Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment

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哺乳动物精原干细胞的研究进展

精原干细胞是雄性体内可以永久维持的成体干细胞,它具有自我更新和分化的能力,保证了雄性个体生命过程中精子发生的持续进行,从而实现将遗传信息传递给下一代。精原千细胞不仅可在体外实现长期培养或诱导分化为各级生精细胞,并且可在特定条件下将其诱导去分化成为多能性干细胞。同样,这种多能性干细胞如同胚胎干细胞,可被诱导形成造血细胞、神经元细胞、肌细胞等多种类型细胞。鉴于其独具的生物学特性,精原干细胞在揭示精子的发生机制、治疗雄性不育和转基因动物等研究中具有重要价值。该文对精原干细胞在生物学特性、纯化培养、移植、体外诱导分化及其相关调控方面的各项研究进行了小结,综述了近年来的研究历程和最新研究成果。     

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.     

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。     

Single-cell analysis of human testis aging and correlation with elevated body mass index

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Evaluation of premeiotic activity in the developing rabbit testis using a modified squash technique

Germ cells in the developing rabbit testis were studied using a modified squash technique. Preleptotene figures present in the postnatal testis were examined and compared with corresponding stages in ovarian germ cells. The findings indicated differences in the pattern of preleptotene changes in the developing ovary and testis.     

Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1‐positive subpopulations in men

Background Isolation of spermatogonial stem cells (SSCs) could enable in vitro approaches for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. SSC isolation from adult testes is difficult due to low cell numbers and lacking cell surface markers. Glial cell‐derived neurotrophic factor family receptor alpha‐1 (GFRα1) plays a crucial role for the maintenance of SSCs in rodents and is expressed in monkey spermatogonia. Methods Magnetic activated cell sorting was employed for the enrichment of GFRα1+ spermatogonia from adult primate testes. Results Magnetic activated cell sorting of monkey cells enriched GFRα1+ cells threefold. 11.4% of GFRα1+ cells were recovered. 42.9% of GFRα1+ cells were recovered in sorted fractions of human testicular cells, representing a fivefold enrichment. Interestingly, a high degree of morphological heterogeneity among the GFRα1+ cells from human testes was observed. Conclusions Magnetic activated cell sorting using anti‐GFRα1 antibodies provides an enrichment strategy for spermatogonia from monkey and human testes.     

探索Sertoli细胞对去除精原细胞的动态反应

目的探索Sertoli细胞对去除小鼠精原细胞后睾丸的动态反应。方法采用15、30和44 mg/kg的白消安腹腔注射法建立不同程度去除精原细胞的动物模型,处理后5 d和28 d时对睾丸进行组织学检测,评价精子发生状态,并运用实时定量荧光PCR技术检测这两个时期睾丸GDNF、PLZF、Nanog和GFRα1基因mRNA的表达量。结果在白消安处理后第5天,GDNF出现显著升高,且呈剂量依赖趋势,而PLZF与GFRɑ1并无显著变化,睾丸组织学观察亦无明显变化。在白消安处理后28 d时,GDNF、PLZF、Nanog、GFRɑ1基因mRNA相对表达量均出现大幅度的升高,睾丸组织学切片观察显示随着给药剂量的增加,精子发生受到的损伤愈加严重。结论 Sertoli细胞早在白消安处理后第5天就对精原细胞的变化发生了反应,Sertoli细胞分泌GDNF的能力发生代偿性增加,进而刺激精原干细胞自我更新速度加快,体现在Nanog和PLZF水平提高,从而实现精子发生的重建。     

In vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells

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Disruption of testis cords by cyclopamine or forskolin reveals independent cellular pathways in testis organogenesis

Most studies to date indicate that the formation of testis cords is critical for proper Sertoli cell differentiation, inhibition of germ cell meiosis, and regulation of Leydig cell differentiation. However, the connections between these events are poorly understood. The objective of this study was to dissect the molecular and cellular relationships between these events in testis formation. We took advantage of the different effects of two hedgehog signaling inhibitors, cyclopamine and forskolin, on gonad explant cultures. Both hedgehog inhibitors phenocopied the disruptive effect of Dhh(-/-) on formation of testis cords without influencing Sertoli cell differentiation. However, they exhibited different effects on other cellular events during testis development. Treatment with cyclopamine did not affect inhibition of germ cell meiosis and mesonephric cell migration but caused defects in Leydig cell differentiation. In contrast, forskolin treatment induced germ cell meiosis, inhibited mesonephric cell migration, and had no effect on Leydig cell differentiation. By carefully contrasting the different effects of these two hedgehog inhibitors, we demonstrate that, although formation of testis cords and development of other cell types normally take place in a tightly regulated sequence, each of these events can occur independent of the others.     

哺乳动物精原干细胞自我更新调控的研究进展

精原干细胞（spermatogonial stem cells,SSCs）具有自我更新和分化的功能,这两种功能的平衡协调不仅能维持其自身数量的稳定,还能满足雄性动物精子生成的需要。近几年,由于细胞培养技术、基因工程技术、生殖细胞移植技术的建立和完善,使SSCs自我更新调控机制的研究取得了许多突破,主要体现在蛋白调控因子和微小RNA分子以及DNA甲基化新作用的发现等方面。该文将着重围绕调控SSCs自我更新的外源性细胞因子和内源性转录因子等蛋白因子进行综述,以期为哺乳动物SSCs的深入研究提供借鉴。     

Concomitant sertoli and leydig cell tumor of the testis: a case report

A rare intratubular gonadal stromal tumor was present in the testis of a 45-year-old man who was admitted to our hospital with the chief complaint of gradual enlargement of the left testis. Tumoral markers were negative and no extension was observed. The tumor comprised an intratubular mixture of two types of tumor cells with intercellular junctions: the predominant tumor cells were consistent with a Sertoli cell origin and cells comprising the minor population consistent with a Leydig cell origin. The patient is disease free after 6-month follow-up. The case is considered to be a testicular mixed tubular Sertoli-Leydig cell tumor. It highlights a rare type of primary tumor of the testis that features a good prognosis.     

哺乳动物精原干细胞的分离富集及检测鉴定

获得高纯度、高活力的具有生物学安全性的精原干细胞(SSCs)并能从功能上对其进行检测鉴定,是SSCs体外生物学研究的关键所在。近年来,借助于SSCs移植这一功能性检测手段,有关SSCs的分离富集及识别鉴定取得了快速进展。本文结合作者的工作实践,就生精上皮细胞悬液的制备、SSCs的分离富集及其检测鉴定方法进行评述。     

RBFOX2缺失抑制雄性小鼠的减数分裂起始

减数分裂起始是配子发生的关键步骤。目前人们已经发现了一些减数分裂起始所必需的基因,但是对于该过程的调控基因及其作用机制还知之甚少。本实验室先前建立了精原干细胞（spermatogonial stem cells,SSCs）体外培养以及体外诱导减数分裂起始的技术体系,为更好地探究减数分裂起始调控基因及作用机理提供了良好的条件。实验室前期研究发现RNA结合蛋白RBFOX2可能调控减数分裂起始,然而RBFOX2在生殖细胞的减数分裂起始过程中的功能及作用机制还需深入研究。本研究利用慢病毒介导的转基因技术产生了RBFOX2敲降的精原干细胞系;发现RBFOX2敲降后,精原干细胞的自我更新、增殖以及分化未发生显著变化,但是减数分裂无法启动,分化型精原细胞发生明显凋亡。这一结果进一步显示了RNA结合蛋白在雄性动物减数分裂起始中的重要功能。     

Activation of Toll like receptor 3 induces spermatogonial stem cell apoptosis

Germ cell apoptosis may be associated with the male infertility. The pathogenesis is to be further understood. Viral infection is one of the causative factors of apoptosis of the body cells. This study aims to investigate the role of activation of Toll like receptor (TLR)3 in the induction of germ cell apoptosis. In this study, spermatogonial stem cells (SSC) were isolated from C57BL/6 mouse testes. The expression of TLR3 on SSC was by RT‐qPCR and Western blotting. polyinosinic–polycytidylic acid (Poly I:C) was employed to activate TLR3 on SSCs. The results showed that re‐activation by PolyI:C induced SSC apoptosis. Exposure to PolyI:C induced interferon regulatory factor 3 (IRF3) phosphorylation in SSCs. TLR3 and IRF3 formed a complex in the SSCs. The complex of TLR3/IRF3 bound to the promoter of Fas ligand and promoted Fas ligand expression in SSC, and thus induced SSC apoptosis. In conclusion, the results of the present study indicate that activation of TLR3 by PolyI:C induces the SSC apoptosis, which implies that viral infection may interfere with the male germ cell development. Copyright © 2015 John Wiley & Sons, Ltd.     

Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.     

Distribution of gelsolin in human testis

Gelsolin, an actin-binding and severing protein present in many mammalian cells, was characterized in human testis. Although abundant in testicular extracts, gelsolin was not detected in purified spermatogenic cells by immunoblot analysis. Immunofluorescence studies of testis sections showed that gelsolin has two main localizations: peritubular cells and the seminiferous epithelium. In peritubular cells, gelsolin was present together with α-SM actin, in agreement with the myoid cell characteristics of these cells. In a large proportion of the tubules, gelsolin was found mainly, together with actin, in the apical part of the seminiferous epithelium. This localization of gelsolin also was observed in seminiferous tubules with a partial or complete absence of germinal cells, which evokes a presence of gelsolin at the apex of Sertoli cells. However, in normal testis, a complex pattern of gelsolin labeling was also present, mostly in the apical third of the epithelium, around cells or groups of cells, mainly spermatids, and, less frequently, in various other localizations from the apical to the basal part of the seminiferous epithelium. Taken together, these observations suggest that gelsolin may play different functions in the seminiferous epithelium: (1) regulation of the dynamic alterations of the actin cytoskeleton in the apical cytoplasm of Sertoli cells, and (2) modification of actin filaments assemblies in specific structures at germ cell-Sertoli cell contacts. Thereby, the actin-modulating properties of gelsolin are probably involved in reorganization of the seminiferous epithelium related to germ cell differentiation. Mol. Reprod. Dev. 48:63–70, 1997. © 1997 Wiley-Liss, Inc.