Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.     

Functional analysis of stem cells in the adult rat testis

Adult stem cells maintain several self-renewing systems and processes in the body, including the epidermis, hematopoiesis, intestinal epithelium, and spermatogenesis. However, studies on adult stem cells are hampered by their low numbers, lack of information about morphologic or biochemical characteristics, and absence of functional assays, except for hematopoietic and spermatogonial stem cells. We took advantage of the recently developed spermatogonial transplantation technique to analyze germ line stem cells of the rat testis. The results indicate that the stem cell concentration in rat testes is 9.5-fold higher than that in mouse testes, and spermatogenic colonies derived from rat donor testis cells are 2.75 times larger than mouse-derived colonies by 3 mo after transplantation. Therefore, the extent of spermatogenesis from rat stem cells was 26-fold greater than that from mouse stem cells at the time of recipient testis analysis. Attempts to enrich spermatogonial stem cells in rat testis populations using the experimental cryptorchid procedure were not successful, but selection by attachment to laminin-coated plates resulted in 8.5-fold enrichment. Spermatogonial stem cells are unique among adult stem cells because they pass genetic information to the next generation. The high concentration of stem cells in the rat testis and the rapid expansion of spermatogenesis after transplantation will facilitate studies on stem cell biology and the introduction of genetic modifications into the male germ line. The functional differences between spermatogonial stem cells of rat vs. mouse origin after transplantation suggest that the potential of these cells may vary greatly among species.     

Epigenetic modifications and self-renewal regulation of mouse germline stem cells

Germline stem (GS) cells were established from gonocytes and spermatogonia of postnatal mouse testes. GS cells proliferate in the presence of several kinds of cytokines, and a small percentage of GS cells also show spermatogonial stem cell (SSC) activity, i.e., they differentiate into sperm after being transplanted into infertile mouse testes without endogenous spermatogenesis. Interestingly, in GS cell culture, we also found that pluripotent stem cells (multipotent germline stem cells (mGS cells)) could be derived and these mGS cells do not have normal androgenetic genomic imprinting marks that are shown in GS cells, e.g., H19 hypermethylation. A new culture system for fetal male germ cells (embryonic GS (eGS) cells) has also been recently developed. Although these cells exhibited SSC potential, the offspring from cultured cells showed heritable imprinting defects in their DNA methylation patterns. In an attempt to understand the self-renewal machinery in SSCs, we transfected H-Ras and cylin D2 into GS cells, and successfully reconstructed the SSC self-renewal ability without using exogenous cytokines. Although these cells showed SSC activity in germ cell transplantation assays, we also found development of seminomatous tumors, possibly induced by excessive self-renewing signal. These stem cell culture systems are useful tools not only for understanding the mechanisms of self-renewal or epigenetic reprogramming but also for clarifying the mechanism of germ cell tumor development.     

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.     

Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0-3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4-5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12-28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3-4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.     

Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W^v mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.     

Stem cell and niche development in the postnatal rat testis

Adult tissue stem cells self-renew and differentiate in a way that exactly meets the biological demand of the dependent tissue. We evaluated spermatogonial stem cell (SSC) activity in the developing rat testis and the quality and accessibility of the stem cell niche in wild type, and two busulfan-treated models of rat pup recipient testes using an SSC transplantation technique as a functional assay. While our results revealed a 69-fold increase in stem cell activity during rat testis development from neonate to adult, only moderate changes in SSC concentration were observed, and stem cells from neonate, pup, and adult donor testes produce spermatogenic colonies of similar size. Analysis of the stem cell niche in recipient rat testes demonstrated that pup testes support high levels of donor stem cell engraftment when endogenous germ cells are removed or compromised by busulfan treatment. Fertility was established when rat pup donor testis cells were transplanted into fetal- or pup-busulfan-treated recipient rat pup testes, and the donor genotype was transmitted to subsequent generations. These results provide insight into stem cell/niche interactions in the rat testis and demonstrate that techniques originally developed in mice can be extended to other species for regenerative medicine and germline modification.     

Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells

Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I(-) Thy-1(+) alpha 6-integrin(+) alpha v-integrin(-/dim) throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.     

Identification, isolation, and in vitro culture of porcine gonocytes

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.     

Retrovirus-mediated modification of male germline stem cells in rats

The ability to isolate, manipulate, and transplant spermatogonial stem cells provides a unique opportunity to modify the germline. We used the rat-to-nude mouse transplantation assay to characterize spermatogonial stem cell activity in rat testes and in culture. Our results indicate that rat spermatogonial stem cells can survive and proliferate in short-term culture, although a net loss of stem cells was observed. Rat spermatogonial stem cells also were susceptible to transduction with a retroviral vector carrying a lacZ reporter transgene. Using a 3-day periodic infection protocol, 0.5% of stem cells originally cultured were transduced and produced transgenic colonies of spermatogenesis in recipient mouse testes. The level of transgenic donor-derived spermatogenesis observed in the rat-to-mouse transplantation was similar to levels that produced transgenic progeny in the mouse-to-mouse transplantation. This work provides a basis for understanding the biology of rat spermatogonial stem cells. Development of an optimal rat recipient testis model and application of these methods for germline modification will enable the production of transgenic rats, potentially valuable tools for evaluating genes and their functions. In addition, these methods may be applicable in other species where existing transgenic methods are inefficient or not available.     

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.     

Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models

Spermatogenesis is a complex and productive process that originates from stem cell spermatogonia and ultimately results in formation of mature spermatozoa. The stem cell undergoes self-renewal throughout life, but study of its biological characteristics has been difficult because a very small number (2 to 3 in 10(4) cells) exist in the testis and they can only be identified by function. Although the development of the spermatogonial transplantation technique has provided an assay system for stem cells, efficient methods to enrich stem cells have not been available. Here, we examined two infertile mouse models, Steel/Steel(Dickie)(Sl/Sl(d)) and experimental cryptorchid, as a source of testis cell populations enriched in stem cells. The Sl/Sl(d) testis showed little enrichment, which raises questions about how adult stem cell number is determined and about the currently accepted belief that adult stem cells are independent of Sl factor. The cells recovered from cryptorchid testes were enriched for stem cells 25-fold (colonies) or 50-fold (area) compared to wild-type testes. The cryptorchid condition does not affect stem cell activity, but eliminates almost all differentiated cells, and about 1 in 200 cells is a stem cell. Thus, cryptorchid testes provide an important approach for purification and characterization of spermatogonial stem cells.     

In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues

Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sl^d mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.     

The role of the tumor suppressor p53 in spermatogenesis

The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in spermatogonia, suggesting that, in contrast to other reports, p53 does have a role in spermatogonia. To determine the possible role of p53 in spermatogonia, histological analysis was performed in testes of both p53 knock out C57BL/6 and FvB mice. The results indicate that p53 is an important factor in normal spermatogonial cell production as well as in the regulation of apoptosis after DNA damage. First, p53 knock out mouse testes contained about 50% higher numbers of A1 spermatogonia, indicating that the production of differentiating type spermatogonia by the undifferentiated spermatogonia is enhanced in these mice. Second, 10 days after a dose of 5 Gy of X-rays, in the p53 knock out testes, increased numbers of giant sized spermatogonial stem cells were found, indicating disturbance of the apoptotic process in these cells. Third, in the p53 knock out testis, the differentiating A2-B spermatogonia are more radioresistant compared to their wild-type controls, indicating that p53 is partly indispensable in the removal of lethally irradiated differentiating type spermatogonia. In accordance with our immunohistochemical data, Western analysis showed that levels of p53 are increased in total adult testis lysates after irradiation. These data show that p53 is important in the regulation of cell production during normal spermatogenesis either by regulation of cell proliferation or, more likely, by regulating the apoptotic process in spermatogonia. Furthermore, after irradiation, p53 is important in the removal of lethally damaged spermatogonia.     

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.     

Isolation of male germ-line stem cells; influence of GDNF

The identification and physical isolation of testis stem cells, a subset of type A spermatogonia, is critical to our understanding of their growth regulation during the first steps of spermatogenesis. These stem cells remain poorly characterized because of the paucity of specific molecular markers that permit us to distinguish them from other germ cells. Thus, the molecular mechanisms driving the first steps of spermatogenesis are still unknown. We show in the present study that GFR alpha-1, the receptor for GDNF (glial cell line-derived neurotrophic factor), is strongly expressed by a subset of type A spermatogonia in the basal part of the seminiferous epithelium. Using this characteristic, we devised a method to specifically isolate these GFR alpha-1-positive cells from immature mouse testes. The isolated cells express Ret, a tyrosine kinase transmembrane receptor that mediates the intracellular response to GDNF via GFR alpha-1. After stimulation with rGDNF, the isolated cells proliferated in culture and underwent the first steps of germ cell differentiation. Microarray analysis revealed that GDNF induces the differential expression of a total of 1124 genes. Among the genes upregulated by GDNF were many genes involved in early mammalian development, differentiation, and the cell cycle. This report describes the first isolation of a pure population of GFR alpha-1-positive cells in the testis and identifies signaling pathways that may play a crucial role in maintaining germ-line stem cell proliferation and/or renewal.     

Effect of the W mutation, for white belly spot, on testicular germ cell differentiation in mice.

The effect of the mutation for white belly spot controlled by the dominant gene W on spermatogenesis in mice was examined by experimental cryptorchidism and its surgical reversal. The course of spermatogenesis from spermatogonia to spermatid was normal in intact testes of W/+ mice. In cryptorchid testes, there was no difference in the number and activity of Type A spermatogonia between the testes of W/+ and +/+ mice, in mitotic and labelling indices. Although surgical reversal of the cryptorchid testis resulted in regenerative differentiation of germ cells in both genotypes, the recovery of cell differentiation in the W/+ testis was slower than in the +/+ testis. There were fewer germ cells, such as intermediate-Type B spermatogonia or more advanced ones, in W/+ testes. On Day 17 after surgical reversal, cell associations in W/+ testes were abnormal and the numbers of intermediate-Type B spermatogonia, spermatocytes and spermatids were approximately 70, 50 and 15%, respectively, of those in +/+ testes. These results indicate that the W gene affects spermatogenic cell differentiation in adult mice.     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.     

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.