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.     

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).     

Arrest of spermatogonial differentiation in jsd/jsd, Sl17H/Sl17H, and cryptorchid mice.

The nature of the spermatogenic arrest in cryptorchid C57Bl mice and in jsd/jsd and Sl17H/Sl17H mutant mice was identified by studying whole mounts of seminiferous tubules. In all three types of mice, virtually only A spermatogonia were found, topographically arranged in clones of 1 to 16 (rarely more) cells. These clonal sizes are typical for undifferentiated spermatogonia. The proportion of these cells lying in chains of more than 2 cells (50-70%) was comparable to that seen in epithelial stages VII-VIII in the normal epithelium. It is concluded that in all three types of mice, spermatogenesis is arrested at the point where the undifferentiated A spermatogonia, specifically A(al) spermatogonia, differentiate into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia. The remaining A spermatogonia were proliferating, but no accumulation of spermatogonia was present, as spermatogonial apoptosis also took place. Spermatogonial clones of all sizes were seen to undergo apoptosis, but there were relatively many large apoptotic clones, indicating that the clones became more vulnerable when they became larger. In contrast to what is seen in the normal epithelium, odd-numbered clones, not composed of 2(n) cells, were present, as well as clumps of 2 or more spermatogonial nuclei in the same cytoplasm, in all three types of mice. This indicates a lack of integrity of spermatogonial clones, also observed in other situations with a relative paucity of cells on the basal membrane. It is concluded that the differentiation of the undifferentiated spermatogonia, affected in all three types of mice as well as in vitamin A-deficient animals, is a rather vulnerable point in the spermatogenic developmental pathway.     

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.     

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.     

The radiation-induced block in spermatogonial differentiation is due to damage to the somatic environment, not the germ cells

Radiation and chemotherapeutic drugs cause permanent sterility in male rats, not by killing most of the spermatogonial stem cells, but by blocking their differentiation in a testosterone-dependent manner. However, it is not known whether radiation induces this block by altering the germ or the somatic cells. To address this question, we transplanted populations of rat testicular cells containing stem spermatogonia and expressing the green fluorescent protein (GFP) transgene into various hosts. Transplantation of the stem spermatogonia from irradiated adult rats into the testes of irradiated nude mice, which do not show the differentiation block of their own spermatogonia, permitted differentiation of the rat spermatogonia into spermatozoa. Conversely transplantation of spermatogonial stem cells from untreated prepubertal rats into irradiated rat testes showed that the donor spermatogonia were able to colonize along the basement membrane of the seminiferous tubules but could not differentiate. Finally, suppression of testosterone in the recipient irradiated rats allowed the differentiation of the transplanted spermatogonia. These results conclusively show that the defect caused by radiation in the rat testes that results in the block of spermatogonial differentiation is due to injury to the somatic compartment. We also observed colonization of tubules by transplanted Sertoli cells from immature rats. The present results suggest that transplantation of spermatogonia, harvested from prepubertal testes to adult testes that have been exposed to cytotoxic therapy might be limited by the somatic damage and may require hormonal treatments or transplantation of somatic elements to restore the ability of the tissue to support spermatogenesis.     

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 1994^1,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal². The use of donor males carrying the bacterial β-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals¹. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located³. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation^4,5, to study Sertoli cell-germ cell interaction^6,7, SSC homing and colonization^3,8, as well as SSC self-renewal and differentiation^9,10.Germ cell transplantation is also feasible in large species¹¹. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species¹². An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm¹³. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice¹⁴.     

Altered cell-surface targeting of stem cell factor causes loss of melanocyte precursors in Steel17H mutant mice.

The normal products of the murine Steel (Sl) and Dominant white spotting (W) genes are essential for the development of melanocyte precursors, germ cells, and hematopoietic cells. The Sl locus encodes stem cell factor (SCF), which is the ligand of c-kit, a receptor tyrosine kinase encoded by the W locus. One allele of the Sl mutation, Sl17H, exhibits minor hematopoietic defects, sterility only in males, and a complete absence of coat pigmentation. The Sl17H gene encodes SCF protein which exhibits an altered cytoplasmic domain due to a splicing defect. In this paper we analyzed the mechanism by which the pigmentation phenotype in Sl17H mutant mice occurs. We show that in embryos homozygous for Sl17H the number of melanocyte precursors is severely reduced on the lateral neural crest migration pathway by e11.5 and can no longer be detected by e13.5 when they would enter the epidermis in wildtype embryos. The reduced number of dispersing melanocyte precursors correlates with a reduction of SCF immunoreactivity in mutant embryos in all tissues examined. Regardless of the reduced amount, functional SCF is present at the cell surface of fibroblasts transfected with Sl17H mutant SCF cDNA. Since SCF immunoreactivity normally accumulates in basolateral compartments of SCF-expressing embryonic epithelial tissues, we analyzed the localization of wildtype and Sl17H mutant SCF protein in transfected epithelial (MDCK) cells in vitro. As expected, wildtype forms of SCF localize to and are secreted from the basolateral compartment. In contrast, mutant forms of SCF, which either lack a membrane anchor or exhibit the Sl17H altered cytoplasmic tail, localize to and are secreted from the apical compartment of the cultured epithelium. We suggest, therefore, that the loss of melanocyte precursors prior to epidermal invasion, and the loss of germ cells from mature testis, can be explained by the inability of Sl17H mutant SCF to be targeted to the basolateral compartment of polarized epithelial keratinocytes and Sertoli cells, respectively.     

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.     

Spermatogonial stem cell transplantation, cryopreservation and culture.

Testis cells of a fertile male mouse can be transplanted to the seminiferous tubules of an infertile male, where the donor spermatogonial stem cells will establish spermatogenesis and produce spermatozoa that transmit the donor haplotype to progeny. In addition, stem cells can be cryopreserved for long periods, thereby making male germ lines immortal. Recently, mouse testis cells have been cultured for longer than 3 months and, following transplantation, produced spermatogenesis. These techniques are likely to be applicable to many species, since rat testis cells can be cryopreserved and generate spermatogenesis in the seminiferous tubules of immunodeficient mice.     

A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse

In whole mounts of seminiferous tubules of C3H/101 F₁ hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis.

The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A₁ plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the A_pr and all of the A_al spermatogonia differentiate into A₁ spermatogonia. It was estimated that there are 2.5 × 10⁶ differentiating spermatogonia and 3.3 × 10⁵ undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycle.     

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.     

Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis

In mice, the juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis followed by failure of type A spermatogonial stem cells to repopulate the testis, rendering male animals sterile. It is not clear whether the defect in jsd resides in a failure of the somatic component to support spermatogenesis or in a failure that is intrinsic to the mutant's germ cells. To determine if the jsd intratesticular environment is capable of supporting spermatogenesis, germ cell transplantation experiments were performed in which C57BL/6 ROSA germ cells were transplanted into jsd recipients. To determine if jsd spermatogonia are able to develop in a permissive seminiferous environment, jsd germ cells were transplanted into W/W(v) and busulfan-treated C57BL/6 animals. The data demonstrate that up to 7 mo after transplantation of normal germ cells, jsd seminiferous tubules are capable of supporting spermatogenesis. In contrast, when jsd germ cells were transplanted into busulfan-treated C57BL/6 testis, or into testis of W/W(v) mice, no jsd-derived spermatogenesis was observed. The data support the hypothesis that the jsd phenotype is due to a defect in the germ cells themselves, and not in the intratubular environment.     

The Missing Niche for Spermatogonial Stem Cells: Do Blood Vessels Point the Way?

Although spermatogonial stem cell niches have been defined in lower organisms, their definitive localization in mammalian seminiferous tubules has been elusive. In a recent Science paper, Yoshida et al. (2007) elegantly demonstrated a vascular and interstitial tissue-associated niche for undifferentiated spermatogonia in the mouse.     

Availability of subfertile transgenic rats expressing the c-myc gene as recipients for spermatogonial transplantation

The spermatogonial transplantation system was applied to evaluate stem cell kinetics and niche quality and to produce gene-modified animals using the stem cells after homologous recombination-based selection. This study was designed to determine whether the transplanted spermatogonia were able to proliferate and differentiate in male rats expressing the c-myc transgene under control of the human metallothionein IIA promoter (MT-myc Tg rats). Donor testicular cells were prepared from heterozygous chicken beta actin (CAG)/enhanced green fluorescent protein (EGFP)-transgenic rats (EGFP Tg rats) during the second week after birth and injected into the seminiferous tubules of the MT-myc Tg rats (line-A and -B; both subfertile) or rats pretreated with busulfan to remove endogenous spermatogonia. Three to four months after transplantation, cell colonies with EGFP fluorescence were detected in 36% (4/11), 40% (8/20), and 71% (5/7) of the transplanted testes in line-A MT-myc Tg rats, line-B MT-myc Tg rats, and busulfan-treated rats, respectively. No EGFP-positive colonies were detected when wild-type male rats were used as recipients (0/7; testis-basis). The histopathological and immunofluorescent examination of the serial sections from the transplanted testes showed normal spermatogenesis of the donor spermatogonia, but atrophy of the recipient seminiferous tubules. Microinsemination with round spermatids and mature spermatozoa derived from EGFP-positive testes in line-A rats resulted 26% (10/39 transferred) and 23% (11/48 transferred) full-term offspring, respectively. Thus, the MT-myc Tg male rats were suitable as potent recipients for spermatogonial transplantation without any chemical pretreatment to remove the endogenous spermatogonia.     

Transplantation of male germ line stem cells restores fertility in infertile mice

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.     

Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes.

Recently a system was developed in which transplanted donor spermatogonial stem cells establish complete spermatogenesis in the testes of an infertile recipient. To obtain insight into stem cell activity and the behavior of donor germ cells, the pattern and kinetics of mouse spermatogonial colonization in recipient seminiferous tubules were analyzed during the 4 mo following transplantation. The colonization process can be divided into three continuous phases. First, during the initial week, transplanted cells were randomly distributed throughout the tubules, and a small number reached the basement membrane. Second, from 1 wk to 1 mo, donor cells on the basement membrane divided and formed a monolayer network. Third, beginning at about 1 mo and continuing throughout the observation period, cells in the center of the network differentiated extensively and established a colony of spermatogenesis, which expanded laterally by repeating phase two and then three. An average of 19 donor cell-derived colonies developed from 10(6) cells transplanted to the seminiferous tubules of a recipient testis; the number of colonized sites did not change between 1 and 4 mo. However, the length of the colonies increased from 0.73 to 5.78 mm between 1 and 4 mo. These experiments establish the feasibility of studying in a systematic and quantitative manner the pattern and kinetics of the colonization process. Using spermatogonial transplantation as a functional assay, it should be possible to assess the effects of various treatments on stem cells and on recipient seminiferous tubules to provide unique insight into the process of spermatogenesis.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

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.