哺乳动物精子发生中减数分裂的起始

生殖细胞的发生、增殖和分化是生命科学领域研究的重要课题之一. 生殖是所有动物赖以生存的基础,精子发生是完成繁殖所必须经历的过程,其最终目的是源源不断地产生单倍体精子.精子发生过程本身是一个复杂特殊的细胞增殖与分化过程,其中减数分裂是精子发生最重要的步骤,但关于减数分裂如何精确起始的分子机制仍知之甚少.已有报道发现,维甲酸（RA）调控Stra8可能是哺乳动物减数分裂起始的机制之一,Nanos2、Boule对RA-Stra8通路具有重要的调控作用. 本文对哺乳动物精子发生中减数分裂起始的相关研究进展进行综述.     

Cytological and expression studies and quantitative analysis of the temporal and stage-specific effects of follicle-stimulating hormone and testosterone during cocultures of the normal human seminiferous epithelium

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

大鼠睾丸曲细精管组织支持细胞/生殖细胞长期共培养与精子发生分析

睾丸体外生殖模型的发展为体外研究睾丸的精子发生分子机制和睾丸毒理学提供了实验工具。很多报道的模型都无法真正地模拟体内复杂的生化分子及功能性相互作用从而导致研究价值有限。该实验拟建立一个体外长期维持睾丸生殖细胞存在,并能持续产生精子细胞的支持细胞/生殖细胞共培养体系。体系中的支持细胞和生殖细胞均由曲细精管组织块迁移到培养皿上,在不添加任何生长因子的情况下维持体外精子发生至圆形精子细胞超过2个月。RT-PCR分析显示,共培养细胞稳定表达cdh1、scp3、tnp2;免疫荧光染色结果显示,CDH1、PLZF、SCP3以及SOX9阳性细胞存在。这些结果例证了体系中同时存在精原干细胞、精母细胞、精子细胞和支持细胞。简单高效的支持细胞/生殖细胞体外共培养体系可用于雄性生殖的分子机制和毒理学研究。     

Ultrastructure of spermatogonia, spermatocytes, and sertoli cells in the testis of the crayfish, Procambarus paeninsulanus

The testes of the crayfish, Procambarus paeninsulanus, were prepared for light and transmission electron microscopy. During early stages of spermatogenesis, when the spermatogonia are dividing, processes from a single Sertoli cell extend between numerous spermatogonia. As the cells enter meiosis, many points of contact can be observed between the Sertoli cell processes and spermatocytes. These desmosome-gap junctions are maintained between the germ and Sertoli cells until the early spermatid stage.     

In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

Long-term culture of male germline stem cells from hamster testes

Spermatogonial stem cells provide the foundation for spermatogenesis in male animals. We recently succeeded in culturing and genetically engineering mouse spermatogonial stem cells, but little is known regarding the culture and growth requirements of spermatogonial stem cells in other animal species. In this study, we report the successful long-term culture of spermatogonial stem cells from hamster testes. Spermatogonial stem cells were purified using an anti-ITGA6 antibody and cultured in the presence of glial cell line-derived neurotrophic factor. The cells continued to proliferate for at least 1 year. During this period, they were genetically modified using a lentivirus and underwent spermatogenesis after transplantation into the testes of immunodeficient nude mice. However, germ cells generated in the surrogate xenogeneic recipients did not differentiate beyond the spermatid stage, and these round spermatids could not produce offspring through in vitro microinsemination. These results suggest that the germ cells may not have acquired characteristics necessary for fertility in the xenogeneic microenvironment. Nevertheless, the successful establishment of culture conditions conducive for hamster spermatogonial stem cell growth and maintenance indicates that this technique can be extended to other animal species in which current genetic modification techniques are impossible or inefficient.     

生精干细胞的研究进展

生精干细胞（spermatogonial stem cells,Sscs）是动物出生后保持分裂能力的生殖细胞,其通过自身复制从而终生存在,并不停地进行减数分裂而分化成精子。然而,最近的研究发现生精干细胞具有一定的多能性,在体外可被培养和诱导成多能性细胞,显示生精干细胞是再生医学和细胞治疗疾病的另一理想祖细胞来源。该综述将着重讨论生精干细胞的多能性研究情况和相关问题。     

Spermatogenesis in heterozygotes for Robertsonian chromosomal rearrangements from natural populations of the common shrew, Sorex araneus

Twenty-two adult male common shrews were collected from 5 sites in the vicinity of Oxford (UK) close to the zone of hybridization between two karyotypic races. The shrews were subdivided into 3 karyotypic categories: homozygotes, simple Robertsonian heterozygotes (which form one or more trivalents at prophase I of meiosis) and complex Robertsonian heterozygotes (which form a quadrivalent). The ratio of primary spermatocytes to round spermatids was determined from transverse sections of seminiferous tubules, to provide an indication of germ cell death. In no individual was there severe germ cells loss. Homozygotes had the highest mean spermatocyte: spermatid ratio and complex heterozygotes the lowest, but there was substantial individual variation and the differences were not significant. Complex heterozygotes also had a higher proportion of defective seminiferous tubules and lower testis weights than did other categories and it is reasonable to propose that, as a population, complex heterozygotes had reduced fitness relative to other categories on the basis of spermatogenic performance. However, there is no evidence from studies of spermatogenesis that simple Robertsonian heterozygotes are less fit than homozygotes.     

Developmental expression of the translocator protein 18 kDa (TSPO) in testicular germ cells

Translocator protein (TSPO) is a high affinity 18 kDa drug- and cholesterol-binding protein strongly expressed in steroidogenic tissues where it mediates cholesterol transport into mitochondria and steroid formation. Testosterone formation by Leydig cells in the testis is critical for the regulation of spermatogenesis and male fertility. Male germ cell development comprises two main phases, the pre-spermatogenesis phase occurring from fetal life to infancy and leading to spermatogonial stem cell (SSC) formation, and spermatogenesis, which consists of repetitive cycles of germ cell mitosis, meiosis and differentiation, starting with SSC differentiation and ending with spermiogenesis and spermatozoa formation. Little is known about the molecular mechanisms controlling the progression from one germ cell phenotype to the next. Here, we report that testicular germ cells express TSPO from neonatal to adult phases, although at lower levels than Leydig cells. TSPO mRNA and protein were found at specific steps of germ cell development. In fetal and neonatal gonocytes, the precursors of SSCs, TSPO appears to be mainly nuclear. In the prepubertal testis, TSPO is present in pachytene spermatocytes and dividing spermatogonia. In adult testes, it is found in a stage-dependent manner in pachytene spermatocyte and round spermatid nuclei, and in mitotic spermatogonia. In search of TSPO function, the TSPO drug ligand PK 11195 was added to isolated gonocytes with or without the proliferative factors PDGF and 17β-estradiol, and was found to have no effect on gonocyte proliferation. However, TSPO strong expression in dividing spermatogonia suggests that it might play a role in spermatogonial mitosis. Taken together, these results suggest that TSPO plays a role in specific phases of germ cell development.     

Dispensable role of PTEN in mouse spermatogenesis

PTEN (phosphatase and tensin homologue deleted on chromosome ten) plays critical roles in multiple cellular processes, including cell proliferation, survival, migration and transformation. A role of PTEN in mammalian spermatogenesis, however, has not been explored. To address this question, we generated a mouse model with PTEN conditional knockout in postnatal male germ cells. We found that spermatogenesis was normal in PTEN-deleted male germ cells. PTEN conditional mutant males produced sperm and sired offspring as competently as wild-type littermates. Moreover, our biochemical analysis also indicated that the Akt (acutely transforming retrovirus AKT8 in rodent T cell lymphoma) signalling pathway was not affected in mutant testis. Taken together, these findings demonstrate that PTEN is dispensable in mouse spermatogenesis.     

In vitro derivation of germ cells from embryonic stem cells in mammals

Previous reports have shown that embryonic stem (ES) cells, derived from the inner cell mass of mouse or human blastocysts, could differentiate in vitro into female and male germ cells as well as into the cell types of all three germ layers. While in one case, the ES cell‐derived germ cells have been reported to give birth to live offspring in the mouse, these cells differ in fertilization capacity from the sperm and oocytes produced in vivo as they cannot complete meiosis under in vitro conditions. The efficiency of functional germ cell isolation from ES cells is also low. According to published reports, factors such as the proper selection of feeder cells, including ovarian granulosa cells and those which could secrete bone morphogenic protein‐4 (BMP4), and the addition of retinoic acid into culture medium, could to some extent establish and improve the microenvironment ES cells rely on for differentiation into germ cells. This review briefly describes the progress of deriving germ cells from ES cells and discusses possible factors that could improve in vitro gamete production. Mol. Reprod. Dev. 77: 586–594, 2010. © 2010 Wiley‐Liss, Inc.