Follistatin and activin A production by the male reproductive tract

Follistatin is a binding protein for the activin and inhibin family of hormones, regulating their biological activity. In the male reproductive tract, the interaction of these factors is likely to be involved in the regulation of the proliferation of several cell types. We have investigated the presence of follistatin and activin A in seminal plasma using specific immunoassays and have localized follistatin and activin/inhibin subunits in the adult human testis, prostate and seminal vesicle to establish their likely sources. High concentrations of immunoreactive follistatin were present in seminal plasma in normal men (mean 97.9 ng/ml; 1.43 ng/ml in peripheral plasma) and were similar in men with oligo/azoospermia and following vasectomy. Follistatin immunoreactivity was localized to both Leydig and Sertoli cells of the testis, and to epithelial cells of the prostate gland and seminal vesicle, which are likely to be the predominant sources of the hormone in seminal plasma. Activin A was also present in seminal plasma in normal men but was undetectable following vasectomy, thus deriving from the testis. Consistent with this finding, the betaA-subunit was immunolocalized in Sertoli and Leydig cells but was not present in seminal vesicle or prostate gland. The functional significance of the high concentrations of follistatin secreted into seminal plasma by the prostate gland and/or seminal vesicle is uncertain, but they may regulate the biological activity of testis-derived activin A and inhibin B.      

Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation and tissue remodelling, we hypothesized that activin A is involved in the paracrine regulation of decidualization. We examined the expression of activin receptors (ActRs) by semi-quantitative and real-time RT-PCR. mRNA for all ActR subtypes (Ia, Ib, IIa and IIb) was detected in endometrium, with maximal expression in the early secretory phase and in early pregnancy. ActR protein was localized exclusively to stromal and endothelial cells. This expression pattern was confirmed by in-situ hybridization. Activin bioavailability is locally regulated by its binding protein, follistatin, and also by the antagonist, inhibin. Inhibin competition for ActRII binding is enhanced by the binding protein, betaglycan. Follistatin and betaglycan were also detected in the endometrium, localized to stromal and epithelial cells. This co-expression of activin subunits, receptors and binding proteins indicates that stromal cells are capable of responding to activin, and that there is tight local regulation of activin action within the endometrium. As activin production is up-regulated in decidual cells, this provides further evidence for an involvement of activins during stromal cell decidualization.     

Experimental Sertoli cell tumors in the mouse and their progression into a mixed germ cell-sex cord proliferation.

Males of transgenic families where the large T protein of polyoma virus is expressed in the seminiferous epithelium of the testis (Sertoli and germ cells) develop bilateral testicular tumors when they become old (15 to 18 months). The histological features of these tumors revealed a neoplastic proliferation of Sertoli cell origin. Occasional isolated germ cells arrested at premeiotic stages were seen in the tumor. They did not participate in tumoral proliferation and their malignant character could not at first be established. Tumor cells injected in athymic (nu/nu) mice generated secondary tumors. In this case, a proliferative component of non-Sertoli origin was clearly evident. Its ultrastructural characteristics and the expression of genes that are transcribed in vivo in male germ cells (c-kit, LDH-X, and Hox a-4) suggest the progression of an initial, apparently pure Sertoli cell tumor into a mixed proliferation.     

成人睾丸支持细胞的分离培养及其免疫豁免机制

目的：建立成人睾丸支持细胞的体外培养方法并探讨其免疫豁免机制。方法：依次用胰蛋白酶、胶原酶、透明质酸酶及脱氧核糖核酸酶消化制备成人睾丸支持细胞，以SABC染色法检测支持细胞上Fas-L和TGF-β1的表达。将其与成人脾细胞共同培养，用MTT比色法检测其对脾细胞增殖的抑制作用。结果：成人睾丸支持细胞占培养细胞总数的80％以上，活性可达90％。睾丸支持细胞上可表达Fas-L和TGF-β1，体外可抑制共培养的脾细胞增殖。结论：建立了培养成人睾丸支持细胞的方法，其免疫豁免机制可能与Fas-L和TGF-β1的表达有关。     

Suppression of activin A in autoimmune lung disease associated with anti-GM-CSF

Pulmonary alveolar proteinosis (PAP) is an autoimmune disorder characterized by neutralizing autoantibodies to granulocyte-macrophage colony stimulating factor (GM-CSF). Surfactant metabolism is severely dysregulated in PAP, resulting in a foam cell appearance of alveolar macrophages. Microarray analysis of RNA from PAP bronchoalveolar lavage (BAL) cells to explore autoimmune-related genes yielded evidence of a deficiency of activin A, a cytokine implicated in regulation of B-cell proliferation and reduction of foam cell formation. Subsequent studies confirmed a severe deficiency of activin A gene expression and protein secretion in PAP BAL cells and marked reduction of activin A protein in PAP BAL fluids compared to healthy controls. PAP cells, however, like those of healthy controls, were capable of elevated activin A production in response to GM-CSF. Treatment with activin A in vitro suppressed proliferation of PAP peripheral blood B-cells in a receptor-dependent manner and also reduced secretion of anti-GM-CSF autoantibody. These studies are the first to suggest that activin A may play a role in autoimmune disease.     

Activin receptor subunits in normal and dysfunctional adult human testis

BACKGROUND: The cellular sites of activin action and its regulation in the normal and dysfunctional adult human testis are unknown. METHODS: Activin type I (ALK2 and ALK4) and type II (ActRIIA and ActRIIB) receptors were detected using immunohistochemistry on Bouins fixed sections of normal, carcinoma in situ (CIS), seminoma, non-seminoma and gonadotropin-deprived human testis. ActRIIA mRNA was localized by in situ hybridization. RESULTS: ALK2, ALK4 and ActRIIB proteins were observed in Sertoli cells, spermatogonia and some spermatocytes within normal and gonadotropin-suppressed adult human testis; all three receptor subunits were also detected in CIS, seminoma and non-seminoma cells. ActRIIA immunoreactivity was faint to absent in the normal testis and in CIS and non-seminoma cells, whereas some seminoma cells displayed a strong signal. Also in contrast to the normal testis, a majority of spermatogonia and Sertoli cells in gonadotropin-deprived samples exhibited a strong ActRIIA immunohistochemical and in situ hybridization signal. CONCLUSIONS: Spermatogonia and Sertoli cells appear as the primary targets of activin action in the adult human testis. Changes in testicular function associated with altered hormone levels may enhance ActRIIA mRNA and protein synthesis, thus modifying signalling by activin or other TGFbeta ligands within specific cells of the seminiferous epithelium.     

Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes.

The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY-positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20-30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation.     

Identity of M2A (D2-40) antigen and gp36 (Aggrus, T1A-2, podoplanin) in human developing testis, testicular carcinoma in situ and germ-cell tumours

Testicular germ-cell tumours of young adults are derived from a pre-invasive intratubular lesion, carcinoma in situ (CIS). In a recent genome-wide gene expression screening using cDNA microarrays, we found PDPN over-expressed in CIS compared to normal adult testis. PDPN encodes podoplanin (Aggrus, human gp36, T1A-2), a transmembrane glycoprotein expressed in lymphatic endothelium and various solid tumours. To examine a potential role for PDPN in testicular neoplasms and during testicular development, we investigated its expression pattern during the development of human testis and in a series of testicular CIS, gonadoblastoma and overt germ-cell tumours. We established by RT-PCR and by immunohistochemistry with a gp36 antibody that PDPN mRNA and the protein product were expressed in testes with germ-cell neoplasms but not in the normal adult testis. We also found gp36 expression in early foetal gonocytes and immature Sertoli cells, similar to the expression pattern of M2A antigen, a previously identified marker for CIS and seminoma. This reinforced our previous proposal that M2A (D2-40) antigen was identical to gp36 (podoplanin, Aggrus, T1A-2). Our findings also suggest that podoplanin has a function in developing testis, most likely at the level of cell–cell interactions among pre-meiotic germ cells and immature Sertoli cells.     

The role of activin a in regulation of hemopoiesis

Activin A, a cytokine member of the transforming growth factor-beta superfamily, is expressed locally by the mesenchymal component of the hemopoietic microenvironment. Its expression is regulated on the mRNA level by different cytokines, and the biological activity of the protein is tightly controlled by several inhibitory molecules. Activin A affects hemopoietic cells of various lineages, as evidenced by in vitro studies of leukemia and lymphoma cell lines, which were used to elucidate the mechanism of its action. In the B-cell lineage, activin A is a cell cycle inhibitor, a mediator of apoptosis, and a cytokine antagonist. Limited information is available on the effects of activin A on normal hemopoietic cells. Recent studies suggest that it might be a negative regulator of normal B lymphopoiesis. Whereas the functions of activin A in vitro are well established, further research tools are needed to elucidate its role within specific hemopoietic microenvironments in vivo.     

Effects of glial cell line-derived neurotrophic factor on isolated developing mouse Sertoli cells in vitro

Cell proliferation is a key factor in sex determination where a size increase relative to the XX gonad is one of the first signs of testis differentiation. Moreover, proliferation of Sertoli cells during development is important in building up the stock of supporting cells necessary for subsequent successful fertility. Because proliferation is such an essential part of testis development, the hypothesis under long-term investigation is that it is under fail-safe control by multiple alternative growth factors. This study was undertaken to investigate the role of glial cell-derived neurotrophic factor (GDNF) on developing mouse Sertoli cells in vitro. Sertoli cells, isolated from mouse embryos at three stages of testis development, were maintained for 2-7 days in vitro (div) in the presence or absence of GDNF at 1, 10 and 100 ng mL(-1). Overall the presence of extracellular matrix gel had little effect on proliferative activity, but encouraged expression of the epithelial phenotype. A statistically significant difference in proliferation, assessed by immunocytochemical staining for proliferating cell nuclear antigen, was seen with GDNF at embryonic day (E)12.5 after 2 div (at both 10 and 100 ng mL(-1), P < 0.001) and 7 div (at both 10 and 100 ng mL(-1), P < 0.05); at E13.5 after 3 div (at both 10 and 100 ng mL(-1), P < 0.05) and at E14.5 after 7 div (100 ng mL(-1), P < 0.01), compared with controls cultured without growth factor. In conclusion, GDNF stimulates mitosis throughout this critical developmental window. The in vitro approach used here is a useful adjunct to the knockout mouse model and has been applied to show that GDNF exerts a proliferative effect on developing mouse Sertoli cells.     

Anti-MIC2 as a tool in examination of testicular biopsies.

MIC2 is a pseudoautosomal gene localized on X and Y chromosomes. The MIC2 gene product is a glycoprotein expressed on the cell membranes of a number of somatic cells, including Sertoli cells of the testis, but not on the cell membranes of germ cells. In cases of cryptorchidism, a testicular biopsy is recommended in order to evaluate future fertility potential. The spermatogonia are identified on histological sections and the number per tubular transverse section is compared with normal values for age. The patient is at 33-100% risk of subsequent infertility when the number of spermatogonia per tubular transverse section is lower than 1% of the lowest normal age-matched value. Besides Sertoli cells the seminiferous tubules in undescended testes contain only a few germ cells, and it may be difficult to pinpoint the germ cells in small biopsies. Especially in nonpalpable testes their number may be heavily reduced. A reliable identification of germ cells may also be difficult in cultures of testicular biopsies from undescended testes. Against this background, we tried the use of an immunohistochemical method with DAKO antibody to the MIC2 gene product (MIC2, 12 E7, code no. M3601) in order to obtain a "negative reaction" of germ cells, contrasting with the stained Sertoli cells. The material comprised: 44 specimens of testicular parenchyma taken at time of surgery for cryptorchidism from 24 cryptorchid boys with nonpalpable testes and 14 testicular biopsies from 13 cryptorchid patients with palpable testes which had been cultured in vitro for 7, 14 or 21 days. In all cases the immunohistochemical method with DAKO antibody to the MIC2 gene product was helpful for identification of Sertoli cells and germ cells, and we therefore recommend the use of anti-MIC2 in all testicular biopsies where it is difficult to pinpoint the germ cells.     

In vitro induction of experimental autoimmune orchitis: characterization of a primary T lymhocyte response against testicular self antigens.

Lymphocytes from normal, young adult rats were autosensitized in vitro against trypsin-dissociated autologous testis cells. The autoimmune response had been assayed both by determination of the lymphocyte proliferation, using a (3H)thymidine incorporation test, as well as by determination of their specific cytostatic activity against monolayer-forming testis cells which was measured in a terminal 51Cr uptake assay. The actively responding cells in this system are T lymphocytes. Non-T cells can exert additional, non-specific cytostasis. The immune specificity of the in vitro experimental autoimmune orchitis (EAO) has been demonstrated on two levels. First, the responding T lymphocytes were found to specifically adhere around cells from one particular testis subpopulation which morphologically resemble Sertoli cells. The rosetting lymphocytes respond only to testicular self antigen, but not to allogeneic lymphocytes. Second, specificity to the effector T lymphocytes was revealed in transfer experiments. Autosensitized EAO effector T cells cytostatically affected only major histocompatibility gene complex (MHC)-compatible testis cells. Their reactivity against MHC-different testis cells or MHC-compatible nontesticular fibroblasts was significantly decreased which suggests both tissue as well as MHC specificity of the EAO response.