Expression Of Inhibin Alpha Research Articles

Inhibin Alpha Expression in Human Tumors: A Tissue Microarray Study on 12,212 Tumors.

As a result of its expression in corresponding normal cell types, inhibin alpha (INHA) is used as an immunohistochemical marker for adrenocortical neoplasms and testicular or ovarian sex cord stromal tumors. However, other tumors can also express INHA. To comprehensively determine INHA expression in cancer, a tissue microarray containing 15,012 samples from 134 different tumor types and subtypes was analyzed by immunohistochemistry. INHA positivity was found in 72 of 134 tumor categories, including 26 categories with ≥1 strongly positive case. A moderate to strong INHA positivity was found in 100% of 37 granulosa cell tumors of the ovary, 100% of 43 other sex cord stromal tumors of the ovary/testis, 100% of 31 granular cell tumors, 78.5% of 28 adenomas, 44% of 25 carcinomas of the adrenal cortex, and 46.7% of 15 pancreatic acinar cell carcinomas. At least a weak INHA positivity was seen in <33% of cases of 46 additional tumor entities. In summary, these data support the use of INHA antibodies for detecting sex cord stromal tumors, granular cell tumors, and adrenocortical neoplasms. Since INHA can also be found in other tumor entities, INHA immunohistochemistry should only be considered as a part of any panel for the distinction of tumor entities.

Deletion of Spata2 by CRISPR/Cas9n causes increased inhibin alpha expression and attenuated fertility in male mice.

As somatic cells in the testis seminiferous tubule, Sertoli cells provide the medium for spermatogenesis. One of the important functions of Sertoli cells is synthesizing and secreting cell factors to affect the production of sperm; however, much of those molecular regulation mechanisms remain unknown. Here, we confirm the localization of protein SPATA2 (spermatogenesis-associated protein 2), which had previously been shown to be highly expressed in Sertoli cells of the adult mouse testis. To further conduct a functional study, we generated SPATA2 global knockout mice via use of the CRISPR/Cas9n gene editing technology. The 120-day-old knockout mice testes showed almost a 40% decrease in size and weight and variations in the histomorphology of the seminiferous epithelium, with a 40% decrease in sperm count. Further examination revealed that the proliferation of germ cells in the seminiferous tubules was attenuated by 28%. In addition, we found that SPATA2 deletion led to an approximately 70% increase in the inhibin alpha-subunit mRNA and protein level in the testes compared to that of wild-type mice. Our data revealed the impact of SPATA2 on male fertility and suggested that SPATA2 ensures the normal secretory function of Sertoli cells.

Characterization of transforming growth factor beta superfamily, growth factors, transcriptional factors, and lipopolysaccharide in bovine cystic ovarian follicles

The process of transformation of growing bovine follicles into cysts is still a mystery. Local expression of proteins or factors, including transforming growth factor β, growth factors, and transcription factors, plays a central role in mammals. Therefore, in abattoir-derived cystic ovarian follicles and follicular fluid, the role of some transforming growth factor β superfamily proteins, insulinlike growth factor-1 (IGF-1) and GATA-4 and GATA-6, were investigated. The relationship between intrafollicular lipopolysaccharide (LPS) and etiopathogenesis of ovarian cysts was also assessed. Data on the preovulatory follicle and the largest follicle (F1) were compared. The number of intrafollicular LPS-positive samples and LPS concentrations were higher in cysts. Immunohistochemical staining was mildly positive for IGF-1, inhibin alpha, and GATA-4 in thecal cells. Staining for anti-Müllerian hormone (AMH), growth differentiation factor-9, bone morphogenetic protein-6 (BMP-6), and GATA-6 was insufficient for their quantitation, and oocytes could not be stained for any of the proteins tested in the cystic follicles. Expression of BMP-6, inhibin alpha, and IGF-1 was moderately higher in granulosa cells of F1 follicles, and all the proteins were moderately expressed in granulosa cells in preovulatory follicles. However, loss of GATA-6 staining was significant in F1 follicles. Intrafollicular progesterone, IGF-1, and AMH concentrations in cysts and F1 follicles were significantly higher than those in preovulatory follicles. Western blot analyses revealed that follicular fluid inhibin-α was strongly expressed, whereas expression of growth differentiation factor-9, BMP-6, GATA-4 and GATA-6 was lower in cysts than in preovulatory follicles. Also, high intrafollicular AMH concentration and low BMP-6 expression were closely associated with cystic degeneration and atresia. In conclusion, immunohistochemical loss of BMP-6 and GATA-6 in the granulosa cells together with high intrafollicular LPS levels may play important roles in disruption of the ovulatory mechanism and steroidogenic reactions in type 2 cyst. Also, high intrafollicular AMH concentration along with low BMP-6 expression may be used as indicators of the bovine degenarative ovarian follicles.

Global Deletion of Trp53 Reverts Ovarian Tumor Phenotype of the Germ Cell–Deficient White Spotting Variant (Wv) Mice

White spotting variant (Wv) mice are spontaneous mutants attributed to a point mutation in the c-Kit gene, which reduces the tyrosine kinase activity to around 1% and affects the development of melanocytes, mast cells, and germ cells. Homozygous mutant mice are sterile but can live nearly a normal life span. The female Wv mice have a greatly reduced ovarian germ cell and follicle reserve at birth, and the remaining follicles are largely depleted soon after the females reach reproductive stage at around 7 weeks of age. Consequently, ovarian epithelial tumors develop in 100% of Wv females by 3 to 4 months of age. These tumors, called tubular adenomas, are benign but can become invasive in older Wv mice.We tested if additional genetic mutation(s) could convert the benign ovarian epithelial tumors to malignant tumors by crossing the Wv mutant into the Trp53 knockout background. Surprisingly, we found that global deletion of Trp53 suppressed the development of ovarian tubular adenomas in Wv mice. The ovaries of Wv/Wv; Trp53 (−/−) mice were covered by a single layer of surface epithelium and lacked excessive epithelial proliferation. Rather, the ovaries contained a small number of follicles. The presence of ovarian follicles and granulosa cells, as indicated by Pgc7 and inhibin-alpha expression, correlated with the absence of epithelial lesions. A reduction of Pten gene dosage, as in Wv/Wv; Pten (+/−) mice, produced a similar, though less dramatic, phenotype. We conclude that deletion of Trp53 prolongs the survival of ovarian follicles in Wv mice and consequently prevents the proliferation of ovarian epithelial cells and development of ovarian tubular adenomas. The results suggest that various cell types within the ovary communicate and mutually modulate, and an intact tissue environment is required to ensure homeostasis of ovarian surface epithelial cells. Especially, the current finding emphasizes the importance of ovarian follicles in suppressing the hyperplastic growth of ovarian epithelial cells, dominating over the loss of p53.

Gastric cancer does not affect the expression of atrophy-related genes in human skeletal muscle

We evaluated the gene expression levels of atrogin-1, MuRF1, myostatin, follistatin, activin A, and inhibin alpha in skeletal muscle samples of patients with gastric cancer and controls. We studied 38 cancer patients and 12 controls who underwent surgery for gastric adenocarcinoma and benign abdominal diseases, respectively. A biopsy specimen was obtained from the rectus abdominis muscle from all participants. The relative gene expression of atrogin-1, MuRF1, myostatin, follistatin, activin A, and inhibin alpha was determined by quantitative real-time polymerase chain reaction analysis. Atrogin-1 and MuRF1 mRNA expression was similar between cancer patients and controls and was unaffected by the disease stage or the severity of body weight loss. Transcript levels of myostatin and follistatin did not differ between cases and controls and were similar across disease stages and categories of weight loss. Finally, no differences were detected in activin A and inhibin alpha gene expression between cancer patients and controls. In skeletal muscle, the gene expression of atrogin-1, MuRF1, myostatin, follistatin, activin A, and inhibin alpha is not affected by the presence of cancer. The expression of atrophy-related genes is unaffected by the disease stage and the degree of weight loss.

FSH modulates the expression of inhibin‐alpha and the secretion of inhibins via orphan nuclear receptor NUR77 in ovarian granulosa cells

It has been previously reported that follicle-stimulating hormone (FSH) regulates the expression of inhibin-alpha in human granulosa cells, but the precise molecular pathway remains unknown. In the present study, we investigated the role of the orphan nuclear receptor, NUR77, in both the transcriptional regulation of the inhibin α-subunit gene and the secretion of inhibins. Our results showed that in a human granulosa cell tumor-derived cell line (KGN) and in human granulosa-lutein cells (hGL), FSH induced the expression of NUR77 and inhibin-alpha, although inhibin-alpha expression did not increased following FSH treatment if NUR77 was knocked down. Furthermore, simply overexpressing or reducing NUR77 levels affected inhibin-alpha expression, while NUR77 overexpression improved the secretion of inhibin A and B from human granulosa cells. In addition, chromatin immunoprecipitation-PCR, avidin-biotin-conjugated DNA precipitation, and luciferase reporter assays confirmed that NUR77 directly regulated the transcription of the inhibin-alpha gene through the specific NGFI-B response element located within its promoter. In the ovarian granulosa cells of the Nur77 knockout mice, the mRNA levels of inhibin-alpha were decreased relative to wild-type mice. These data indicate a role of NUR77 in the regulation of inhibin-alpha in ovarian granulosa cells.

Expression of Wnt and TGF-β pathway components and key adrenal transcription factors in adrenocortical tumors: Association to carcinoma aggressiveness

Factors controlling benign and malignant adrenocortical tumorigenesis are largely unknown, but several mouse models suggest an important role for inhibin-alpha (INHA). To show that findings in the mouse are relevant to human tumors and clinical outcome, we investigated the expression of signaling proteins and transcription factors involved in the regulation of INHA in human tumor samples⋅ Thirty-one adrenocortical tumor samples, including 13 adrenocortical carcinomas (ACCs), were categorized according to Weiss score, hormonal profile, and patient survival data and analyzed using immunohistochemistry and RT-PCR.Expression of the TGF-β signaling mediator SMAD3 varied inversely with Weiss score, so that SMAD3 expression was lowest in the most malignant tumors. By contrast, SMAD2 expression was upregulated in most malignant tumors. Wnt pathway co-receptors LRP5 and LRP6 were predominantly expressed in benign adrenocortical tumors. In ACCs, expression of transcription factors GATA-6 and SF-1 correlated with that of their target gene INHA. Moreover, the diminished expression of GATA-6 and SF-1 in ACCs correlated with poor outcome.We conclude that the factors driving INHA expression are reduced in ACCs with poor outcome, implicating a role for INHA as a tumor suppressor in humans.

Expression of Inhibin-Alpha Is Regulated Synergistically by Wilms' Tumor Gene 1 (Wt1) and Steroidogenic Factor-1 (Sf1) in Sertoli Cells

Wt1 encodes a zinc finger nuclear transcriptional factor, which is specifically expressed in testicular Sertoli cells and knockdown of Wt1 in Sertoli cells causes male mice subfertility. However, the underlying mechanism is still unclear. In this study, we found that expression of inhibin-α is significantly reduced in Wt1-deficient Sertoli cells. Luciferase assays using the inhibin-α promoter indicated that the inhibin-α promoter is transactivated by the Wt1 A, and B isoforms (−KTS), but not the C, and D isoforms (+KTS). Analysis of the Wt1 responsive element of the inhibin-α promoter region using site-directed mutagenesis showed that the nucleotides between −58 and −49 are essential for Wt1-dependent transactivation of the inhibin-α promoter. ChIP assays indicated that Wt1 directly interacts with the inhibin-α promoter. In addition, the inhibin-α promoter is activated synergistically by Wt1 and Sf1. Mutation of the ligand binding domain (LBD) of Sf1 (residues 235–238) completely abolished the synergistic action between Wt1 and Sf1, but did not affect the physical interaction between these two proteins, suggesting that other factor(s) may also be involved in the regulation of inhibin-α in Sertoli cells. Further studies demonstrated that β-catenin enhances the synergistic activation of Wt1 and Sf1 on the inhibin-α promoter. Given the fact that inhibin-α, a subunit of inhibin, is known to be involved in the regulation of spermatogenesis and testicular steroidogenesis, this study reveals a new regulatory mechanism of inhibin-α in Sertoli cells and also sheds light on the physiological functions of Wt1 in gonad development and spermatogenesis.

Cross Talk Between Estradiol and mTOR Kinase in the Regulation of Ovarian Granulosa Proliferation

Treatment of ovarian granulosa cells and follicles with the mammalian target of rapamycin (mTOR) kinase inhibitor results in biphasic effects where nanomolar rapamycin (RAP) results in reduced proliferation, mitotic anomalies, and attenuated follicle growth, while the picomolar RAP results in accelerated follicle growth. Here, we tested whether such effects are specific to RAP or could be mimicked by 2 alternative mTOR inhibitors, everolimus (EV) and temsirolimus (TEM), and whether these effects were dependent on the presence of estradiol (E2). Spontaneously immortalized rat granulosa cells (SIGCs) were cultured in dose curves of RAP, EV, TEM, or vehicle with or without E2. Proliferation and phosphorylation of mTOR targets p70S6 kinase and 4E-binding protein (BP) were determined. Cell cycle gene array analysis and confirmatory quantitative reverse transcriptase polymerase chain reaction were performed upon cells treated with picomolar RAP versus controls. Nanomolar RAP, EV, and TEM reduced SIGC proliferation and decreased phospho-p70 and 4E-BP. Picomolar concentrations accelerated proliferation without affecting mTOR substrate phosphorylation. Acceleration of growth by picomolar inhibitor required E2. Picomolar drug treatment altered the transcription of cell cycle regulators, increasing Integrin beta 1 and calcineurin expression, and decreasing inhibin alpha, Chek1, p16ARF, p27/Kip1, and Sestrin2 expression. At nanomolar concentrations, mTOR inhibitors attenuated granulosa proliferation. Accelerated growth and alterations in cell cycle gene transcription found with picomolar concentrations required E2 within the intrafollicular concentration range. The low concentrations of inhibitors required to increase granulosa proliferation suggest a novel use to support the growth of ovarian follicles.

Differential responses to oxidative stress and calcium influx on expression of the transforming growth factor‐β family in myoblasts and myotubes

Changes in gene expression of TGF-beta family members and their receptors in response to treatment with H(2)O(2) and a calcium ionophore, A23187, were examined in C2C12 myoblasts and myotubes. The expression of Myf5, an initial regulator of myogenesis, was increased by A23187, and H(2)O(2) inhibited the up-regulation of Myf5. Treatment with H(2)O(2) decreased the expression of MHC IIb, a protein component of the myofibrils, irrespective of the presence of A23187, suggesting an inhibitory role of oxidative stress for myogenesis. Expression of ligands and receptors for the TGF-beta family was modulated in response to H(2)O(2) and A23187. Treatment with H(2)O(2) decreased expression of TGF-beta3, BMP-4, ALK4, ALK5, and ActRIIB, and increased expression of inhibin alpha and inhibin betaA in either the myoblast stage or the myotube stage, or both. A23187 potentiated down-regulation of BMP-4 and ALK4 expression, and up-regulation of TGF-beta1, TGF-beta2, inhibin alpha, inhibin betaA, ALK2, and ALK3 expression. These results indicate that oxidative stress and Ca(2+) influx affect expression of the TGF-beta family in C2C12 myoblasts and myotubes.

Effects of Diethylstilbestrol on Programmed Oocyte Death and Induction of Polyovular Follicles in Neonatal Mouse Ovaries1

In mice, neonatal exposure to a synthetic estrogen, diethylstilbestrol (DES), induces polyovular follicles, which contain two or more oocytes per ovarian follicle. We reported previously that the estrogen receptor beta (ESR2) mediates DES signaling in polyovular follicle induction. However, the specific mechanism of polyovular follicle induction has not yet been clarified. Folliculogenesis in rodents begins soon after birth, accompanied by programmed oocyte death and germ cell loss. In this study, the effects of DES on oocyte death and on mRNA expression of genes thought to be involved in polyovular follicle induction were analyzed during a crucial period of folliculogenesis in the ovary of C57BL/6J, Fas(lpr/lpr) (lacking cell death receptor, FAS), and Esr2 knockout (Esr2 KO) mice. Neonatal DES exposure reduced programmed oocyte death in C57BL/6J mice; however, this reduction was not observed in Esr2 KO mice. In control Fas(lpr/lpr) mice, the oocyte apoptotic index was significantly lower than that in the control C57BL/6J mice. However, the polyovular follicle incidence in control 20-day-old Fas(lpr/lpr) mice was similar to that in the control C57BL/6J mice. Moreover, DES exposure changed mRNA expression of inhibin-alpha (Inha) in 2-day-old C57BL/6J mice. These results suggest that inhibition of oocyte death by DES through ESR2 may be one of the triggers for polyovular follicle induction. The FAS system is also involved in neonatal oocyte death; however, reduction of oocyte death is not sufficient for polyovular follicle induction. The combination of increased Inha mRNA and reduction of oocyte death in the ovaries of mice by DES through ESR2 might be correlated with polyovular follicle induction.

Physiological Androgen Insensitivity of the Fetal, Neonatal, and Early Infantile Testis Is Explained by the Ontogeny of the Androgen Receptor Expression in Sertoli Cells

Although gonadotropins and testosterone are high in the fetal/early postnatal periods, Sertoli cells remain immature and spermatogenesis does not progress. We hypothesized that Sertoli cells do not respond to testosterone because they do not express the androgen receptor. The objective of the study was to describe the precise ontogeny of androgen receptor expression in the human testis from fetal life through adulthood. This was an immunohistochemical study on testicular biopsies from fetal, neonatal, prepubertal, pubertal, and adult human testes. Quantification of androgen receptor expression in Sertoli cells was measured. Evaluation of androgen receptor expression in peritubular and interstitial cells as well as anti-Müllerian hormone and inhibin-alpha was also performed. Androgen receptor expression was first observed in the nuclei of few Sertoli cells at the age of 5 months. Labeling was weak in 2-15% of Sertoli cells until 4 yr of age and progressively increased thereafter. High levels of androgen receptor expression were observed in more than 90% from the age of 8 yr through adulthood. Androgen receptor was positive in peritubular cells and variable in interstitial cells. Anti-Müllerian hormone immunolabeling was strong in all Sertoli cells from fetal life throughout prepuberty and weakened progressively as spermatogenesis developed. Inhibin-alpha expression was detected in all Sertoli cells from fetal life through adulthood. A lack of androgen receptor expression could explain a physiological Sertoli cell androgen insensitivity during fetal and early postnatal life, which may serve to protect the testis from precocious Sertoli cell maturation, resulting in proliferation arrest and spermatogenic development.

Impaired Follicle Development and Infertility in Female Mice Lacking Steroidogenic Factor 1 in Ovarian Granulosa Cells1

The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.

The expression and localization of inhibin isotypes in mouse testis during postnatal development

Inhibin, which is important for normal gonadal function, acts on the pituitary gonadotropins to suppress follicle-stimulating hormone (FSH) secretion. The level and cellular localization of the inhibin isotypes, α, βA and βB, in the testis of mice were examined during postnatal development in order to determine if inhibin expression is related to testicular maturation. Mouse testes were sampled on postnatal days (PNDs) 1, 3, 6, 18, 48 and 120, and analyzed by Western blotting and immunofluorescence. Western blot analysis showed very low levels of inhibin α, βA and βB expression in the testes at days 1 to 6 after birth. The levels then increased gradually from PND 18 to 48-120, and there were significant peaks at PND 48. Inhibin α, βA and βB were detected in testicular cells during postnatal development using immunohistochemistry. The immunoreactivity of inhibin α was rarely observed in testicular cells during PND 1 to 6, or in the cytoplasmic process of Sertoli cells surrounding the germ cells and interstitial cells during PND 18 to 120. Inhibin βA and βB immunoreactivity was rarely observed in the testis from PND 1 to 6. On the other hand, it was observed in some spermatogonial cells, as well as in the interstitial space between PND 48 and PND 120. We conclude that the expression of inhibin isotypes increases progressively in the testis of mice with increasing postnatal age, suggesting that inhibin is associated with a negative feedback signal for FSH in testicular maturation.

Comparison of Immunohistochemical Markers in the Differential Diagnosis of Adrenocortical Tumors

Most adrenocortical tumors (ACTs) can be diagnosed directly by a combination of morphologic features and clinical findings. However, sometimes it may be difficult to distinguish ACTs from other neoplasms such as pheochromocytomas and some metastatic tumors, particularly for small biopsy specimens because they may be morphologically similar. Expression of calretinin has recently been suggested as a valuable immunomarker for the differential diagnosis between ACTs and other tumors; however, its diagnostic value is still under debate. To determine the diagnostic value of calretinin in Chinese patients with adrenocortical and non-ACTs, we employed both polyclonal and monoclonal anticalretinin to characterize the expression of calretinin in adrenal tissues and compared its expression with that of inhibin alpha, Melan-A, cytokeratin, or CD99 by immunohistochemistry in tissue microarrays and standard tissue sections of 414 specimens. Our results revealed that calretinin was expressed by adrenocortical cells, but not by the other cells tested and the percentage of calretinin-positive ACTs reached 99% when stained with polyclonal antibodies, which was higher than that with monoclonal anticalretinin (91.3%), anti-Melan-A (90.3%), antiinhibin alpha (81.6%). In addition, our results also revealed that ACTs were stained by cytokeratin (AE1/AE3) with variable degrees (58.7%). Furthermore, unlike anti-Melan-A that stained all metastatic malignant melanoma, anticalretinin did not recognize other tested tumors. Therefore, immunohistologic staining with polyclonal anticalretinin is more sensitive than other antibodies tested for the diagnosis of ACTs. However, monoclonal anticalretinin appeared to be more specific. Importantly, our data suggested that the fried-egg-like staining pattern, but not the mere cytoplasmic staining, was characteristic of anticalretinin staining in adrenocortical tissues. Notably, a few anticalretinin negative-ACTs were stained by other immunomarkers that we tested. Thus, the combinational characterization of calretinin (either by polyclonal or monoclonal antibody), inhibin alpha, and Melan-A expression is of great significance in the differential diagnosis of ACTs.

Interaction of testosterone with inhibin α and βA subunits to regulate prostate gland growth

Testosterone regulation of prostate gland growth has been shown to involve reciprocal interaction with inhibin and activin. This study was therefore conducted to correlate the effect of testosterone on prostate gland proliferation and differentiation with the level of expression of inhibin alpha and betaA subunits. Immature dogs were treated with testosterone for 0, 3, 7, and 14 days and prostate gland growth was assessed by morphological and immunohistological localization of differentiation and proliferation markers. The results showed that testosterone treatment resulted in an initial significant increase in PCNA proliferation index by days 3 and 7, followed by a significant decrease by day 14 post-treatment. Interestingly, the decrease of cell proliferation was associated with structural and biochemical changes characteristic of glandular and stromal differentiation of the prostate gland. These changes include progressive glandular ductal canalization and inter-ductal stroma differentiation which were apparent from a gradual shift from vimentin expression to vimentin and alpha-actin expression. Testosterone also had a differential effect on inhibin alpha and beta subunits. Although testosterone treatment resulted in significant and constant inhibition of alpha subunit mRNA expression, it resulted in a significant increase of betaA mRNA expression by day 3, followed by a decrease by days 7 and 14. These results indicated that testosterone acts first to drive proliferation of undifferentiated prostatic cells and then to maintain a low proliferation turnover of differentiated cells. Because it has been shown that activin is an antagonistic regulator of androgens, the attenuated stimulatory effect of testosterone on cell proliferation by day 14 might be mediated, at least in part, by interplay between testosterone and activin.

Diagnose und Differenzialdiagnose des Granulosazelltumors

Clinically and morphologically, two types of granulosa cell tumor can be distinguished, the more frequent adult type and the juvenile type. In the adult type, different growth patterns can be observed: microfollicular (most frequent, characterized by Call-Exner bodies), macrofollicular, trabecular, insular, solid-tubular, gyriform and diffuse (sarcomatoid). The juvenile type is characterized by solid and follicular structures. The neoplastic granulosa cells in the adult type have limited cytoplasm and haphazardly arranged angular, pale, mostly grooved nuclei. In the juvenile type, the cells have ample eosinophilic cytoplasm and polymorphic, sometimes bizarre nuclei, which are usually non-grooved. The number of mitoses in the adult type usually does not exceed 2/10 HPF, whereas it is considerably higher in the juvenile type, including atypical mitoses. A common feature in both types of tumor is the expression of inhibin-alpha, calretinin and CD 99. Epithelial membrane antigen is negative. Molecular genetics has demonstrated loss of heterozygosity at 19p13.3 in 52% of the cases. Besides chromosomal aberrations, there are a large number of cytogenetic anomalies. The most important prognostic factor in both types of tumor is tumor stage. Whereas recurrences in the adult type can develop even after decades, in the juvenile type they usually occur during the first 3 years after diagnosis.

Expression of estrogen and progesterone receptors and inhibin-α in hepatobiliary cystadenoma: an immunohistochemical study

To further characterize the immunohistochemical features of hepatobiliary cystadenoma with mesenchymal stroma, a battery of stains was performed on nine typical cases. All nine tumors had been resected from female patients who ranged in age from 30 to 59 years. Freshly cut sections were stained with antibodies to estrogen receptor (ER), progesterone receptor (PR), alpha-smooth muscle actin (SMA), inhibin-alpha, and cytokeratins (CK) 7, 8, 18, and 19. Nuclear staining of the mesenchymal stromal cells for ER and PR was present in all and seven out of nine cases, respectively. A strong cytoplasmic staining of the mesenchymal stromal cells for SMA was seen in all cases. A patchy pale cytoplasmic staining of the tumor epithelium for ER and PR was seen in five out of nine and four out of nine cases, respectively. Immunoreactivity of luteinized stromal cell for inhibin-alpha was documented in three out of nine cases. All tumors (nine out of nine) demonstrated strong cytoplasmic positivity of the epithelial lining of the cysts to CK7, CK8, CK18, and CK19, typical of biliary-type epithelium. The expression of ER, PR, and inhibin-alpha in the ovarian-like stroma supports the likely hormonal dependence of this tumor and probably explains its almost exclusive occurrence in women.

Characterization of cAMP/PKA/CREB signaling cascade in the bonnet monkey corpus luteum: expressions of inhibin-α and StAR during different functional status

Luteinizing hormone mediates its nuclear action primarily by activating cAMP/Protein kinase A (PKA) pathway leading to phosphorylation of cAMP response element binding (CREB) family of transcription factors. Earlier studies have documented altered cAMP responsiveness of luteal cells during maturation, and in the rhesus monkey, extinction of CREB expression following luteinization and ovulation. In the course of studies aimed at characterizing LH-cAMP signaling pathway, we serendipitously discovered that CREB is after all present in the monkey corpus luteum (CL). The present experiments were carried out to examine the PKA activity, CREB expression and RT-PCR expression of inhibin-alpha (Inh-alpha) subunit and steroidogenic acute regulatory protein (StAR) in CL obtained from a variety of model systems. PKA activity in the CL was maintained throughout the luteal phase. Messenger RNA expression by RT-PCR and Northern analyses and protein levels employing antibodies specific to total- and phospho-forms demonstrated presence of CREB in the CL. Additionally, immuno-histo/cytochemical analyses, Electrophoretic mobility shift assays and chromatin immunoprecipitation assays for Inh-alpha and StAR genes further confirmed the presence of CREB in the CL. The present study, contrary to an earlier report, demonstrates the presence of CREB (both transcript and protein) in the monkey CL. Also, analysis of expression of Inh-alpha and StAR genes (considered to be cAMP responsive), during different functional status of CL suggests that LH regulates their expression perhaps by cAMP/PKA/CREB pathway.

Expression of activin and inhibin subunits, receptors and binding proteins in human pheochromocytomas: a study based on mRNA analysis and immunohistochemistry

Pheochromocytomas are uncommon tumours arising from chromaffin cells of the adrenal medulla and related paraganglia. So far, one of the few reported markers to discriminate malignant from benign tumours is the betaB-subunit of inhibin and activin, members of the transforming growth factor (TGF)-beta superfamily of growth and differentiation factors. We investigated the expression of the mRNAs coding for activin and inhibin subunits, their receptors and binding proteins by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and studied the presence of the inhibin betaB-subunit in human pheochromocytomas by immunohistochemistry. Samples from resected pheochromocytomas of patients operated between 1973 and 2003 were used for experiments. The immunohistochemical investigations revealed that staining of the inhibin betaB-subunit was positive in 12 of 36 (33%) benign and 5 of 34 (15%) malignant pheochromocytomas (P > 0.05). Therefore, it was not possible to discriminate between benign and malignant tumours solely on the basis of inhibin betaB-subunit immunohistochemistry. Quantitative real-time RT-PCR in nine benign and four malignant tumours showed expression of inhibin alpha-, betaA- and betaB-subunits, the activin receptors Alk-4, ActRIIA, and ActRIIB, and the inhibin- and activin-binding proteins betaglycan and follistatin in all samples. No correlations were detected between individually coupled expression of mRNAs of these activin- and inhibin-related genes in the 13 pheochromocytomas. Only inhibin betaA-subunit expression was different in malignant compared to benign pheochromocytomas (P = 0.020). No clear role for activin and inhibin was found in discriminating between benign and malignant pheochromocytomas.