Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

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.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

Increment of murine spermatogonial cell number by gonadotropin-releasing hormone analogue is independent of stem cell factor c-kit signal

Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steeldickie (Sl/Sld) mutant mice, which lack expression of membrane bound form SCF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.     

Gfra1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation.     

Stem cell protein Piwil2 modulates expression of murine spermatogonial stem cell expressed genes

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ cells of adult testis, suggesting that this gene functions in spermatogonial stem cell self-renewal. In order to find molecular mechanisms underlying stem cell activity mediated by Piwil2 gene, an in vitro gain of function cell culture model was established. Messenger RNAs isolated from cells expressing Piwil2 and mRNAs isolated from cells without Piwil2 expression were compared using a stem cell array technique. It was shown that Piwil2 modulates expression of stem cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and spermatogonial cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in stem cells proliferation (Pdgfrb), energy metabolism (Slc2a1), cell adhesion, cell-cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ cell differentiation (Stra8). The expression of these markers in spermatogonial stem cells and other nongerminal stem cells suggests that these cells share elements of common molecular machinery with stem cells in other tissues which are modulated by stem cell protein Piwil2.     

Morphological characteristics of the spermatogonial stem cells in man

The present investigation is concerned with establishing morphological criteria of spermatogonial stem cells in man. Testicular biopsies from patients having undergone semicastration for malignant tumors and radio- and chemotherapy for one year following the operation were studied light and electron microscopically. Those spermatogonial types that survived the treatment were regarded as stem cells in view of the fact that the stem cells, in contrast to the more differentiated spermatogonia, are radiation resistant and less sensitive to various noxious agents. In 7 out of 28 cases examined, a small number of spermatogonia was found adjacent to the basement membrane. The majority of these cells show the characteristic features of pale type A spermatogonia, while a few cells may represent variants of this cell type. The dark type A spermatogonia are almost completely eliminated from the seminiferous tubules. A concept is proposed that the stem cells of the human testis may be derived from the pale type A spermatogonia or the variants of this cell type.     

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.     

Study of the potential spermatogonial stem cell compartment in dogfish testis, <Emphasis Type="Italic">Scyliorhinus canicula</Emphasis> L.

In the lesser-spotted dogfish (Scyliorhinus canicula), spermatogenesis takes place within spermatocysts made up of Sertoli cells associated with stage-synchronized germ cells. As shown in testicular cross sections, cysts radiate in maturational order from the germinative area, where they are formed, to the opposite margin of the testis, where spermiation occurs. In the germinative zone, which is located in a specific area between the tunica albuginea of the testis and the dorsal testicular vessel, individual large spermatogonia are surrounded by elongated somatic cells. The aim of this study has been to define whether these spermatogonia share characteristics with spermatogonial stem cells described in vertebrate and non-vertebrate species. We have studied their ultrastructure and their mitotic activity by 5′-bromo-2′-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) immunodetection. Additionally, immunodetection of c-Kit receptor, a marker of differentiating spermatogonia in rodents, and of α- and β-spectrins, as constituents of the spectrosome and the fusome, has been performed. Ultrastructurally, nuclei of stage I spermatogonia present the same mottled aspect in dogfish as undifferentiated spermatogonia nuclei in rodents. Moreover, intercellular bridges are not observed in dogfish spermatogonia, although they are present in stage II spermatogonia. BrdU and PCNA immunodetection underlines their low mitotic activity. The presence of a spectrosome-like structure, a cytological marker of the germline stem cells in Drosophila, has been observed. Our results constitute the first step in the study of spermatogonial stem cells and their niche in the dogfish. G.L. is supported by a CIFRE grant (ANRT and C.RIS Pharma).     

Bcl-2 inhibits apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner

The growth, differentiation, and death/survival of spermatogonia are precisely regulated for the proper production of spermatozoa. We have previously shown that Bcl-2 ectopically expressed in spermatogonia caused the inhibition of normal spermatogonial apoptosis and the subsequent failure of differentiation in transgenic mice. In addition, the growth of spermatogonial stem cells seemed to be temporally arrested in the transgenic mice. In the present study, we attempted to examine whether the abnormality of spermatogonia described above was caused by Bcl-2 misexpression in the spermatogonia or by an abnormal spermatogenic environment of the transgenic mice. We transplanted testicular cells of transgenic mice to seminiferous tubules of W/Wv mice in which transplanted normal testicular cells can undergo spermatogenesis. We found that the transplanted spermatogonia of the transgenic mice reproduced a series of abnormal changes including temporal growth arrest of spermatogonial stem cells and abnormal accumulation of spermatogonia in tubules, which were also observed in the testes of the transgenic mice. The results indicated that Bcl-2 inhibited apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner. We also cultured testicular cells of transgenic mice and found that the spermatogonia of the transgenic mice were better able to survive than were those of wild-type mice but that their differentiation was not affected. The result suggested that failure of differentiation of the accumulated spermatogonia in the transgenic testes is not due to the abnormality of the bcl-2 misexpressing spermatogonia, but may be caused by extrinsic problems including improper interaction of spermatogonia with supporting cells.     

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 radiosensitivity of spermatogonial stem cells in C3H/101 F1 hybrid mice

The radiosensitivity of spermatogonial stem cells of C3H/HeH × 101/H F₁ hybrid mice was determined by counting undifferentiated spermatogonia at 10 days after X-irradiation. During the spermatogenic cycle, differences in radiosensitivity were found, which were correlated with the proliferative activity of the spermatogonial stem cells. In stage VIII_irr, during quiescence, the spermatogonial stem cells were most radiosensitive with a D₀ of 1.4 Gy. In stages XI_irr−V_irr, when the cells were proliferatively active, the D₀ was about 2.6 Gy. Based on the D₀ values for sensitive and resistant spermatogonia and on the D₀ for the total population, a ratio of 45:55% of sensitive to resistant spermatogonial stem cells was estimated for cell killing.

When the present data were compared with data on translocation induction obtained in mice of the same genotype, a close fit was obtained when the translocation yield (Y; in % abnormal cells) after a radiation dose D was described by Y = e^τD, with τ = 1 for the sensitive and τ = 0.1 for the resistant spermatogonial stem cells, with a maximal e^τD of 100.     

Akt mediates self-renewal division of mouse spermatogonial stem cells

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.