小鼠睾丸TERT表达的增龄变化

应用免疫组织化学和图像分析方法研究了10,20,30,60,90天龄的小鼠睾丸不同分化类型生殖细胞中端粒酶催化亚基TERT的表达.结果发现在精子发生过程中精原细胞及成熟精子中均未见TERT阳性表达,而初级精母细胞尤其是粗线期精母细胞和精子细胞是高表达TERT的细胞类群,附睾管上皮细胞也呈TERT阳性表达;同时TERT在小鼠个体发育过程中的表达存在着从弱到强再到弱的动态变化趋势.结果表明TERT在小鼠睾丸中的表达特点与精子发生过程密切相关,可能对维持生殖细胞遗传物质的完整性和减数分裂的正常进行发挥着重要作用.     

小鼠睾丸发育全过程的组织学观察

为系统观察小鼠睾丸组织发育过程,将生后1～57d处于不同发育时期的17组昆明种正常小鼠睾丸组织制备石蜡切片、进行H.E染色分析。结果表明:小鼠生后初期睾丸曲细精管中只有支持细胞和原始生精细胞;至生后8d时出现B型精原细胞;15d时出现初级精母细胞;23d时出现次级精母细胞及少量圆形精子细胞,此后圆形精子细胞逐渐增多;30d时圆形精子细胞发生变态;36d时大量精子开始稳定出现于曲细精管管腔中,并延续至此后各期。这意味着小鼠生精细胞在出生后经过一个相对连续的发育分化过程,至生后约36d发育成熟。这为细化小鼠睾丸组织发育和精子发生过程提供了详细资料。     

睾丸支持细胞功能的研究进展*

摘要: 支持细胞位于睾丸曲细精管内皮,作为与生精细胞接触的惟一体细胞,在生精过程中起着至关重要的调控作 用。支持细胞在精子发生过程中可为精子细胞提供结构支持; 他分泌的各种蛋白质参与精子发生所需物质的转 运; 同时支持细胞表达一些膜蛋白,促进局部免疫豁免形成,为精子发生提供有利的微环境; 再者,支持细胞通过发 挥粘附与吞噬作用,清除精子发生中凋亡的精子细胞。随着研究的深入,近年来对支持细胞又有了新的认识。     

大天鹅睾丸和附睾的组织结构及EGF,Bax,Bcl-2蛋白和iNOS的表达

为了解大天鹅(Cygnus cygnus)睾丸和附睾的组织结构特征及相关活性物质的表达情况,用生物显微技术观察了大天鹅睾丸和附睾的组织结构,用免疫组织化学方法检测了EGF,Bax蛋白,Bcl-2蛋白和iNOS在睾丸和附睾中的表达.结果表明,大天鹅睾丸内生精小管主要由支持细胞和各级生精细胞构成,众多生精小管被睾丸间质隔开;附睾由输出小管和附睾管组成;大天鹅睾丸生精小管直径、生殖细胞大小、附睾管直径及上皮厚度等与其他脊椎动物存在差异;EGF,Bax蛋白,Bcl-2蛋白在睾丸生精小管管周的肌样细胞、精原细胞、精母细胞、精子细胞、间质细胞和附睾管上皮细胞等处呈免疫反应阳性;iNOS在睾丸间质内的血管壁及间质细胞、附睾管上皮细胞、附睾结缔组织等处有阳性表达;EGF可能参与其精子发生、获能及激素生成的调控过程;Bax蛋白和Bcl-2蛋白可能共同参与调控正常大天鹅睾丸和附睾细胞的凋亡;iNOS可能也参与雄性大天鹅各种生殖活动的调节.     

间二硝基苯对小鼠睾丸生殖细胞DNA损伤作用的SCGE研究

采用小鼠睾丸支持细胞/ 生殖细胞共培养法以及单细胞凝胶电泳技术,研究了间二硝基苯对小鼠睾丸生殖细胞DNA的损伤作用.结果表明:间二硝基苯能够导致生殖细胞DNA链断裂,而且其受损率与剂量具有明显的剂量效应关系,与对照组相比均具有显著性差异(P＜0.01,P＜0.05);间二硝基苯可以引起离体小鼠睾丸生殖细胞DNA损伤,具有生殖毒性.     

雄激素对小鼠睾丸支持细胞Akt和MEK/Erk磷酸化水平的影响

磷酸化信号通路在精子发生过程中有重要作用,本文研究小鼠睾丸支持细胞中雄激素诱导的蛋白磷酸化。研究中分离培养小鼠睾丸支持细胞,建立了稳定的培养体系。蛋白磷酸化抗体芯片结果显示,睾酮快速激活Akt及MEK/Erk的磷酸化,蛋白免疫印迹验证了芯片分析结果,且激酶磷酸化的激活是由AR介导。结果表明睾酮诱导小鼠睾丸支持细胞中Akt和MEK/Erk磷酸化。     

棕色田鼠睾丸和附睾胚后发育中的雌二醇表达

应用免疫组织化学方法研究了产后1日龄、10日龄、25日龄、45日龄及60日龄(成体)5个发育阶段的棕色田鼠(Microtus mandarinus)睾丸和附睾中雌二醇(estradiol,E2)的表达.结果表明:1日龄,睾丸生殖母细胞有E2阳性表达.10日龄,生殖母细胞E2表达减弱(P<0.05).25日龄,E2表达主要见于精子细胞.45日龄,精母细胞和精子细胞中有E2表达.60日龄,精原细胞、精母细胞、精子细胞及精子中均有E2表达,精母细胞和精子细胞表达较强,精原细胞表达较弱.在胚后发育过程中,1日龄和10日龄间质区细胞的E2表达最强,25日龄和45日龄E2表达减弱,60日龄E2表达最弱(P<0.05).1日龄至60日龄附睾上皮细胞和连接组织均有E2表达,60日龄附睾管内有大量精子,且有明显的E2表达.这些结果说明,棕色田鼠从出生到性成熟过程中,生殖细胞和间质细胞有E2生成.在精子发生的各阶段,雌激素通过其受体可能对生精细胞的分化和增殖有直接调控作用,对间质细胞的发育也存在着调节作用.同时,附睾的发育和精子的成熟也受到雌激素的调节.     

小鼠精原细胞体外培养分化的研究

目的 探讨小鼠精原细胞的分离纯化及体外培养条件。方法采用组合酶消化法分离纯化小鼠精原细胞和支持细胞,在添加FSHC和TC的基本培养基中进行体外培养。采用反转录PCR方法检测精子细胞特异性基因过渡蛋白9（Tnp2）的表达以对分化细胞进行鉴定。结果培养5d后有分化的圆形精子细胞出现,培养7d后收集的细胞可见Tnp2基因表达。结论通过体外培养小鼠精原细胞可迅速完成减数分裂过程和减数分裂后的分化过程,并可获得更接近成熟阶段的精子细胞。     

5种细胞周期调控基因在小鼠生殖细胞发育过程中的表达研究

 以成熟(10周龄以上)的昆明种正常小鼠的精巢和卵巢为材料,利用地高辛标记的基因探针进行组织切片上的DNA-mRNA分子原位杂交,研究了PCNA,cdc2,cyclin D1,p2 1和 p16 5种细胞周期调控基因在生殖细胞发育过程中的表达.结果表明:PCNA基因在睾丸组织的精原细胞和精母细胞中有强杂交信号,而在雌性生殖细胞及滤泡细胞的发育过程都没有杂交信号;cyclin D1,cdc2,p2 1,p16基因在生殖细胞的发育过程中都没有,表明这些基因并没有参与小鼠生殖细胞的生长和分化调控.这些事实表明在生殖细胞发育过程中,控制细胞增殖和增殖抑制的基因与培养细胞有不同的机制,它们可能采用了不同的调控系统.     

CREBBP在小鼠睾丸发育过程中表达模式的研究

睾丸是雄性动物最为重要的生殖器官,主要由曲细精管和间质构成。雄性哺乳动物持续的精子发生依赖于精原干细胞。精原干细胞通过有丝分裂自我更新,并在一些因子的诱导下启动分化进一步发育为精母细胞。研究睾丸中各细胞基因的表达调控对男性不育和睾丸生殖细胞肿瘤的治疗具有十分重要的意义。本研究利用RT-PCR、免疫组织化学及免疫荧光双染的方法主要研究了CREBBP在小鼠睾丸中的表达模式,结果显示CREBBP主要在精原细胞中表达,并在增殖状态的精原细胞中有表达,但在分化后启动了减数分裂的细胞中未检测到阳性信号。因此我们推测CREBBP可能参与了精原细胞的增殖,为后期精原细胞自我更新机制的研究提供理论基础。     

Germ cell transplantation in infertility mouse

This work investigated the spermatogenesis in an infertility BALB/c-nu mouse model by reinfusing germline stem cells into seminiferous tubules. Donor germ cells were isolated from male FVB/NJ-GFP trensgenic mice. Seminiferous tubule microinjection was applied to achieve intratubular germ cell transfer. The germ cells were injected into exposed testes of the infertility mice. We used green fluorescence and DNA analysis of donor cells from GFP transgenic mice as genetic marker. The natural mating and Southern blot methods were applied to analyze the effect of sperm cell transplantation and the sperm function after seminiferous tubule microinjection. The spermatogenesis was morphologically observed from the seminiferous tubules in 41/60 （68.33%） of the injected recipient mice using allogeneic donor cells. In the colonized testes, matured spermatozoa were seen in the lumen of the seminiferous tubules. In this research, BALB/c-nu infertility mouse model, the recipient animal, was used to avoid immunological rejection of donor cells, and germ cell transplantation was applied to overcome infertility caused by busulfan treatment. These results demonstrate that this technique of germ cell transplantation is of great use. Germ cell transplantation could be potentially valuable to oncological patients.     

小鼠精原干细胞的分离、分选、移植和培养

精原干细胞具有自我增殖和分化为精子的能力。精原干细胞的培养、移植和体外诱导分化等研究将使我们最终阐明精原干细胞的自我更新机制和精子发生机理。经过一年多的研究,我们不仅建立了用含血清培养基对分离的乳鼠睾丸细胞进行差异贴壁分选来富集生殖细胞的简单方法,而且成功地对分选出的精原干细胞进行了近一个月的无血清培养。流式分析结果表明,差异贴壁分选能将精原干细胞富集11倍以上。移植分析表明,分选出的精原干细胞具有在受体鼠的曲精小管中产生克隆的能力和正常生精作用。免疫细胞化学和RT-PCR检测表明,培养的细胞表达精原干细胞标志基因。该研究为体外长期培养小鼠或其它哺乳动物的精原干细胞提供了一个范例,也为利用基因修饰的精原干细胞通过移植产生转基因动物奠定了基础。     

Tyro-3 family receptors are essential regulators of mammalian spermatogenesis

We have generated and analysed null mutations in the mouse genes encoding three structurally related receptors with tyrosine kinase activity: Tyro 3, Axl, and Mer. Mice lacking any single receptor, or any combination of two receptors, are viable and fertile, but male animals that lack all three receptors produce no mature sperm, owing to the progressive death of differentiating germ cells. This degenerative phenotype appears to result from a failure of the tropic support that is normally provided by Sertoli cells of the seminiferous tubules, whose function depends on testosterone and additional factors produced by Leydig cells. Tyro 3, Axl and Mer are all normally expressed by Sertoli cells during postnatal development, whereas their ligands, Gas6 and protein S, are produced by Leydig cells before sexual maturity, and by both Leydig and Sertoli cells thereafter. Here we show that the concerted activation of Tyro 3, Axl and Mer in Sertoli cells is critical to the role that these cells play as nurturers of developing germ cells. Additional observations indicate that these receptors may also be essential for the tropic maintenance of diverse cell types in the mature nervous, immune and reproductive systems.     

In vitro production of functional sperm in cultured neonatal mouse testes

Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation. The whole process, therefore, has never been reproduced in vitro in mammals, nor in any other species with a very few exceptions in some particular types of fish. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2?months in tissue fragments positioned at the gas-liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.     

Abnormal sexual development in transgenic mice chronically expressing müllerian inhibiting substance

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein normally secreted by the Sertoli cells of the fetal and adult testis and by granulosa cells of the postnatal ovary. The production of MIS in the male fetus brings about the regression of the Müllerian ducts, the anlagen of the uterus, oviducts, and upper vagina. In addition, purified MIS induces the formation of seminiferous cord-like structures in fetal rat ovaries cultured in vitro, suggesting that MIS may influence testicular differentiation. We have produced transgenic mice chronically expressing human MIS under the control of the mouse metallothionein-1 promoter to investigate its role during sexual development. In females, chronic expression led to the inhibition of Müllerian duct differentiation, resulting in a blind vagina and no uterus or oviducts. At birth the ovaries had fewer germ cells than normal; during the next two weeks germ cells were lost and the somatic cells became organized into structures resembling seminiferous tubules. Apparently, these structures degenerate as they are undetectable in adult females. The majority of transgenic males developed normally. But in two lines with the highest levels of MIS expression, some males showed feminization of the external genitalia, impairment of Wolffian duct development, and undescended testes. These results suggest that MIS has several distinct roles in mammalian sexual development.     

Sperm from neonatal mammalian testes grafted in mice

Spermatogenesis is a productive and highly organized process that generates virtually unlimited numbers of sperm during adulthood. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells. Many complex aspects of testis function in humans and large animals have remained elusive because of a lack of suitable in vitro or in vivo models. Germ cell transplantation has produced complete donor-derived spermatogenesis in rodents but not in other mammalian species. Production of sperm in grafted tissue from immature mammalian testes and across species has not yet been accomplished. Here we report the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. This approach maintains structural integrity and provides the accessibility that is essential for studying and manipulating the function of testes and for preserving the male germ line. Our results indicate that this approach is applicable to diverse mammalian species.