的减数分裂后形成单倍体圆形精细胞。后者经过变态发育过程,最终形成精子并释放到曲细精管管腔中~([1])。研究表明,成千上万的基因参与了精子发生过程,然而如何实现对这     

貉精子发生的研究

本文应用光镜和电镜技术,研究了雄貉生殖细胞,支持细胞的细胞学变化及发育规律。结果表明,貉生精细胞的细胞结构和精子发生与其它哺乳动物相似,从精子细胞经过四个阶段的变态而形成分化完善的精子,支持细胞内微管参与生精上皮的构筑,精子细胞的运动和释放,在附睾内随着精子的迁移,胞质脂滴逐渐减少,同时在附睾内有假顶体反应精子。     

哺乳动物精子发生过程中的基因表达

在精子发生过程中,体细胞表达的结构性基因静止,而生精细胞特异的基因被激活[1].研究表明,在每个精子发生的不同阶段,至少生成有5%的具有阶段特异性的多肽(Boitani C.,1980).因此精子发生过程中伴随着许多睾丸特异性基因表达,包括原癌基因(proto-oncogenes)、细胞骨架蛋白基因(actin和tubulin等)、无精子因子基因(azoospermia factor,AZF)、热休克蛋白基因(heat shock protein,HSP)、同工酶基因(磷酸甘油激酶PGK-2,乳酸脱氢酶LDH-C等)等等.     

支持细胞及其对精子发生的内分泌调控

支持细胞（ＳｅｒｔｏｌｉＣｅｌ）以多种途径影响精子发生。随着研究手段、方法的改进，支持细胞的诸多作用，如细胞联系、内分泌调控以及与间质细胞、生精细胞之间的交互作用等，已经或正在逐步被证实和揭示出来。这些功能的精细协调，为精子发生创造了适宜的微环境，保证了精子发生的有序进程。     

羊驼睾丸细胞凋亡及凋亡相关蛋白的定位

本研究旨在观察羊驼睾丸的出生后发育和精子发生过程中的细胞凋亡及凋亡相关蛋白Bcl2和Caspase3 的定位.取材新生、12月龄和24月龄羊驼的睾丸,用TUNEL法检测睾丸发育和精子发生过程的细胞凋亡,用免疫组织化学技术检测凋亡相关蛋白Bcl2和Caspase3在羊驼出生后发育和精子发生过程中的定位.结果显示在新生羊驼睾丸未检测到TUNEL阳性细胞,Caspase3和Bcl2表达于间质细胞,提示在新生期凋亡蛋白参与间质细胞凋亡的调节,为曲精小管的发育提供空间;12月龄羊驼睾丸TUNEL阳性细胞定位于曲精小管中央部分,Caspase3 和Bcl2定位于间质细胞和曲精小管中央生殖细胞,提示在青春期(12月龄)羊驼睾丸,细胞凋亡和凋亡相关蛋白参与曲精小管管腔形成的调节;24月龄羊驼睾丸TUNEL阳性细胞定位于精原细胞、精母细胞和精子细胞,Caspase3 和Bcl2定位于间质细胞和各个发育阶段的生精细胞,Caspase3阳性细胞在精原细胞最高,向精母细胞和精子细胞逐渐减少,Bcl2在精原细胞弱阳性表达,在血睾屏障以内的曲精小管近腔室部分呈弥散性强阳性表达,提示在性成熟(24月龄)羊驼睾丸精子发生过程中,细胞凋亡主要发生于精原细胞和早期精母细胞,Bcl2可能抑制精母细胞之后生殖细胞的凋亡.结果提示在羊驼睾丸出生后发育和精子发生过程中存在细胞凋亡现象;凋亡蛋白Caspase3和Bcl2参与羊驼睾丸发育和精子发生过程中细胞凋亡的调节.     

PGP9.5和神经肽Y在双峰驼正常睾丸和隐睾的分布比较

探讨PGP9.5和神经肽Y在双峰驼正常睾丸和隐睾的表达及在精子发生中的作用机制。采集性成熟未交配2~3岁成年双峰驼正常睾丸及隐睾,应用免疫组织化学SP法检测PGP9.5和神经肽Y在睾丸的定位,并通过图像分析技术进行定量分析。结果表明:PGP9.5在正常睾丸支持细胞、各级生精细胞和动静脉血管都有高密度阳性反应;神经肽Y在支持细胞呈中等阳性,各级生精细胞和动静脉血管呈弱阳性,隐睾组中PGP9.5和神经肽Y表达位置相似于正常组,但表达量显著降低。PGP9.5和神经肽Y在正常组和隐睾组Leydig细胞表达无差异,均为强阳性。可见PGP9.5及神经肽Y参与了双峰驼正常睾丸和隐睾生精功能的调节,二者通过支持细胞及管周肌样细胞对于隐睾生精微环境的调控能力降低,但隐睾内间质细胞的分泌并未受明显影响。本研究为进一步研究双峰驼睾丸神经递质变化与隐睾症发生关系及调控机制提供了参考。     

支持细胞及其对精子发生的内分泌调控

支持细胞以（ＳｅｒｔｏｌｉＣｅｌｌ）以多种途径影响精子发生，随上研究手段，方法的改进，支持细胞的诸多作用，如细胞联系，内分泌调控以及与间质细胞，生精细胞之间的交互作用等，已经或正逐步被证实和揭示出来。这些功能的精细协调，为精子发生创造子适宜的微环境，保证精子发生的有序进程。     

支持细胞对精原干细胞增殖、分化调控的研究进展

哺乳动物精子发生是在睾丸支持细胞(Sertoli cells,SCs)调节下完成的,SCs不但为精子发生提供物理支撑和稳定微环境,而且通过分泌多种蛋白对生殖细胞发挥增殖、分化、凋亡、吞噬、免疫豁免等多种调节作用。生理状态下,SCs调控精子发生的确切机制还不是很清楚。因此,本文对SCs与精原干细胞(spermatogonial stem cells,SSCs)及系列生精细胞特殊的结构关系以及二者间的调控关系进行了综述,以期为推动二者间调控机理的深入研究及提高动物育种繁殖效率提供参考。     

支持细胞及其对精子发生的内分泌调控

支持细胞（ＳｅｒｔｏｌｉＣｅｌｌ）以多种途径影响精子发生。随着研究手段，方法的改进，Ｓｅｒｔｏｌｉ细胞的诸多作用，如细胞联系，内分泌调控以及与间质细胞，生精细胞之间的交互作用等，已经或正在逐步被证实和揭示出来。这些功能的精细协调，为精子发生创造了适宜的微环境，保证了精子发生的有序进程。     

精原干细胞标记及活体追踪的研究进展

研究表明,精原干细胞（spermatogonial stem cells,SSCs）具有重建不育个体精子发生的能力。采用细胞标记试剂标记精原干细胞,可以活体追踪移植至睾丸的精原干细胞命运,即在发育过程中的增殖、运动及分化情况。标记和追踪精原干细胞将有助于了解精子发生恢复的机理并选择最优的移植策略。     

睾丸支持细胞的功能、分离纯化及鉴定研究进展

支持细胞对维持精子形成过程中的微环境起决定作用,它可以通过分泌功能、细胞间连接形成的血睾屏障功能以及吞噬功能等来促进精子的形成过程,其发育异常会导致不同程度的雄性生殖缺陷。基于支持细胞在雄性动物生殖过程中的作用,体外培养高纯度支持细胞可成为研究睾丸两大核心功能-精子发生和性激素分泌功能相关调节机制重要的细胞模型。此外,体外培养睾丸支持细胞也可作为生殖毒理学等新兴热点领域的细胞模型,为评估和研究环境因素对雄性生殖的影响提供便利。因此,作者系统地归纳、总结了目前关于动物支持细胞生物功能的研究及常用的体外分离纯化、培养及鉴定方法,以期为利用动物支持细胞开展雄性生殖领域的研究提供参考。     

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood–testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3′-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.     

支持细胞中促卵泡素信号通路研究进展

支持细胞在精子生成过程中起重要作用,支持细胞的数量决定睾丸大小、生精细胞数量以及最终生成精子数量。促卵泡素作为保证雄性生育能力的重要激素之一,通过与支持细胞上的促卵泡素受体结合,诱导5种信号途径,导致支持细胞中各类转录因子被激活,引起基因表达发生变化,从而保证生精细胞顺利发育成精子。文章主要围绕支持细胞中促卵泡素信号途径,及其对细胞发育过程中相关基因表达的影响等方面进行了综述。     

The Sertoli cell nucleolus in domestic and wild animals

Sertoli cells produce special microenvironment for developing germ cells; therefore it is assumed that they play primary role in the onset and control of spermatogenesis. In this connection we extended our previous study on the ultrastructure of Sertoli cells in different domestic and wild animals with special regard to nucleolus. Sertoli cells of domestic and wild ruminants possess the typical vesicular nucleolus except for fallow deer, in this species no vesicular nucleolus occurs in Sertoli cells even during the rut. In roe.buck, another wild ruminant with seasonal spermatogenesis, cyclic changes were found in the nucleolus of Sertoli cells. If no spermatogenesis is present, the Sertoli cells have a reticular nucleolus. Membranous vesicles appear in the nucleolus of Sertoli cells of roe-buck at the onset of spermatogenesis 1-2 months before rut. In domestic ruminants with continuous spermatogenesis the vesicular nucleolus in Sertoli cells is present permanently. During postnatal development of bull and ram the vesicular nucleolus appears in Sertoli cells just before the onset of spermatogenesis. In experimental cryptorchidism of bulls a vesicular nucleolus is found in the Sertoli cells. Our observations and experiments support a hypothesis that Sertoli cells have primary role at the onset and the maintenance of spermatogenesis.     

LncWNT3-IT affects the proliferation of Sertoli cells by regulating the expression of the WNT3 gene in goat testis

The proliferation and differentiation ability of testicular Sertoli cells directly affects spermatogenesis and male reproductive development. WNT proteins are involved in the regulation of cell proliferation, differentiation and spermatogenesis. Therefore, to study whether lncRNAs, which regulate the expression of WNT proteins during cell proliferation and differentiation, are worthwhile. In this study, testicular tissue from the Dazu black goat (Capra, goat, Chongqing, China) at neonatal time (less than 7 days old), early puberty time (45 days old) and sexual maturity time (90 days old) at three ages was subjected to high-throughput sequencing to predict testicular growth and development associated with WNT lncRNA. The final screening of lncWNT3-IT may be targeted to regulate the expression of WNT3. At the same time, the expression of WNT3 was verified by lncWNT3-IT by paraffin sectioning, fluorescence in situ hybridization, interference, overexpression, cytotoxicity assay, Western blotting and qPCR. The following results were obtained: lncWNT3-IT was expressed in the testicular Sertoli cells and played a role in the Sertoli cell cytoplasm. Fluorescence in situ hybridization localization analysis showed that lncWNT3-IT positively regulated the expression of WNT3, and through cell viability and cell proliferation experiments, it was found that the expression of lncWNT3-IT assisted in Sertoli cell proliferation. In summary, lncWNT3-IT can influence the proliferation of Sertoli cells by positively regulating the expression of WNT3.     

Immunohistochemical analysis of cyclins in canine normal testes and testicular tumors

The expression of cyclins A, D1, D2 and E were examined immunohistochemically in 5 canine normal testes and 31 testicular tumors, including 14 seminomas, 11 Sertoli cell tumors and 6 Leydig cell tumors. In canine normal testes, cyclin A expression was detected in spermatogonia and primary spermatocytes. This suggests that A-type cyclins may play some role in canine spermatogenesis. Cyclin A expression was also observed in 13/14 (92.9%) seminomas and 2/11 (18.2%) Sertoli cell tumors, but no positive reaction was observed in Leydig cell tumors. Parallel examinations for cyclins D1, D2 and E gave negative results in canine normal testes and testicular tumors. High levels of cyclin A expression in canine seminomas indicate that the neoplastic germ cells may be arrested at the spermatogonia and primary spermatocyte stages of differentiation.     

Numbers of Sertoli cells, quantitative rates of sperm production, and the efficiency of spermatogenesis in relation to the daily sperm output and seminal quality of young beef bulls

Data from 34 yearling Hereford or Angus bulls were used to investigate relationships of testicular size, quantitative rates of sperm production, Sertoli cell numbers, numbers of germ cells supported per Sertoli cell, and the efficiency of spermatogenesis to daily sperm output and seminal quality. Two ejaculates were collected by electroejaculation from each bull on each of 2 days/week throughout the study. The percentage of progressively motile sperm and the percentage of morphologically normal sperm were determined from aliquots of fresh semen. Additional aliquots of semen were frozen in glass ampules or plastic straws and subsequently evaluated for postthaw motility and percentage of sperm with intact acrosomes. Sertoli cell numbers, the numbers of germ-cells per Sertoli cell, and the efficiency of spermatogenesis were unrelated to the quality of fresh or frozen semen (P greater than 0.05). In first ejaculates, the numbers of sperm and motile sperm were related (P less than 0.05) to testicular parenchymal weight (r = 0.38 and 0.50), daily sperm production (r = 0.45 and 0.53), and spermatids per gram of testicular parenchyma (r = 0.35 and 0.34). Testicular parenchymal weight and daily sperm production also were related to daily sperm output and to the average daily motile sperm output of these bulls (P less than 0.05), but could account for less than 25% of the variability in these end points among bulls.     

miR-362 knock-down promotes proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting the RMI1 gene

Immature Sertoli cell proliferation determines the total number of mature Sertoli cells and further regulates normal spermatogenesis. Accumulating evidence demonstrates that microRNAs (miRNAs) play regulatory roles in immature Sertoli cell proliferation, while the functions and mechanisms of the Sertoli cells of domestic animals are poorly understood. In the present study, we aimed to investigate the roles of miR-362 in cell proliferation and apoptosis of porcine immature Sertoli cells. The results showed that miR-362 inhibition promoted the entrance of cells into the S phase and increased the expressions of cell cycle-related genes c-MYC, CNNE1, CCND1 and CDK4. Knock-down of miR-362 also promoted cell proliferation and inhibited apoptosis, which was demonstrated by the results from cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) and Annexin V-FITC/PI staining assays. The recQ-mediated genome instability protein 1 (RMI1) gene was identified as a potential target gene of miR-362 via luciferase reporter assay, and miR-362 repressed the protein expression of RMI1 in porcine immature Sertoli cells. siRNA-induced RMI1 knock-down further abolished the effects of miR-362 inhibition on porcine immature Sertoli cells. Collectively, we concluded that miR-362 knock-down promotes proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting the RMI1 gene, which indicates that miR-362 determines the fate of immature Sertoli cells.     

小鼠睾丸支持细胞分离培养及生物学特性鉴定

为了揭示睾丸支持细胞在精子发生及睾丸免疫豁免中的生物学作用,采用胰蛋白酶、胶原酶分步消化法和1/3浓度的Hank′s低渗液、50mmol/L Tris-HC(l pH7.1)液分别低渗处理及油红O脂质染色法对16～22d的幼鼠睾丸进行分离、纯化和生物学特性鉴定。结果表明:分离、纯化后的睾丸支持细胞损伤少、活率高、睾丸支持细胞占培养细胞总数的90%以上;根据形态学观察及油红O脂质染色法鉴定,睾丸支持细胞的生长状态良好,细胞突起很多,核仁清晰,胞质中可见大小不等的红色空泡状脂质小滴。