INHBA Expression Research Articles

The Interactions Between the Stimulatory Effect of Follicle-Stimulating Hormone and the Inhibitory Effect of Estrogen on Mouse Primordial Folliculogenesis1

The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pregranulosa cells. The mechanisms that trigger primordial follicle formation likely are influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary, and decrease of estrogen after birth is permissive of follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E(2)) levels during the critical window of primordial follicle formation (Postnatal Days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, mouse fetal ovaries (17.5 days postcoitus) were cultured in vitro at two concentrations of E(2) (meant to reflect maternal and fetal levels of E(2)) and FSH for 6 days. High levels of E(2) (10(-6) M) inhibited germline nest breakdown, and this effect was significantly reduced when fetal ovaries were cultured in the low E(2) concentration (10(-10) M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E(2) culture conditions. Low E(2) was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin beta A subunit (Inhba) expression, pregranulosa cell proliferation, and oocyte survival. The decrease of E(2) plus the presence of FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox) in that inappropriate exposure to FSH or E(2) during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E(2) level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling pathways and the expression of oocyte-specific transcription factors.

Inhibin βB expression in murine adipose tissue and its regulation by leptin, insulin and dexamethasone

Inhibin betaB (INHBB; coding for the activin betaB subunit) has previously been identified in both human and rodent adipose tissue and using Taqman real-time PCR with specific primers we confirm the expression of INHBB mRNA in rodent adipose tissue. Expression of INHBB in murine epididymal adipose tissue was higher than in any of the other tissues studied and appears to be regulated by changes in energy balance and leptin. It was increased fourfold in the epididymal fat depot of ob/ob mice compared with the same fat depot in lean mice. The i.p. administration of leptin in obese ob/ob mice decreases the expression of INHBB. In human adipose tissue, INHBB is reduced by weight loss. In keeping with this, we demonstrate that INHBB expression in murine adipose tissue is decreased in fasting and increased upon refeeding. We show that INHBB is expressed in both the mature adipocyte and the stromal vascular fraction of adipose tissue. INHBB increases with the differentiation of pre-adipocytes into mature adipocytes in the 3T3-L1 cell line. In differentiated 3T3-L1 adipocytes, where receptors to activin have been previously reported, insulin increases the expression of INHBB, while dexamethasone decreases the expression of INHBB when compared with untreated control cells. Taken together, these results suggest that the regulation of INHBB expression in adipose tissue may play a physiological role in energy balance or the insulin insensitivity associated with obesity.

Regulation of inhibin/activin expression in rat early antral follicles

The aim of the present study was to determine the endocrine activity of cultured early antral follicles (EAF) isolated from prepubertal diethylstilbestrol-treated rats. The effect of steroidogenic substrates and FSH on steroid, inhibin A and B, Pro-αC and activin A production was evaluated. Androsterone was the predominant steroid produced by EAF. The addition of androstenedione, androstenedione + FSH and progesterone stimulated oestradiol production, whereas 25-hydroxycholesterol (25-OH-Chol) increased progesterone production. Inhibin A, B, Pro-αC, and activin A were produced under basal conditions. The predominance of inhibin B over inhibin A was not affected by the addition of androstenedione or progesterone. Inhibin A and activin A production was stimulated by FSH. 25-OH-Chol increased Inha, Inhba and Inhbb mRNA expression and the production of the three molecular forms of inhibins but decreased activin A production. These results show that FSH and the steroid follicular microenvironment differentially modulate the gene expression of inhibin/activin subunits, their assembly and secretion.

Upregulated INHBA Expression May Promote Cell Proliferation and Is Associated with Poor Survival in Lung Adenocarcinoma

The expression, mechanisms of regulation, and functional impact of INHBA (activin A) in lung adenocarcinoma (AD) have not been fully elucidated. INHBA expression was examined in 96 lung samples (86 ADs, 10 normal lung) using oligonucleotide microarrays and 187 lung samples (164 ADs, 6 bronchioalveolar carcinomas, and 17 normal lung) using immunohistochemistry. The proliferation of AD cell lines H460 and SKLU1 was examined with WST-1 assays after treatment with recombinant activin A, follistatin, and INHBA-targeting small-interfering RNA. Cells were also treated with 5-aza-2' deoxycytidine and trichostatin A to investigate the role of epigenetic regulation in INHBA expression. Primary ADs expressed 3.1 times more INHBA mRNA than normal lung. In stage I AD patients, high levels of primary tumor INHBA transcripts were associated with worse prognosis. Immunohistochemistry confirmed higher inhibin betaA protein expression in ADs (78.7%) and bronchioalveolar carcinomas (66.7%) compared with normal lung (11.8%). H460 and SKLU1 demonstrated increased proliferation when treated with exogenous activin A and reduced proliferation when treated with follistatin or INHBA-targeting small-interfering RNA. INHBA mRNA expression in H460 cells was upregulated after treatment with trichostatin A and 5-aza-2' deoxycytidine. INHBA is overexpressed in AD relative to controls. Inhibin betaA may promote cell proliferation, and its overexpression is associated with worse survival in stage I AD patients. In addition, overexpression of INHBA may be affected by promoter methylation and histone acetylation in a subset of lung ADs.

Several Apoptosis Related Genes Differentially Expressed in Human Granulosa Cells of the Embryos With Different Developmental Potential

Background: In humans, growing oocytes derive most substrates for energy metabolism and biosynthesis from cumulus granulosa cells. Abnormal patterns of gene expression in cumulus cells could represent either causes or consequences of abnormal oocyte development. Studies of gene expression in cumulus cells might help to determine molecular markers for the developmental potential of their enclosed oocyte. Objective: To compare differential expression of INHBB, INHBA, PTX3, PCNA, BAX, BIRC5, PUM1 and 2, INT1B, and BAG3 genes in cumulus granulosa cells derived from human oocytes that: 1) either resulted in pregnancy vs. failed to develop; and 2) were from patients with normal vs. decreased ovarian reserve. Methods: 68 patients undergoing IVF were enrolled in the study. After oocyte retrieval, cumulus granulosa cells were trimmed from the oocytes and processed for RNA extraction. Real-time PCR was used to quantify the expression of the genes listed above. Oocytes were cultured individually, and monitored for fertilization and embryo development. Embryos were selected for transfer based on morphology. Results: Levels of PCNA and BIRC5 gene mRNA were two and four times up-regulated in the cumulus cells of the embryos that failed to develop in vivo when compared to the cumulus cells of the embryos that resulted in pregnancy. mRNA levels of PCNA, INT1B, and PUM1 genes were more than two times upregulated (P<0.05) in embryos derived from the patients with decreased ovarian reserve (FSH above 7 mU/mL). Removal of the cumulus granolusa cells did not affect embryo development, as the pregnancy rates in the study group were not significantly different from the other patients in the practice. Conclusions: In this study we determined that PCNA and BIRC5 gene are upregulated in the cumulus granulosa cells of the embryos that failed to develop in vivo when compared to the cumulus cells of the embryos that resulted in pregnancy. We also found that PCNA, INT1B, and PUM1 genes are upregulated in the cumulus granulosa cells in patients with decreased ovarian reserve. With further research, we hope to confirm molecular targets that will be predictive of successful embryo development and pregnancy to increase elective single embryo transfer rates and to decrease the rate of multiple births with ART.

Weaning and the Developmental Changes in Follicle-Stimulating Hormone, Pituitary Adenylate Cyclase-Activating Polypeptide, and Inhibin B in the Male Rat1

Pituitary Fshb concentrations increase markedly and selectively beginning on Postnatal Day 20 in the male rat. To evaluate the factors potentially responsible for this rise in FSH, we adjusted the time of weaning, which is generally also on Day 20. Male rat pups were provided nutrients by suckling only and were weaned to laboratory chow earlier (Day 17) or later (Day 23) than normally performed in animal facilities (Day 20). Between ages 17 and 29 days, significant increases were seen in serum LH (1.4-fold) and FSH (2.4-fold) levels; pituitary expression of Lhb (5.4-fold), Fshb (21.3-fold), and inhibin beta B (Inhbb, 2.26-fold) mRNAs; and testicular expression of Inhbb (10-fold) mRNA. Concurrently, significant decreases occurred in serum inhibin B levels (1.8-fold); pituitary adenylate cyclase-activating polypeptide (Adcyap1, 4.2-fold), total follistatin (Fst, 3.5-fold), and Fst isoform 288 (5.6-fold) mRNAs; and testicular expression of inhibin beta A (8.2-fold) mRNA. Early weaning significantly increased serum FSH but not LH and increased pituitary expression of Fshb and GnRH receptor (Gnrhr) mRNAs but not Lhb. Early weaning also significantly decreased serum inhibin B but increased testicular expression of the Inhbb subunit. Early weaning also caused pituitary expression of Fst and Adcyap1 to decline earlier than in the control group. Immediately after weaning, growth accelerated substantially, and the time of weaning produced significant and differential effects on circulating leptin levels that were not related to indices of FSH production. From these observations, we propose the novel hypothesis that the increase in growth rate subsequent to weaning signals circulating inhibin B levels to fall and pituitary Adcyap1 and consequently Fst expression to decrease, and that these events together facilitate the rise in Fshb and Gnrhr expression by increasing pituitary activin signaling.

INHIBIN BETA A AND HEDGEHOG: A NOVEL REGULATORY LOOP IN FETAL TESTIS CORD DEVELOPMENT

In most mammals, testis development is initiated by the male-specific gene Sry. The function of Sry is to establish the Sertoli cell lineage, which is responsible for the recruitment of the remaining testis cell lineages and their subsequent organization into testis cords. The mechanisms involved in initial testis cord organization have become clear; however, it remains uncertain whether maintenance and further expansion of the testis cords requires additional signals. To identify players involved in later testis cord growth, we searched for molecules expressed in mouse testes after embryonic day 12.5 (e12.5), when the testis cords have formed. We found that inhibin beta A (Inhba), but not inhibin beta B (Inhbb), was upregulated after e12.5 and expressed specifically in the testis interstitium. The exclusive presence of Inhba suggests that the possible protein products in the interstitium are inhibin A and/or activin A. Activins, but not inhibins, are known to trigger cellular response. Therefore we examined whether activins were active in the testis by immunostaining for phospho-SMAD2/3, the intracellular effectors of activins. Phospho- SMAD2/3 were found in the nuclei of Sertoli cells after e12.5, leading to our hypothesis that Inhba or its protein product activin A from the interstitium regulates Sertoli cell development after initial testis cord formation. To test this hypothesis, we examined Inhba-null embryos and found that initial testis cord formation was normal without Inhba. However, testis cord defects arose after e15.5. In wild type males, testis cords developed from straight tubes at e12.5 to highly convoluted structures at birth. In Inhba-null newborns, testis cords failed to elongate and remained straight tubes as at e12.5. Inhba-null testis cords contained fewer Sertoli cells and some Sertoli cells were located within the lumen of the testis cords, apparently sloughed off from their usual position encircling the germ cells. Despite these testis cord defects, Leydig cells in Inhba-null testes developed normally and all parameters for androgen action were similar to those in wild type testes. These findings indicate that interstitium-derived Inhba is essential for proper development of the testis cords via regulation of Sertoli cell expansion and differentiation. The novelty of an interstitial product promoting Sertoli cell development prompted us to investigate how Inhba is regulated. The testis interstitium is known to express Patched-1, a receptor for the signaling molecules of the hedgehog family. Sertoli cells are the source of hedgehog ligands in the developing testis, suggesting that Inhba expression by the interstitium could be stimulated by the hedgehog pathway. We therefore inhibited hedgehog signaling by culturing fetal testes with cyclopamine, an inhibitor of the hedgehog pathway. We found that cyclopamine treatment decreased Inhba expression, indicating that expression of Inhba in the testis interstitium requires an active hedgehog pathway. Based on these findings, we propose that hedgehog ligands from the Sertoli cells induce interstitial expression of Inhba, which in turn regulates Sertoli cell differentiation and further development of the testis cords. Our findings have identified the first regulatory loop that is responsible for maintenance and expansion of testis cords in embryonic testes. (Supported by NIH-HD46861 and the March of Dimes Birth Defects Foundation). (platform)

THE GH-IGF AXIS AND ITS POTENTIAL ROLE IN THE OVARY OF ZEBRAFISH, Danio rerio

Growth hormone (GH) from the pituitary is the major hormone responsible for body growth in vertebrates. However, there has been increasing evidence that GH is also involved in several reproductive functions including steroidogenesis and gametogenesis in the gonads. In view of this, GH has also been termed co-gonadotropin. It is well established that most effects of GH are mediated by insulin-like growth factors (IGFs) produced in different tissues. In the ovary, however, both IGF-dependent and independent mechanisms have been proposed for GH actions, and one of the factors that work downstream of GH signaling appears to be activin. Interestingly, the expression of GH itself has been detected in the ovary, suggesting the existence of an intraovarian GH-IGF axis. Using zebrafish as the model, the present project was undertaken to characterize the mini-GH-IGF-I axis in the ovary with particular reference to its relationship with the local activin-follistatin system. RTPCR analysis showed that Gh (gh), Igf1 (igf1), Igf2 (igf2), and their receptors were all expressed in the ovary, indicating that a potential intraovarian GH-IGF system also exists in the zebrafish. Further experiment on spatial distribution using isolated follicle layers and denuded oocytes revealed that gh was exclusively expressed in the fullgrown oocytes, while its receptor Ghr (ghr) could be detected in both oocytes and follicle cells. Igf1 and its receptor igf1r as well as igf2 could also be detected in both oocytes and follicle cells. Analysis of temporal expression patterns demonstrated that gh and ghr were differentially expressed during folliculogenesis. The expression level of gh in the follicles seemed to be the highest at the primary growth (PG) stage, but it gradually decreased when PG follicles developed to full-grown (FG) follicles. On the contrary, the expression of ghr was low at the PG stage but significantly increased in the previtellogenic (PV) stage and remained high in later stages. Similarly, the expression of igf1r gradually increased from PG to mid-vitellogenic (MV) stage, but decreased significantly in the final FG stage. Igf1 expression increased from PG to EV stage and decreased afterwards till FG stage. While expression of igf2 maintained constant during folliculogenesis. When tested in cultured follicle cells, recombinant human Igf1 increased the expression of activin beta A (inhba) but decreased that of activin beta B (inhbb) in a clear time and dose-dependent manner. GH (bream and human GH), however, seemed to increase the expression of both inhba and inhbb, which supports our previous observation that GH-induced zebrafish oocyte maturation could be blocked by follistatin, an activin-binding protein. Although we have not obtained convincing evidence for IGFs as the downstream mediators of GH in the zebrafish ovary, our preliminary data did suggest a potential role for the local ovarian activin system in GH as well as IGF signaling. (platform)

Transcriptional Cooperation between the Transforming Growth Factor-β and Wnt Pathways in Mammary and Intestinal Tumorigenesis

Transforming growth factor-beta (TGF-beta) and Wnt ligands function in numerous developmental processes, and alterations of both signaling pathways are associated with common pathologic conditions, including cancer. To obtain insight into the extent of interdependence of the two signaling cascades in regulating biological responses, we used an oligonucleotide microarray approach to identify Wnt and TGF-beta target genes using normal murine mammary gland epithelial cells as a model. Combination treatment of TGF-beta and Wnt revealed a novel transcriptional program that could not have been predicted from single ligand treatments and included a cohort of genes that were cooperatively induced by both pathways. These included both novel and known components or modulators of TGF-beta and Wnt pathways, suggesting that mutual feedback is a feature of the coordinated activities of the ligands. The majority of the cooperative targets display increased expression in tumors derived from either Min (many intestinal neoplasia) or mouse mammary tumor virus (MMTV)-Wnt1 mice, two models of Wnt-induced tumors, with nine of these genes (Ankrd1, Ccnd1, Ctgf, Gpc1, Hs6st2, IL11, Inhba, Mmp14, and Robo1) showing increases in both. Reduction of TGF-beta signaling by expression of a dominant-negative TGF-beta type II receptor in bigenic MMTV-Wnt1/DNIIR mice increased mammary tumor latency and was correlated with a decrease in expression of Gpc1, Inhba, and Robo1, three of the TGF-beta/Wnt cooperative targets. Our results indicate that the TGF-beta and Wnt/beta-catenin pathways are firmly intertwined and generate a unique gene expression pattern that can contribute to tumor progression.

The expression of inhibin beta B is high in human adipocytes, reduced by weight loss, and correlates to factors implicated in metabolic disease

Adipose tissue is an endocrine organ that produces and secretes adipokines. The aim of this study was to identify genes predominantly expressed in human subcutaneous adipocytes. For this purpose, an algorithm was developed and DNA microarray expression profiles from 33 human tissues and cell types were used to select genes. Inhibin beta B (INHBB; coding for the activin βB subunit) was identified and high expression in adipocytes was confirmed by real-time PCR and immunohistochemistry. INHBB expression in adipose tissue was down regulated by diet-induced weight loss ( p < 0.001). Furthermore, INHBB expression was positively correlated to total ( p < 0.001) and subcutaneous ( p < 0.01) adipose tissue areas and serum levels of fasting insulin ( p < 0.01) and cholesterol ( p < 0.05). In conclusion, INHBB expression was high in human adipocytes, reduced by weight loss and adipose tissue INHBB mRNA levels correlated to metabolic risk factors. This suggests that activin B produced in adipocytes may play a role in the metabolic syndrome.