Previous studies have shown that transcription factors GATA-4 and GATA-6 are expressed in granulosa and thecal cells of the mouse ovary and that GATA-4 expression in ovarian tissue is regulated by gonadotropins. Given the emerging role of GATA-4 and GATA-6 in gonadal cells, we have now studied the expression and regulation of these factors in the mouse testis and testicular cell lines. In situ hybridization demonstrated GATA-4 messenger RNA (mRNA) in the fetal testis at 13.5 days postcoitum. Both GATA-4 and GATA-6 transcripts were observed in late fetal, neonatal, juvenile, and adult Sertoli cells. In addition, GATA-4 mRNA was detected in interstitial cells throughout development. Immunohistochemistry demonstrated GATA-4 protein in both Sertoli and Leydig cells in postnatal animals. The regulation of GATA-4 and GATA-6 expression was explored using established testicular cell lines. Treatment of Leydig tumor cell lines with hCG resulted in a modest, but statistically significant, increase in the steady state level of GATA-4 mRNA, comparable to the previously described effect of FSH on GATA-4 expression in Sertoli cell lines. Gonadotropin or androgen action was not, however, a prerequisite for the basal expression of GATA-4 or GATA-6 in the testis, as their presence in Sertoli and Leydig cells was demonstrated in genetically hypogonadal hpg mice, in rats treated with GnRH receptor antagonist, and in Sertoli cells after chemical abolition of Leydig cells. Cotransfection studies using a GATA-4 expression plasmid and an inhibin alpha promoter/reporter gene construct in Leydig and granulosa tumor cell lines revealed that the inhibin alpha promoter harboring essential GATA-binding sites can be trans-activated by GATA-4. In light of these results, we propose that transcription factors GATA-4 and GATA-6 play differing roles in the maturation and function of testicular somatic cells.

Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.

Transcription factor GATA-4 has been suggested to have a role in mammalian gonadogenesis, e.g., through activation of the Müllerian-inhibiting substance (MIS) gene expression. Although the expression of GATA-4 during gonadogenesis has been elucidated in detail, very little is known about FOG-2, an essential cofactor for GATA-4, in ovarian development. We explored in detail the expression of FOG-2 and GATA-4 in the fetal and postnatal mouse ovary and in the fetal testis using Northern blotting, RNA in situ hybridization, and immunohistochemistry. GATA-4 and FOG-2 are evident in the bipotential urogenital ridge, and their expression persists in the fetal mouse ovary; this result is different from earlier reports of GATA-4 downregulation in the fetal ovary. In contrast to ovary, FOG-2 expression is lost in the fetal Sertoli cells along with the formation of the testicular cords, leading to the hypothesis that FOG-2 has a specific role in the fetal ovaries counteracting the transactivation of the MIS gene by GATA-4. In vitro transfection assays verified that FOG-2 is able to repress the effect of GATA-4 on MIS transactivation in granulosa cells. In postnatal ovary, granulosa cells of growing follicles express FOG-2, partially overlapping with the expression of MIS. These data suggest an important role for FOG-2 and the GATA transcription factors in the developing ovary.

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Objective: The transcription factors GATA-1 and GATA-4 have been implicated in the regulation of testicular development and function. Their cofactors FOG-1 and FOG-2 are expressed in the gonads, but their cell-specific and developmental expression in the testis remains unresolved. Therefore, we analyzed GATA-1, GATA-4, FOG-1 and FOG-2 expression in detail, from undifferentiated male urogenital ridge to adult testis. Methods: Immunohistochemistry and in situ hybridization were applied on mouse testicular samples. Results: GATA-4 and FOG-2, but not GATA-1 or FOG-1, were expressed as early as in the male urogenital ridge. FOG-2 expression was localized in the Sertoli cells at embryonal day 12.5 (E12.5), but it diminished with advancing fetal testicular development. In E17.5 testis, FOG-2 was present only in the testicular capsule and a subset of fetal Leydig cells. FOG-1 was expressed from E15.5 Sertoli cells onwards, whereas GATA-1 was not detected during the fetal period at all. In the postnatal testis, FOG-2 was abundantly expressed immediately after birth, but in adult testis its expression was predominantly restricted to stage VII -XII seminiferous tubules. Stage specificity was also found for FOG-1, which, similarly to GATA-1, was abundantly expressed in stage VII -XII tubules during adulthood. Conclusions: Our results indicate that FOG-2, in addition to GATA-4, has a role in early gonadal development and sexual differentiation, and FOG-1 at later fetal stages, while GATA-1 executes its action postnatally. The findings suggest that, in contrast to the hematopoietic system and the heart, GATA-1 and GATA-4 do not use FOG-1 and FOG-2 respectively as their only cofactors during the early stages of testicular development.

SUMMARY T helper cell type 1 (Th1) response to gluten has been implicated in the pathogenesis of coeliac disease (CD). To characterize immunological activation and mild inflammations leading to overt CD in potential coeliac patients, jejunal biopsies were obtained from family members of patients with CD or dermatitis herpetiformis (DH). Nine family members and one latent CD, eight CD patients and eight normal controls furnished jejunal biopsy specimens. Immunohistochemical staining of sections for interleukin‐1α (IL‐1α), IL‐2, IL‐4, interferon‐γ (IFN‐γ), tumour necrosis factor α (TNF‐α), CD3, γδ‐T cell receptor (γδ‐TCR), and αβ‐TCR was carried out with monoclonal antibodies. Further, expression of IL‐4 and IFN‐γ messenger RNA was detected by radioactive in situ hybridization in these same samples. In lamina propria, CD patients and potential CD patients had higher densities of IL‐2 (P = 0·028, P = 0·043), IL‐4 (P = 0·021, P = 0·034) and IFN‐γ positive cells (P = 0·000, P = 0·009) than did controls. Moreover, CD patients showed a higher density of TNF‐α positive cells (P = 0·012, P = 0·001) than the other two groups, and expression of IFN‐γ mRNA (P = 0·035) was higher in them than in the other two study groups. Additionally, higher densities of TNF‐α and IFN‐γ positive cells occurred in potential CD patients with high γδ‐TCR+ intraepithelial lymphocytes (IELs). Our findings support the hypothesis that lamina propria T cells and macrophages, through their secretion of cytokines, play a central role in the pathogenesis of coeliac disease. The inflammatory cytokines found in potential CD specimens strongly suggest that these inflammatory markers can be identified long before visible villous changes have occurred.

The GATA family of transcription factors have been implicated in regulating the development and function of many organs. Furthermore, they have been linked to signaling cascades regulating cell fate through apoptosis. GATA-6 has been shown to be expressed in the gonads, but its cell-specific expression in the testis has remained unclear. We have studied GATA-6 expression in human fetal testis using in situ hybridization and immunohistochemistry and compared these results with the expression of the apoptosis-related proteins Bcl-2 and Bax. Furthermore, apoptosis was studied by thymidine deoxyribose-mediated deoxy-UTP nick end labeling assay, and cell proliferation by Ki-67 immunohistochemistry. GATA-6 mRNA and protein were expressed in Sertoli and Leydig cells early in gestation. Apoptotic cells were scanty between wk 16 and 40, and proliferation significantly ceased during the third trimester, supporting the view that only a little tissue remodeling occurs in the late fetal testis. Bax was present throughout the fetal period, whereas Bcl-2 expression decreased toward term. Neither of these factors correlated to the extent of apoptosis, and thus their role in the regulation of apoptosis in the fetal testis remains open. Despite strong expression, GATA-6 did not correlate with apoptosis or cell proliferation and is therefore unlikely to be directly involved in these processes in the human fetal testis.

Previous studies have shown that transcription factors GATA-4 and GATA-6 are expressed in granulosa and thecal cells of the mouse ovary and that GATA-4 expression in ovarian tissue is regulated by gonadotropins. Given the emerging role of GATA-4 and GATA-6 in gonadal cells, we have now studied the expression and regulation of these factors in the mouse testis and testicular cell lines. In situ hybridization demonstrated GATA-4 messenger RNA (mRNA) in the fetal testis at 13.5 days postcoitum. Both GATA-4 and GATA-6 transcripts were observed in late fetal, neonatal, juvenile, and adult Sertoli cells. In addition, GATA-4 mRNA was detected in interstitial cells throughout development. Immunohistochemistry demonstrated GATA-4 protein in both Sertoli and Leydig cells in postnatal animals. The regulation of GATA-4 and GATA-6 expression was explored using established testicular cell lines. Treatment of Leydig tumor cell lines with hCG resulted in a modest, but statistically significant, increase in the steady state level of GATA-4 mRNA, comparable to the previously described effect of FSH on GATA-4 expression in Sertoli cell lines. Gonadotropin or androgen action was not, however, a prerequisite for the basal expression of GATA-4 or GATA-6 in the testis, as their presence in Sertoli and Leydig cells was demonstrated in genetically hypogonadal hpg mice, in rats treated with GnRH receptor antagonist, and in Sertoli cells after chemical abolition of Leydig cells. Cotransfection studies using a GATA-4 expression plasmid and an inhibin alpha promoter/reporter gene construct in Leydig and granulosa tumor cell lines revealed that the inhibin alpha promoter harboring essential GATA-binding sites can be trans-activated by GATA-4. In light of these results, we propose that transcription factors GATA-4 and GATA-6 play differing roles in the maturation and function of testicular somatic cells.