Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-, is used by SCF/ckit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation.

The objective of this study was to examine the expression and activation of the c-kit receptor, a specific receptor for kit ligand (stem cell factor, steel factor), in rat type A spermatogonia. Testes were obtained from 9-day-old rats, decapsulated, and then subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location similar to that found in the in vivo 9-day-old testis. The synthesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Localization of the c-kit receptor in the isolated cells was determined by immunocytochemistry using an antibody to c-kit protein. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the c-kit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the c-kit receptor at approximately 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-kit receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may lead to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation.

We studied the distribution of messenger RNA (mRNA) that encodes for vascular endothelial growth factor (VEGF) within the primate ovary by in situ hybridization and Northern analysis to determine if the presence of mRNA for this angiogenic factor is associated with structures within the ovary in which angiogenesis is thought to play a role in development and/or function. In situ hybridization to sections of cynomolgus ovaries with a 35S-labeled antisense RNA probe revealed specific tissue localization within the follicle as well as the corpus luteum, but not stromal tissue. Intense expression of mRNA for VEGF during the late follicular phase was confined to the maturing follicle which, we presume, was destined for ovulation. Hybridization within the corpus luteum exhibited a punctate pattern suggesting that there may be specific cells within the corpus luteum that express mRNA for VEGF. The expression of mRNA for VEGF during the early and late luteal phase of the menstrual cycle was studied by Northern analysis. Messenger RNAs were detectable at approximately 3.7 and 5.0 kb positions in corpora lutea collected during the early luteal phase of the menstrual cycle (days 3-5 postovulation). No hybridization signals were observed with RNA prepared from regressing corpora lutea (1-2 days following the onset of menses). The gonadotropic regulation of the expression of mRNA for VEGF in the corpus luteum was studied by treating monkeys with a potent GnRH antagonist during the midluteal phase of the menstrual cycle. Administration of the antagonist for 1 or 2 days did not alter the expression of mRNA for VEGF in comparison to corresponding controls. However, a 3-day treatment regimen brought about a significant reduction in the levels of mRNA for VEGF (P less than 0.01). These studies demonstrate a development-related expression of mRNA for VEGF in the ovary during the menstrual cycle and are consistent with the hypothesis that VEGF may play important roles in follicle selection and corpus luteum function in primates.

Spermatogenesis is initiated with the divisions of the type A spermatogonial stem cells; however, the regulation of this stem cell population remains unknown. In order to obtain a better understanding of the biology of these cells, type A spermatogonia were isolated from 80-day-old pig testes by sedimentation velocity at unit gravity. The cells were cultured for up to 120 h in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12) or a potassium-rich medium derived by the simplex optimization method (KSOM). At the end of the 120-h culture period, 30-50% of the spermatogonia were viable in KSOM, whereas in DMEM/F12 very few cells survived. Using KSOM as the culture medium, the effects of stem cell factor (SCF) and granulocyte macrophage-colony stimulating factor (GM-CSF) were studied. SCF significantly enhanced the percentage of cell survival at 100 ng/ml but not at lower concentrations. In comparison, GM-CSF promoted survival at relatively low concentrations (0.01, 0.1, and 1 ng/ml). At a higher dose (10 ng/ml), a significant reduction in percentage of cell survival was observed. The combination of SCF with GM-CSF had no significant effect on the percentage survival of type A spermatogonial cells. These data indicate that SCF and GM-CSF play a role in the regulation of survival and/or proliferation of type A spermatogonia.

Overexpression of vascular endothelial growth factor (VEGF) in the testis of transgenic mice induces infertility, suggesting a potential role for VEGF in the process of spermatogenesis. Spermatogenesis occurs within the confines of the seminiferous tubules, and the seminiferous epithelium lining these tubules consists of Sertoli cells and germ cells in various stages of maturation. We investigated the source of VEGF and VEGF-target cells within the seminiferous tubules of the normal mouse testis. Sections of testes fixed in Bouin solution and embedded in paraffin were subjected to immunofluorescent staining with specific antibodies against VEGF, and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Total RNA was extracted from isolated populations of Sertoli cells, type A spermatogonia, pachytene spermatocytes, and spermatids. Primer pairs specific for VEGF and its receptors were designed and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. Immunofluorescent studies indicated that VEGF is strongly expressed in the cytoplasm of Sertoli cells. VEGFR-1 and VEGFR-2 were not expressed by the Sertoli cell. In contrast, a differential expression of VEGF receptors was observed in germ cells. Although VEGFR-2 was expressed in the cytoplasm of type A spermatogonia, VEGFR-1 was expressed in the acrosomal region of spermatids and spermatozoa. Pachytene spermatocytes did not exhibit any staining. Further, we examined the transcription of VEGF and its receptors by RT-PCR. VEGF was actively transcribed only in Sertoli cells. The transcription of VEGFR-2 was confined to type A spermatogonia. Interestingly, VEGFR-1 was transcribed both in pachytene spermatocytes and round spermatids. The mRNA expression of VEGFR-1 and VEGFR-2 in germ cells was inversely correlated during postnatal development of the mouse testis. Thus, VEGF may play a potential role in regulating the initial stages of the process of spermatogonial proliferation through VEGFR-2 and spermiogenesis through VEGFR-1.

Members of the Notch gene family have been shown to play an important role in the control of cell fate in many developmental systems. We hypothesized that the fate of the male germ line stem cells may also be mediated through the Notch signaling pathway. We therefore sought to determine whether the components of the Notch pathway are expressed in the mouse testis. Western blot analysis revealed the expression of three Notch receptors (Notch 1, Notch 2, and Notch 3), Notch ligands (Jagged 1, Jagged 2, and Delta 1), and presenilin 1 (PS1) in neonatal mouse testis. We then examined their cellular localization by immunohistochemical analysis of cocultures of spermatogonia and Sertoli cells. The 3 Notch receptors were found to be expressed in spermatogonia. Sertoli cells expressed only Notch 2 receptor. Among the Notch ligands, Delta 1 and Jagged 1 were localized exclusively in spermatogonia and Sertoli cells, respectively. PS1 was apparent in both spermatogonia and Sertoli cells. The presence of Notch receptors and Notch ligands in spermatogonia and Sertoli cells indicates that these cells are capable of responding to and eliciting Notch signaling during the process of spermatogenesis. Key words: Cell fate, delta, jagged, presenilin, spermatogenesis.

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man.

Gene expression profiling was performed using the National Institute on Aging 15,000-cDNA microarray to reveal the differential expression pattern of 160 genes between meiotic pachytene spermatocytes and postmeiotic round spermatids of the mouse. Our results indicate that more genes are expressed in spermatids than in spermatocytes. Genes participating in cell cycle regulation and chromatin structure and dynamics are preferentially expressed in spermatocytes, while genes for protein turnover, signal transduction, energy metabolism, and intracellular transport are prevalent in spermatids. This suggests that a switch of functional requirement occurs when meiotic germ cells differentiate into haploid spermatids. Concordant expression patterns were obtained when quantitative real-time polymerase chain reaction was performed to verify the microarray data. Interestingly, the majority of the differentially expressed genes were underrepresented in mitotic type A spermatogonia, and they were preferentially expressed in the testis. Our results suggest that an even higher proportion of the mouse genome is devoted to male gamete development from meiosis than was previously estimated. We also provide evidence that underscores the advantage of using purified germ cells over whole testes in profiling spermatogenic gene expression to identify transcripts that demonstrate stage-specific expression patterns. gamete biology; testis; spermatogenesis; gametogenesis; developmental biology