We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis.

The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a Cterminal CAAX motif. The 3.9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.The inositol-polyphosphate 5-phosphatases (5-phosphatases) 1 are a large family of enzymes that remove the 5-position phosphate from the inositol ring of phosphatidylinositols including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), and the inositol phosphates, inositol 1,4,5-trisphosphate (Ins(1,4,5)-P 3 ) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ). Nine distinct mammalian 5-phosphatases have been identified and characterized, while four 5-phosphatases have been described in Saccharomyces cerevisiae. All 5-phosphatases are defined by the presence of a conserved 300-amino acid domain, which contains two signature motifs, proposed to mediate substrate binding and catalysis (1, 2). Although all 5-phosphatase enzymes contain these signature motifs, there is considerable diversity in the substrate specificity of 5-phosphatase isoforms. The enzyme family has been subclassified on this basis into four types, I-IV. The type I enzymes, characterized by the 43-kDa 5-phosphatase (also called 5-phosphatase I), hydrolyze Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 but not any of the 5-position phosphoinositide substrates; the type II 5-phosphatases, including synaptojanin, 5-phosphatase II (originally designated the 75-kDa 5-phosphatase), and the protein product of the oculocerebrorenal syndrome (OCRL) gene, hydrolyze PtdIns-(4,5)P 2 , PtdIns(3,4,5)P 3 , Ins(1,3,4,5)P 4 ...

Expression of bcl-w, a close relative of bcl-2 is essential for male fertility in mice. Although the initial wave of spermatogenesis in bcl-w 7/7 mice proceeds normally until 3 ± 4 weeks of age, adults fail to produce sperm. To clarify why bcl-w is essential for adult but not juvenile spermatogenesis, we investigated the expression pattern of eight bcl-2 family members. We found that both the level and pattern of expression varied in different cell types during juvenile and adult spermatogenesis. Anti-apoptotic genes bcl-w, bcl-2 and bcl-x L were all expressed in spermatogonia during juvenile spermatogenesis, but only bcl-w was detected in spermatogonia of adult mice. A similar shift was evident in Sertoli cells. This developmental regulation may co-ordinate physiological germ cell apoptosis in wild type mice and account for the time of onset for pathological germ cell apoptosis in bcl-w 7/7 animals. Cell Death and Differentiation (2001) 8, 225 ± 233.

Members of the TGF beta superfamily may compete for receptor occupancy and intracellular signaling molecules in specific developmental circumstances. We explored the potential importance of the TGF beta family inhibitor, Bambi (Bmp and activin membrane-bound inhibitor) by examining its pattern of mRNA expression in juvenile and adult rat tissues, with a focus on reproductive organs. The 1.8-kb transcript was ubiquitous, whereas a 3-kb transcript was unique to enriched spermatocyte and spermatid cell fractions and adult testis. The full-length rat cDNA is 89% (nucleic acid) and 95% (amino acid) identical to its human homolog, hnma. Using in situ hybridization, Bambi mRNA was detected in granulosa and thecal cells of adult ovaries and in spermatogonia, spermatocytes, round spermatids, and Sertoli cells of adult testes. In addition to a persistent signal in Sertoli cells in juvenile testes, this mRNA within germ cells appeared dramatically increased as gonocytes matured into spermatogonia immediately after birth. These data indicate that TGF beta superfamily signaling within male germ cells is down-regulated at the onset of spermatogenesis. The addition of exogenous activin A to 24-h cultures of newborn rat testis fragments decreased the Bambi mRNA level. Regulated Bambi mRNA synthesis may contribute to TGF beta superfamily signaling modulation in several organs, as suggested by its discrete expression switch in male germ cells.