Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell–cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.     

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。     

Indirect effects of Wnt3a/β-catenin signalling support mouse spermatogonial stem cells in vitro

Proper regulation of spermatogonial stem cells (SSCs) is crucial for sustaining steady-state spermatogenesis. Previous work has identified several paracrine factors involved in this regulation, in particular, glial cell line-derived neurotrophic factor and fibroblast growth factor 2, which promote long-term SSC self-renewal. Using a SSC culture system, we have recently reported that Wnt5a promotes SSC self-renewal through a β-catenin-independent Wnt mechanism whereas the β-catenin-dependent Wnt pathway is not active in SSCs. In contrast, another study has reported that Wnt3a promotes SSC self-renewal through the β-catenin-dependent pathway, as it can stimulate the proliferation of a spermatogonia cell line. To reconcile these two contradictory reports, we assessed Wnt3a effects on SSCs and progenitor cells, rather than a cell line, in vitro. We observed that Wnt3a induced β-catenin-dependent signalling in a large subset of germ cells and increased SSC numbers. However, further investigation revealed that cell populations with greater β-catenin-signalling activity contained fewer SSCs. The increased maintenance of SSCs by Wnt3a coincided with more active cell cycling and the formation of germ cell aggregates, or communities, under feeder-free conditions. Therefore, the results of this study suggest that Wnt3a selectively stimulates proliferation of progenitors that are committed to differentiation or are in the process of exiting the SSC state, leading to enhanced formation of germ cell communities, which indirectly support SSCs and act as an in vitro niche.     

Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate

Normal spermatogenesis is essential for reproduction and depends on proper spermatogonial stem cell (SSC) function. Genes and signaling pathways that regulate SSC function have not been well defined. We report that glial cell-line-derived neurotrophic factor (GDNF) signaling through the RET tyrosine kinase/GFRA1 receptor complex is required for spermatogonial self-renewal in mice. GFRA1 and RET expression was identified in a subset of gonocytes at birth, was restricted to SSCs during normal spermatogenesis, and RET expressing cells were abundant in a cryptorchid model of SSC self-renewal. We used the whole-testis transplantation technique to overcome the limitation of neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice and found that each of these genes is required for postnatal spermatogenesis and not for embryological testes development. Each mutant testis shows severe SSC depletion by Postnatal Day 7 during the first wave of spermatogenesis. These defects were due to lack of SSC proliferation and an inability of SSCs to maintain an undifferentiated state. Our results demonstrate that GDNF-mediated RET signaling is critical for the fate of undifferentiated spermatogonia and that abnormalities in this pathway may contribute to male infertility and testicular germ cell tumors.     

H3K27 Demethylase,JMJD3, Regulates Fragmentation of Spermatogonial Cysts

The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of Jmjd3 in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific Jmjd3 null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.     

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.     

Capacity for stochastic self-renewal and differentiation in mammalian spermatogonial stem cells

Mammalian spermatogenesis is initiated and sustained by spermatogonial stem cells (SSCs) through self-renewal and differentiation. The basic question of whether SSCs have the potential to specify self-renewal and differentiation in a cell-autonomous manner has yet to be addressed. Here, we show that rat SSCs in ex vivo culture conditions consistently give rise to two distinct types of progeny: new SSCs and differentiating germ cells, even when they have been exposed to virtually identical microenvironments. Quantitative experimental measurements and mathematical modeling indicates that fate decision is stochastic, with constant probability. These results reveal an unexpected ability in a mammalian SSC to specify both self-renewal and differentiation through a self-directed mechanism, and further suggest that this mechanism operates according to stochastic principles. These findings provide an experimental basis for autonomous and stochastic fate choice as an alternative strategy for SSC fate bifurcation, which may also be relevant to other stem cell types.     

Role for rodent Smc6 in pericentromeric heterochromatin domains during spermatogonial differentiation and meiosis

Chromatin structure and function are for a large part determined by the six members of the structural maintenance of chromosomes (SMC) protein family, which form three heterodimeric complexes: Smc1/3 (cohesin), Smc2/4 (condensin) and Smc5/6. Each complex has distinct and important roles in chromatin dynamics, gene expression and differentiation. In yeast and Drosophila, Smc6 is involved in recombinational repair, restarting collapsed replication forks and prevention of recombination in repetitive sequences such as rDNA and pericentromeric heterochromatin. Although such DNA damage control mechanisms, as well as highly dynamic changes in chromatin composition and function, are essential for gametogenesis, knowledge on Smc6 function in mammalian systems is limited. We therefore have investigated the role of Smc6 during mammalian spermatogonial differentiation, meiosis and subsequent spermiogenesis. We found that, during mouse spermatogenesis, Smc6 functions as part of meiotic pericentromeric heterochromatin domains that are initiated when differentiating spermatogonia become irreversibly committed toward meiosis. To our knowledge, we are the first to provide insight into how commitment toward meiosis alters chromatin structure and dynamics, thereby setting apart differentiating spermatogonia from the undifferentiated spermatogonia, including the spermatogonial stem cells. Interestingly, Smc6 is not essential for spermatogonial mitosis, whereas Smc6-negative meiotic cells appear unable to finish their first meiotic division. Importantly, during meiosis, we find that DNA repair or recombination sites, marked by γH2AX or Rad51 respectively, do not co-localize with the pericentromeric heterochromatin domains where Smc6 is located. Considering the repetitive nature of these domains and that Smc6 has been previously shown to prevent recombination in repetitive sequences, we hypothesize that Smc6 has a role in the prevention of aberrant recombination events between pericentromeric regions during the first meiotic prophase that would otherwise cause chromosomal aberrations leading to apoptosis, meiotic arrest or aneuploidies.     

Spermatogonial stem cells, infertility and testicular cancer

The spermatogonial stem cells (SSCs) are responsible for the transmission of genetic information from an individual to the next generation. SSCs play critical roles in understanding the basic reproductive biology of gametes and treatments of human infertility. SSCs not only maintain normal spermatogenesis, but also sustain fertility by critically balancing both SSC self-renewal and differentiation. This self-renewal and differentiation in turn is tightly regulated by a combination of intrinsic gene expression within the SSC as well as the extrinsic gene signals from the niche. Increased SSCs self-renewal at the expense of differentiation result in germ cell tumours, on the other hand, higher differentiation at the expense of self-renewal can result in male sterility. Testicular germ cell cancers are the most frequent cancers among young men in industrialized countries. However, understanding the pathogenesis of testis cancer has been difficult because it is formed during foetal development. Recent studies suggest that SSCs can be reprogrammed to become embryonic stem (ES)-like cells to acquire pluripotency. In the present review, we summarize the recent developments in SSCs biology and role of SSC in testicular cancer. We believe that studying the biology of SSCs will not only provide better understanding of stem cell regulation in the testis, but eventually will also be a novel target for male infertility and testicular cancers.     

The generation of spermatogonial stem cells and spermatogonia in mammals

Spermatogenesis is a complex series of cellular changes leading to the formation of haploid male gametes (spermatozoa) and includes mitotic, meiotic and post-meiotic phases. Spermatogonial stem cells (SSCs) are essential for the continuous lifelong production of spermatozoa. Spermatogenesis is initiated when SSC is triggered to undergo mitosis that gives rise to progenitors, which further differentiate into spermatogonia. In this review, we describe the origin of SSCs and other spermatogonia populations and summarize the knowledge concerning their markers.     

Spermatogonial Stem Cell Niche and Spermatogonial Stem Cell Transplantation in Zebrafish

Background

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and reside within a specific microenvironment in the testes called “niche” which regulates stem cell properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate.

Methodology/Principal Findings

Here, we introduce zebrafish as a new model for the study of SSCs in vertebrates. Using 5′-bromo-2′-deoxyuridine (BrdU), we identified long term BrdU-retaining germ cells, type A undifferentiated spermatogonia as putative stem cells in zebrafish testes. Similar to rodents, these cells were preferentially located near the interstitium, suggesting that the SSC niche is related to interstitial elements and might be conserved across vertebrates. This localization was also confirmed by analyzing the topographical distribution of type A undifferentiated spermatogonia in normal, vasa::egfp and fli::egfp zebrafish testes. In the latter one, the topographical arrangement suggested that the vasculature is important for the SSC niche, perhaps as a supplier of nutrients, oxygen and/or signaling molecules. We also developed an SSC transplantation technique for both male and female recipients as an assay to evaluate the presence, biological activity, and plasticity of the SSC candidates in zebrafish.

Conclusions/Significance

We demonstrated donor-derived spermato- and oogenesis in male and female recipients, respectively, indicating the stemness of type A undifferentiated spermatogonia and their plasticity when placed into an environment different from their original niche. Similar to other vertebrates, the transplantation efficiency was low. This might be attributed to the testicular microenvironment created after busulfan depletion in the recipients, which may have caused an imbalance between factors regulating self-renewal or differentiation of the transplanted SSCs.     

Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance

p53 protects cells from DNA damage by inducing cell-cycle arrest upon encountering genomic stress. Among other pathways, p53 elicits such an effect by inhibiting mammalian target of rapamycin complex 1 (mTORC1), the master regulator of cell proliferation and growth. Although recent studies have indicated roles for both p53 and mTORC1 in stem cell maintenance, it remains unclear whether the p53-mTORC1 pathway is conserved to mediate this process under normal physiological conditions. Spermatogenesis is a classic stem cell-dependent process in which undifferentiated spermatogonia undergo self-renewal and differentiation to maintain the lifelong production of spermatozoa. To better understand this process, we have developed a novel flow cytometry (FACS)-based approach that isolates spermatogonia at consecutive differentiation stages. By using this as a tool, we show that genetic loss of p53 augments mTORC1 activity during early spermatogonial differentiation. Functionally, loss of p53 drives spermatogonia out of the undifferentiated state and causes a consistent expansion of early differentiating spermatogonia until the stage of preleptotene (premeiotic) spermatocyte. The frequency of early meiotic spermatocytes is, however, dramatically decreased. Thus, these data suggest that p53-mTORC1 pathway plays a critical role in maintaining the homeostasis of early spermatogonial differentiation. Moreover, our FACS approach could be a valuable tool in understanding spermatogonial differentiation.     

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu)

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.