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.     

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.      

Distribution and spatiotemporal relationship of activin a and follistatin in mouse decidual and placental tissue

Problem Cells responsible for the synthesis of follistatin and activin A in the pregnant mouse endometrium have not been characterized. Method of study Immunocytochemistry was used to determine the distribution of follistatin and activin A in the pregnant mouse uterus. Results Follistatin was detected in the endometrium prior to decidualization and embryo implantation. Follistatin was not seen in fully decidualized cells, being restricted to non-decidualized fibroblasts and cells in the process of decidualization. In contrast, activin A was detected exclusively in mature antimesometrial decidual cells during involution. After day eleven of pregnancy, both substances were identified in the extracellular matrix of the spongiotrophoblast. Conclusion As previously described for decidual prolactin-related protein and the proteoglycan perlecan, follistatin and activin A were detected in the extracellular matrix of the spongiotrophoblast, suggesting that this region acts as reservoir for these growth factors in the mouse placenta.     

Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma

BACKGROUND: Activin A is a member of the transforming growth factor-beta superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin. OBJECTIVE: To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2-driven mucosal inflammation in a murine model of allergic asthma. METHODS: Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin-sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes. RESULTS: Follistatin was released concurrently with activin A suggesting it acts as an endogenous regulator: peak BAL concentrations coincided with maximal airway eosinophilia, and frequency of IL-4, IL-5 and IL-13 producing cells in mediastinal lymph nodes but induction lagged behind the onset of inflammation. Follistatin and activin A immunoreactivity were lost in airway epithelial cells in parallel with goblet cell metaplasia. Exogenous follistatin inhibited the allergen-specific Th2 immune response in mediastinal lymph nodes and mucus production in the lung. CONCLUSION: Follistatin is preformed in the normal lung and released in concert with activin A suggesting it serves as an endogenous regulator. Disturbance of the fine balance between activin A and its endogenous inhibitor follistatin may be a determinant of the severity of allergic inflammation or tissue phenotypic shift in asthma.     

Immunocytochemical localization of rat intestinal 15 kDa protein, a member of cytoplasmic fatty acid-binding proteins.

Rat intestinal 15 kDa protein (I-15P) is a member of the family of cytoplasmic fatty acid-binding proteins. Using a specific antiserum against I-15P, we studied the tissue distribution and subcellular localization of this protein in the entire rat body. By immunoblot analysis of cytosolic proteins, I-15P was detected not only in the distal portion of small intestine but also in the ovary and adrenal gland. Immunohistochemically, I-15P was localized to the absorptive epithelial cells as well as a subpopulation of enterochromaffin cells in the intestine, the lutein cells in the ovary, and subpopulations of cortical cells in the adrenal gland. Furthermore, I-15P-like immunoreactivity was also demonstrated in the surface mucous cells of stomach and the granular convoluted tubule cells of submandibular gland. Immuno-electron microscopy showed that the immunoreactivity was confined to the cytoplasmic matrix region, except in the enterochromaffin cells and granular convoluted tubule cells, where it was localized in the secretory granules. The present findings suggest that I-15P plays a role in the cellular metabolism of steroids.     

Follistatin and activin A serum concentrations in obese and non-obese patients with polycystic ovary syndrome.

BACKGROUND: Activin promotes ovarian follicular development, inhibits androgen production and increases FSH and insulin secretion. Follistatin, an activin-binding protein, neutralizes activin bioactivity. Therefore, a decrease in the ratio of activin/follistatin might encourage characteristic features of polycystic ovary syndrome (PCOS). We investigated whether women with PCOS showed disordered follistatin and/or activin serum concentrations. METHODS: The study group included 24 obese and 20 non-obese (body mass index vertical line and <27 kg/m2 respectively) clomiphene-failure PCOS patients. The control group included 16 obese and 46 non-obese patients with normal ovulatory cycles. Blood samples were obtained from the patients on day 3-5 of a progesterone-induced or spontaneous cycle and were assayed for LH, FSH, testosterone, 17-hydroxy-progesterone, androstenedione, follistatin, activin A, fasting glucose and insulin. RESULTS: Follistatin concentrations were comparable between obese and non-obese PCOS patients (mean +/- SE; 1171 +/- 103 and 1045 +/- 159 pg/ml respectively) and significantly higher than their respective controls (628 +/- 61 and 592 +/- 49 pg/ml, P < 0.0001 and P < 0.02 respectively). Activin A concentrations were comparable between the four groups (590 +/- 35, 513 +/- 74, 661 +/- 87 and 595 +/- 43 pg/ml in obese and non-obese PCOS and controls respectively). Stepwise regression analyses for relationships between follistatin or activin A levels and all other variables indicated that follistatin was significantly and independently positively affected by PCOS (P < 0.0001), age (P < 0.02), androstenedione (P < 0.03) and weight (P < 0.05). Activin A was significantly and independently negatively affected by PCOS (P < 0.003) and FSH (P < 0.03), and positively affected by weight (P < 0.009) and androstenedione (P < 0.02). CONCLUSIONS: Serum follistatin is increased in PCOS patients, regardless of obesity. PCOS is the most significant variable that relates to high follistatin and low activin A serum concentration. A high follistatin/activin ratio could well contribute to the pathophysiology of PCOS.     

Immunocytochemical localization of rat intestinal 15 kDa protein,a member of cytoplasmic fatty acid-binding proteins

Rat intestinal 15 kDa protein (I-15P) is a member of the family of cytoplasmic fatty acid-binding proteins. Using a specific antiserum against I-15P, we studied the tissue distribution and subcellular localization of this protein in the entire rat body. By immunoblot analysis of cytosolic proteins, I-15P was detected not only in the distal portion of small intestine but also in the ovary and adrenal gland. Immunohistochemically, I-15P was localized to the absorptive epithelial cells as well as a subpopulation of enterochromaffin cells in the intestine, the lutein cells in the ovary, and subpopulations of cortical cells in the adrenal gland. Furthermore, I-15P-like immunoreactivity was also demonstrated in the surface mucous cells of stomach and the granular convoluted tubule cells of submandibular gland. Immuno-electron microscopy showed that the immunoreactivity was confined to the cytoplasmic matrix region, except in the enterochromaffin cells and granular convoluted tubule cells, where it was localized in the secretory granules. The present findings suggest that I-15P plays a role in the cellular metabolism of steroids.© Willey-Liss, Inc.     

Endocrinology: Presence and possible function of activin-like substance in human follicular fluid

We assessed the presence of an activin-like substance in humanfollicular fluid that was obtained from women undergoing in-vitrofertilization using a bioassay for activin A. Activin activitywas not detected in crude follicular fluids; the bioactivityof standard activin A was inhibited by the addition of follicularfluid. After the follistatin (binding protein of activin A)was removed from follicular fluid using a purification procedure,activin activity was detected in the follicular fluids (meanconcentration: 131 ± 40 ng/ml). Activin activity wasinhibited by the addition of follistatin to fluid. The concentrationof activin activity was substantially higher (100-fold) thanthat reported in serum. The concentration negatively and significantlycorrelated with the number of developed follicles in the ovary(r = 0.501, P < 0.01). These results suggest that activinA and its binding protein are present in follicular fluid inlarge amounts and that they may have a role in local ovarianregulation.     

Expression of cortistatin and MrgX2, a specific cortistatin receptor, in human neuroendocrine tissues and related tumours

Cortistatin (CST), a novel hormone originally described in the rat, mouse, and human cerebral cortex, displays structural and functional similarities to somatostatin (SRIF). CST binds to all five somatostatin receptors and, differently from SRIF, also binds to MrgX2, which has recently been identified as its specific receptor. Little is known about the distribution of CST and MrgX2 in peripheral non-tumour and neoplastic tissues. The aim of the present study was therefore to determine by immunohistochemistry and mRNA analysis (RT-PCR) the distribution of CST and MrgX2 in 56 human non-tumour and 108 tumour tissues, with special reference to neuroendocrine tissue types. Despite the high level of CST mRNA expression in non-tumour and tumour (both neuroendocrine and non-neuroendocrine) tissues, the presence of immunoreactive CST was confirmed in a subset of gastroenteropancreatic, parathyroid, and pituitary non-tumour cells only, and showed a predominantly focal pattern in most neuroendocrine tumours. Co-localization experiments in the gastroenteropancreatic system demonstrated that the normal CST-producing cells are delta cells, while in the adenohypophysis no preferential co-localization of CST with any of the pituitary hormones was observed. MrgX2 mRNA was variably detected in the hypothalamus, pituitary, thyroid, lung, gastroenteropancreatic tract, testis, and ovary, and was negative in the cerebral cortex, parathyroid, and adrenal, as well as in a variety of tumour types. Conversely, immunolocalization of MrgX2 protein was restricted to neurohypophysis and testis, whilst all tumours analysed were negative. A possible explanation for the discrepancy between RT-PCR and immunohistochemistry is that MrgX2 protein was widely detected in blood vessels, scattered lymphocytes, and gastrointestinal ganglia in both normal and neoplastic samples. The findings demonstrate a selective distribution of CST in normal and neoplastic neuroendocrine tissues, suggesting that CST might have a broader functional role than previously assumed, whereas possible autocrine/paracrine actions via its recently described specific receptor MrgX2 are restricted to selected tissues.     

Distinctive Effects of Rat Fibroblast Growth Factor-2 Isoforms on PC12 and Schwann Cells

Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during postnatal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC 12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC 12 cells.     

Identification of activin A and follistatin in human milk

Activin A is a dimeric protein member of the transforming growth factor-beta (TGF-beta) family: it is synthesized by a variety of organs and follistatin is an activin-binding protein. A sensitive and specific assays for bioactive dimeric activin A and follistatin have recently allowed to measure these proteins in blood and other biological fluids, giving a new insights into their possible physiological role. Since human breast is able to produce activin A, the aim of the present study was to evaluate whether it and follistatin are measurable in breast milk of women during lactation. Concentrations of activin A and follistatin were measured in milk samples collected at 3, 5 and 30 days after delivery by using specific and sensitive two-site ELISAs. For the first time the presence of immunoreactive activin A and follistatin in human milk has been shown; no significant different concentration between the third and the fifth day after delivery was found. Furthermore, no difference of activin A and follistatin concentration between the whole and the skim milk or between spontaneous delivery and cesarean section was found. Milk activin A and follistatin concentrations after 1 month of lactation were significantly decreased (P < 0.01). Activin A and follistatin are present in human milk in high concentrations in the first week of lactation, while decrease after a month suggesting a possible role as growth factors in human milk.     

Identification of Activin A and Follistatin in Human Milk

Activin A is a dimeric protein member of the transforming growth factor- &#103 (TGF- &#103 ) family: it is synthesized by a variety of organs and follistatin is an activin-binding protein. A sensitive and specific assays for bioactive dimeric activin A and follistatin have recently allowed to measure these proteins in blood and other biological fluids, giving a new insights into their possible physiological role. Since human breast is able to produce activin A, the aim of the present study was to evaluate whether it and follistatin are measurable in breast milk of women during lactation. Concentrations of activin A and follistatin were measured in milk samples collected at 3, 5 and 30 days after delivery by using specific and sensitive two-site ELISAs. For the first time the presence of immunoreactive activin A and follistatin in human milk has been shown; no significant different concentration between the third and the fifth day after delivery was found. Furthermore, no difference of activin A and follistatin concentration between the whole and the skim milk or between spontaneous delivery and cesarean section was found. Milk activin A and follistatin concentrations after 1 month of lactation were significantly decreased (P <0.01). Activin A and follistatin are present in human milk in high concentrations in the first week of lactation, while decrease after a month suggesting a possible role as growth factors in human milk.     

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.     

Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia

Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.     

中心体蛋白centrin在大鼠精子发生过程中的表达

目的：研究中心体蛋白centrin在大鼠生精细胞中的表达情况,以深入了解centrin在精子发生过程中的作用。方法：通过重力沉降法分离大鼠不同发育阶段的生精细胞,用免疫荧光和蛋白印迹实验检测各级生精细胞中centrin蛋白的表达,用定量RT－PCR检测centrin同源基因centrin1和centrin2mRNA的表达水平。结果：间接免疫荧光和蛋白印迹显示精母细胞、圆形、长形精子细胞均有centrin蛋白存在,位于中心粒上,而在附睾的成熟精子中centrin则消失。RT－PCR研究发现,centrin在睾丸组织中特异性表达,centrin2在多种组织中均有表达。在睾丸中,centrin1仅在生精细胞进入减数分裂后转录,其mRNA水平在圆形精子细胞中最高,而centrin2在精原细胞中即有表达,减数分裂后其mRNA难以检测到。结论centrin蛋白在大鼠雄性配子的发育过程中最终丢失;该基因家族中同源基因centrin1和centrin2表达呈现组织特异性和发育阶段特性,在精子发生过程中发挥不同功能,centrin1蛋白可能与减数分裂及鞭毛生成相关,centrin2则参与细胞有丝分裂过程。     

Serum activin A and follistatin concentrations during human pregnancy: a cross-sectional and longitudinal study

Activin A, a dimer of the betaA-subunit of inhibin, has been shown to have multiple biological activities and sites of production. Follistatin is a high-affinity binding protein for activin, which neutralizes its activity. This study provides the first data, using a cross-sectional design, on the measurement of both these proteins in the maternal circulation of a large cohort of women (6-39 weeks of gestation, n = 2-20 women/time point) during normal pregnancies, and confirms that similar patterns are seen in nine women studied longitudinally during pregnancy. The concentrations of total activin A were measured using a specific two-site enzyme-linked immunosorbent assay (ELISA), and a new radioimmunoassay for measuring total follistatin in serum utilizing dissociating reagents to eliminate the interference of activin is described. At 38-39 weeks gestation, both activin A and follistatin concentrations rose to a peak (4.59 +/- 0.54 ng/ml and 72.7 +/- 3.31 ng/ml, respectively). The activin A and follistatin concentrations were highly correlated both in the cross-sectional study (P <0.0001) and in individual women in the longitudinal study (P <0.05-0.0001). Concentrations of follistatin showed a greater increase in the second trimester of pregnancy relative to activin A concentrations. The parallel increase in the secretion of these two proteins throughout pregnancy probably reflects feto-placental secretion.     

Activin receptor expression and induction of apoptosis in rat blastocysts in vitro

BACKGROUND: Apoptosis, a process of normal embryonic development, is enhanced in blastocyst from diabetic rats. Nevertheless, glucose seems not to be the only factor involved. Activin A, a TGF-beta family member, is also increased in maternal serum from diabetic pregnancy. METHODS: Flushing medium, blastocysts and uterine cells were obtained from 5 day old pregnant rats. The presence of activin A in flushing medium was investigated by western blotting. RT-PCR was used to test for the presence of activin betaA subunit mRNA in cultured uterine cells. Blastocysts were stained by immunohistochemistry for activin receptor types IIA and IIB, and chromatin degradation (apoptosis) was investigated by terminal transferase-mediated dUTP nick end labelling in blastocysts exposed in vitro to activin. RESULTS: In this study, we demonstrate the presence of activin A protein in fluid from rat uterine horns at day 5 of pregnancy, as well as the presence of activin A receptors type IIB in the trophectoderm and inner cell mass and activin A receptor type IIA in trophectoderm cells only. Activin A increases the chromatin degradation level in vitro. CONCLUSIONS: Activin A protein was found in fluid from uterine horns, and mRNA expression of betaA activin subunit in cultured uterine cells suggests probable secretion from decidual cells. Moreover, activin A increases specifically the apoptosis level in rat blastocyst in vitro.     

Immunofluorescence studies on autoantibodies to steroid-producing cells, and to germline cells in endocrine disease and infertility

This study was aimed at comparing the clinical significance of antibodies to steroid-producing cells with reactions to gonadal germline cells in patients with autoimmune polyendocrine diseases and isolated infertility or amenorrhoea respectively. Indirect immunofluorescence was used on human adrenal, ovary and testis. The gonad substrates were compared with rat, rabbit and monkey glands.