Inhibin Alpha Research Articles

025.Ovaries: up in a POF of smoke

Premature ovarian failure (POF) or premature menopause is a common disorder, defined by the occurrence of menopause under the age of 40 years and is characterised by amenorrhoea, hypoestrogenism and elevated gonadotrophins. Worldwide it affects 1% of all women and occurs in 0.1% before the age of 30 years. The major problems associated with POF are the loss of fertility at an early age and the psychological problems associated with this. In addition there are the physiological effects of reduced oestrogen, which include an increased risk of osteoporosis. POF is a heterogeneous disorder and the cause of most cases is unknown. A significant proportion (20-30%) of women with POF have a genetic predisposition. Our primary goal is to identify genes involved in POF. In most cases, the menopause is due to the loss of follicles, and it stands to reason that suitable candidate genes for POF development would be genes that regulate the rate of follicle loss. We have identified two common gene mutations, a 769G>A transition in the inhibin alpha gene in approximately 5% of POF patients associated with POF at a very early age, and mutations in FOXL2 in approximately 5% of patients. FOXL2 is thought to act downstream of inhibin which suggests that other candidate genes may arise from the analysis of the activin signalling pathway. Functional studies will help us to understand more about the molecular basis of POF. Each mutation has been associated with less than 10% of POF cases and POF is likely to be caused by mutations at many different loci. It is hoped that determining the molecular basis of POF will lead to the development of genetic tests to predict the development of POF, and eventually lead to treatment that will return fertility to these women.

G769A variation of inhibin alpha-gene in korean women with premature ovarian failure.

Premature ovarian failure (POF) is menopause before the age of 40 years. The frequency of POF is about 1% of all women. Recently inhibin alpha gene (INHalpha) has been indicated as candidate in POF pathogenesis. Inhibin, a glycoprotein, is a gonadal hormone, which can inhibit the synthesis and secretion of pituitary follicle-stimulating hormone (FSH), which has an important role in the recruitment and development of ovarian follicles during the folliculogenesis. G769A variation of INHalpha, alanine, is highly conserved across species, and has an important role of its receptor binding. We screened a G769A transition in the INHalpha from the total population of the patients of 84 women with POF and 100 normal fertile women. We found no variation between the normal subjects and the POF patients. G769A variation of INHalpha is rare in Korea women with POF.

Effect of Active Immunization of Pony Mares against Recombinant Porcine Inhibin .ALPHA. Subunit on Ovarian Follicular Development and Plasma Steroids and Gonadotropins

Two pony mares were immunized against recombinant porcine inhibin alpha subunit three times with 39 day intervals. Clinical findings and endocrinological changes before immunization were taken as the control. The first significant rise in the anti-inhibin titre (P<0.05) in the circulation was found 27 days after the first injection. Maximum binding activity was reached by the 12th day after the second booster dose. The number of small, medium and large sized follicles had increased significantly compared to before immunization (11.75 +/- 4.30, 2.75 +/- 0.69 and 2.51 +/- 0.63 vs 6.50 +/- 1.43, 1.83 +/- 0.44 and 1.33 +/- 0.38, respectively), but the ovulation rate remained unchanged after immunization. The average plasma concentration of FSH and estradiol-17beta during the estrous cycle increased significantly (P<0.05) after immunization. These results suggest that immunization against inhibin is a useful tool to increase the number of ovarian follicles during the estrous cycle of pony mares. Moreover, the present study supported the concept that inhibin plays a major role in the control of follicular growth through its inhibitory effect on FSH secretion synergistically with steroid hormones.

Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary.

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2, and 3 ligand immunoreactivity is observed in both small and large follicles, whereas the TGFbeta type II receptor (TGFbeta RII) is detected in the oocyte and granulosa cells of antral follicles. The intracellular signaling proteins Smad2 and Smad4 are present in the granulosa cell cytoplasm of all follicle size classes. Smad3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicle size classes. These data suggest that the complete activin signal transduction pathway is present in small follicles and that large follicles primarily produce the inhibins. Our data also suggest that TGFbeta ligands and receptors are colocalized to large antral follicles. Taken together, the ligands, receptors, and signaling proteins for the TGFbeta superfamily are present at distinct points throughout feline folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation.

Pituitary follistatin gene expression in female rats: evidence that inhibin regulates transcription.

Follistatin (FS), along with the members of the transforming growth factor beta family activin and inhibin, are important regulators of FSH secretion and messenger RNA production. While activin and inhibin appear to function as tonic modulators of FSH (stimulatory and inhibitory, respectively), dynamic changes in FS are noted through the estrous cycle and under varying physiological experimental paradigms. This suggests that FS is a major contributor to the precisely coordinated secretion of FSH that maintains reproductive function. The aim of this study was to investigate changes in FS, in particular the early (<12 h) rise observed after ovariectomy (OVX), and to determine whether these changes were as a consequence of variations in gene transcription rates. FS primary transcript (PT) and mRNA were found to increase 3-fold 12 h post-OVX, indicating increased gene transcription during this time period. Replacement with estradiol and/or blockade of GnRH had only modest effects on FS PT concentration. Inhibin immunoneutralization of intact rats resulted in a 3-fold increase in FS PT 12 h after administration of inhibin alpha antisera. Significant increases in FS mRNA at both 2 and 12 h also suggested that inhibin also may have effects on message stability. After administration of recombinant human inhibin A, there was a prompt decline in both FS PT and mRNA. These results indicate that inhibin is a major regulator of FS, both by transcriptional and nontranscriptional mechanisms.

Inhibin alpha distinguishes hemangioblastoma from clear cell renal cell carcinoma.

Inhibin alpha subunit (inhibin A) expression in hemangioblastomas has not been previously reported in the literature. We analyzed the expression of inhibin A in 25 hemangioblastomas from 22 patients. Eleven cases were from 8 patients with von Hippel-Lindau disease, and these tumors were multicentric and/or recurrent. The remaining 14 cases from 14 patients were sporadic. The male-to-female ratio was 8:3, and the age at presentation ranged from 19 to 78 years (mean 35 years; median 45 years). Eighteen tumors were located in the cerebellum/posterior fossa, 1 in the medulla, 1 in the occipital lobe, and 5 in the spinal cord. Four metastatic renal cell carcinomas in brain, 10 renal cell carcinomas from 8 patients with von Hippel-Lindau disease, and 5 sporadic clear cell renal cell carcinomas were also included. Two patients with von Hippel-Lindau disease had both renal cell carcinoma and hemangioblastoma. The stromal cells of all 25 cases of hemangioblastoma expressed inhibin A. Strong, moderate, and weak cytoplasmic immunoreactivity was noted in 17, 5, and 3 cases, respectively. In contrast, none of the 19 renal cell carcinomas, primary as well as metastatic, expressed inhibin A. There was no difference in the inhibin A staining pattern between the sporadic hemangioblastoma and those associated with VHL. These findings demonstrate inhibin A to be a useful marker in distinguishing hemangioblastoma from metastatic clear cell renal cell carcinoma. While the diagnostic importance is evident, the pathophysiology of inhibin A expression by the stromal cells of hemangioblastoma remains unknown and further investigation is required.

Sexually dimorphic roles of steroid hormone receptor signaling in gonadal tumorigenesis.

Sex steroids control cellular phenotypes by binding to receptor proteins that in turn regulate downstream gene expression. They are important tropic factors in hormonally responsive tissues and have been implicated in the pathogenesis of both benign proliferations and malignancies at some of these sites. Knockout mice lacking inhibins, alpha:beta heterodimeric peptide hormones of the TGFbeta superfamily, develop gonadal tumors that produce sex steroids and depend on pituitary gonadotropin hormones. To better appreciate how sex steroid receptor signaling pathways contribute to the loss of granulosa/Sertoli cell proliferation in the ovary and testis of inhibin alpha (Inhalpha) knockout mice, we are using both pharmacologic and genetic approaches. Roles of androgens in testicular tumor development have been investigated in our previous studies using double-mutant mice lacking inhibins and carrying the null testicular feminization (tfm) mutation of the androgen receptor. Herein, we report that androgens also participate in the development of ovarian tumors, as tumor development is forestalled in mice treated with flutamide, a nonsteroidal inhibitor of androgen actions. Additionally, we generated double-mutant mice lacking estrogen receptor alpha (ERalpha) and Inhalpha or ERbeta and Inhalpha, as well as triple-mutant mice lacking ERalpha, ERbeta, and Inhalpha to determine the effects of individual and combined ER signaling pathways on tumor development. Although estrogens may have proliferative effects during follicle development and are important in specifying the granulosa cell phenotype, ERalpha and ERbeta signaling are not essential for timely granulosa cell tumor development or granulosa cell-like morphological features in ovarian tumors. However, redundant ER signaling through ERalpha and ERbeta in males is critical for testicular tumor formation, as triple-knockout, but not double-knockout, males are protected from early Sertoli cell tumorigenesis and death. Together, these studies indicate important and sexually dimorphic functions of estrogens and androgens in tumor development in this mouse model and indicate, for the first time, overlapping functions of ERalpha and ERbeta in Sertoli cell pathophysiology.

Expression profiling analyses of gonadotropin responses and tumor development in the absence of inhibins.

Transgenic mice with engineered disruptions in bidirectional endocrine signaling between the pituitary and gonad have shed light on the specific effects of the loss of function of gonadotropins and inhibins. These models are valuable tools for studying ovarian biology because they phenocopy specific pathological states and have variations in ovarian tissue composition that allow us to identify genes expressed in specific cell types. We have used emerging mRNA expression profiling technologies to gain a more comprehensive view of genes that are expressed in the mammalian ovary and adrenal gland in the FSHbeta and inhibin alpha knockout mouse models. Oligonucleotide array hybridization experiments using Affymetrix GeneChip technology and NIA 15K murine cDNA microarray studies identified hundreds of transcripts differentially expressed compared with wild type, over 30 of which were selected for further characterization by Northern blot analyses. Additionally, we performed in situ hybridization studies to localize 10 mRNAs, melanocyte-specific gene 1, amino acid transporter SN2, overexpressed and amplified in teratocarcinoma (Bcat1), Forkhead box protein FOXO1, 24p3, vascular cell adhesion molecule, epiregulin, Bcl2-like10, PC3B, and retinoblastoma binding protein 7. These 10 genes have expression patterns and postulated functions suggesting that they mediate important processes in the physiology and pathology of ovarian and adrenal tissue.

The activin-follistatin system in the neonatal ovine uterus.

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding and tubulogenesis followed by coiling and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (Postnatal Day [PND] 0) and PND 56. Activins, which are members of the transforming growth factor beta superfamily, and follistatin, an inhibitor of activins, regulate epithelial branching morphogenesis in other organs. The objective of the present study was to determine effects of postnatal age on expression of follistatin, inhibin alpha subunit, betaA subunit, betaB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin alpha subunit mRNA or protein was detected in the neonatal uterus. Expression of betaA and betaB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.

Effects of passive immunization of goats against inhibin on follicular development, hormone profile and ovulation rate

This study was conducted to investigate the effect of immunoneutralization against endogenous inhibin on FSH secretion and ovulation rate, with the aim of developing a new superovulation method using inhibin antiserum in goats. Two groups of goats received an i.v. injection of either 10 ml normal goat serum (control; n = 6) or 10 ml inhibin antiserum developed against [Tyr(30)]-inhibin alpha (1-30) (n = 6) 48 h before treatment with prostaglandin F(2alpha) (PGF(2alpha)). Blood samples were collected at 6 h intervals and ovaries were examined each day using a B-mode ultrasound scanner equipped with a 7.5 MHz transducer during the experimental period. Immunization against inhibin resulted in a four- to fivefold increase (P < 0.01) in plasma concentrations of FSH. After luteolysis, plasma concentrations of oestradiol increased markedly to reach a preovulatory peak, which was about two times higher (P < 0.01) than that of the controls. The treatment was accompanied by a significant increase in the total number of follicles of > or = 3 mm in diameter at 24 (8.2 +/- 0.4 in inhibin antiserum group versus 4.8 +/- 0.3 in control group) and 96 h later (13.5 +/- 1.0 in inhibin antiserum group versus 5.3 +/- 0.6 in control group). The ovulation rate was significantly (P < 0.01) higher in goats treated with inhibin antiserum (4.2 +/- 0.5; n = 6) than in control goats (1.8 +/- 0.3; n = 6). These results indicate that inhibin is an important factor in the regulation of FSH secretion in goats and demonstrate that passive immunization against inhibin at 48 h before treatment with PGF(2alpha) induces the development of more follicles and increases ovulation rate. Thus, inhibin antiserum treatment may be an alternative to the use of exogenous gonadotrophins for induction of superovulation in goats.

Effects of passive immunization of goats against inhibin on follicular development, hormone profile and ovulation rate

This study was conducted to investigate the effect of immunoneutralization against endogenous inhibin on FSH secretion and ovulation rate, with the aim of developing a new superovulation method using inhibin antiserum in goats. Two groups of goats received an i.v. injection of either 10 ml normal goat serum (control; n = 6) or 10 ml inhibin antiserum developed against [Tyr(30)]-inhibin alpha (1-30) (n = 6) 48 h before treatment with prostaglandin F(2alpha) (PGF(2alpha)). Blood samples were collected at 6 h intervals and ovaries were examined each day using a B-mode ultrasound scanner equipped with a 7.5 MHz transducer during the experimental period. Immunization against inhibin resulted in a four- to fivefold increase (P < 0.01) in plasma concentrations of FSH. After luteolysis, plasma concentrations of oestradiol increased markedly to reach a preovulatory peak, which was about two times higher (P < 0.01) than that of the controls. The treatment was accompanied by a significant increase in the total number of follicles of > or = 3 mm in diameter at 24 (8.2 +/- 0.4 in inhibin antiserum group versus 4.8 +/- 0.3 in control group) and 96 h later (13.5 +/- 1.0 in inhibin antiserum group versus 5.3 +/- 0.6 in control group). The ovulation rate was significantly (P < 0.01) higher in goats treated with inhibin antiserum (4.2 +/- 0.5; n = 6) than in control goats (1.8 +/- 0.3; n = 6). These results indicate that inhibin is an important factor in the regulation of FSH secretion in goats and demonstrate that passive immunization against inhibin at 48 h before treatment with PGF(2alpha) induces the development of more follicles and increases ovulation rate. Thus, inhibin antiserum treatment may be an alternative to the use of exogenous gonadotrophins for induction of superovulation in goats.

Inhibin, activin, follistatin, activin receptors and beta-glycan gene expression in the placental tissue of patients with pre-eclampsia.

The objective of this study was to quantify the relative expression of inhibin alpha, inhibin/activin beta(A), beta(B), beta(C), follistatin, activin receptors and beta-glycan genes in placental tissue of term pre-eclamptic patients and controls to investigate if these genes are up-regulated in the placenta in pre-eclampsia. Seven women with pre-eclampsia symptoms were matched with 10 normal pregnant controls for gestational age, maternal age, and parity. Total RNA was isolated from each sample. Complementary DNA samples produced by reverse transcription were used in the real time PCR to quantify the expression of inhibin alpha subunit, inhibin/activin beta(A), beta(B), beta(C) subunits, follistatin, ACTRIA, ACTRIB, ACTRIIA, ACTRIIB, beta-glycan and GAPDH genes. The ratio between the target and GAPDH expression was calculated to provide relative gene expression. Inhibin alpha:GAPDH and inhibin/activin beta(A): GAPDH ratios were significantly higher in placental tissue from women with pre-eclampsia (P = 0.04 and P = 0.01 respectively) compared with matched control placental gene expression. Placental samples from both groups expressed beta(B), beta(C), follistatin, activin receptors and beta-glycan genes. However, there was no significant difference in the relative expression of these genes between the groups. Increases in the placental expression of inhibin alpha and inhibin/activin beta(A) subunit genes could contribute to the rise in circulating levels of inhibin A and activin A in pre-eclampsia. The mechanism(s) involved in increased gene expression in pre-eclampsia is as yet unclear.

Activin and Follicle-Stimulating Hormone Signaling Are Required for Long-Term Culture of Functionally Differentiated Primary Granulosa Cells from the Chicken Ovary1

Follicle-stimulating hormone, activin A, and transforming growth factor (TGF) alpha are important regulators of chicken granulosa cell (cGC) function. Hence, we aimed to test whether these growth factors are useful for establishing a suitable in vitro cell culture model system of primary cGC. Although cGC are easily isolated from distinct follicular stages, a long-term cGC culture system for in vitro studies has been unavailable. Here, we report a novel, long-term cell culture system that allows for cGC proliferation in vitro while maintaining the epithelial phenotype that granulosa cells exhibit in vivo. The cGC rapidly lose their epithelial morphology and acquire a mesenchymal or fibroblastoid phenotype when cultured in the absence of activin A. This process is strongly enhanced by TGFalpha, a well-known granulosa cell mitogen. However, FSH stimulates cGC proliferation without enhancing morphological changes and dedifferentiation. Interestingly, a combination of both activin A and FSH stimulates cGC proliferation and supports maintenance of differentiated epithelial morphology. Furthermore, activin A and FSH synergistically induce granulosa cell-specific differentiation markers such as inhibin alpha and chicken zona pellucida protein C, suggesting that cultured cGC resemble functionally differentiated granulosa cells. Our data demonstrate that activin signaling is necessary to sustain a morphologically differentiated phenotype of cGC in vitro. The results also suggest a pivotal importance of activin signaling for granulosa cell function in vivo.

Serum inhibin A and B concentrations during the menstrual cycle in mothers of spontaneous dizygotic twins.

Dizygotic twinning in humans is influenced by genetic factors suggesting inherited variation affects follicle development and predisposes to double ovulations. In a previous study, we conducted a detailed examination of follicle development and variation in hormone concentrations during the menstrual cycle in mothers of DZ twins (MODZT) compared with an age-matched control group of mothers of singletons. We did not detect differences in FSH concentrations between mothers of twins and mothers of singletons. Serum inhibin concentrations were measured by a radioimmunoassay that did not distinguish between dimeric inhibin A and B forms and free inhibin alpha subunit. We therefore analyzed the samples from this study with specific assays to determine whether concentrations of inhibin A and B were different between MODZT and controls and therefore contribute to the twinning phenotype. There were no significant differences between MODZT with single ovulations and control women in inhibin A and B concentrations during the cycle, including the critical period for the selection of the dominant follicle. These data suggest that the genetic cause of twinning is not associated with changes in FSH concentrations or recognised feedback mechanisms regulating FSH release.

GATA-4 and GATA-6 transcription factors: expression, immunohistochemical localization, and possible function in the porcine ovary.

The expression and localization of GATA-4 and GATA-6 mRNAs and proteins were assessed in porcine ovaries at different stages of the estrous cycle. Reverse transcription polymerase chain reaction and Western blot analyses revealed that GATA-4 and GATA-6 transcripts and proteins were strongly expressed in granulosa cells isolated from antral follicles, intact antral follicles, corpora hemorrhagica (CH), and midluteal phase corpora lutea (CL). Immunoblot analyses showed two predominant proteins with molecular masses of approximately 53 and 55 kDa for GATA-4 and one 55-kDa protein for GATA-6. Immunohistochemical studies revealed GATA-4 and GATA-6 nuclear staining in granulosa cells of healthy primordial and primary antral follicles and antral follicle of various sizes. The percentage of immunopositive thecal cell nuclei increased with follicular development. In CH and CL, luteal cells displayed nuclear immunoreactivity for both transcription factors. Regressing CL showed a decrease in GATA-immunopositive cells. Immunoreactivity for GATA-4 and GATA-6 was present in most blood vessels. In electrophoretic mobility shift assays, nuclear protein extracts isolated from granulosa cells and CL exhibited both GATA-4 and GATA-6 binding to a GATA consensus oligonucleotide, with GATA-4 the predominant binding protein. GATA-4 and GATA-6 DNA binding was elevated in granulosa cell nuclear extracts from preovulatory (8-10 mm) follicles. Cotransfection of primary cultures of luteinizing granulosa cells with GATA-4 or GATA-6 expression vectors increased the activity of the porcine steroidogenic acute regulatory protein gene promoter significantly but did not significantly activate the inhibin alpha gene promoter. The detection of GATA-4 and GATA-6 mRNAs and proteins in porcine ovaries and the identification of at least one possible target gene may help to establish roles for these GATA factors in follicular development and luteal function.

Applications of monoclonal anti-human inhibin α subunit in endometrial curettings

Using archival material, we studied the immunoreactivity and utility of monoclonal anti-human inhibin alpha subunit in the identification of chorionic villi (CV) and trophoblastic subpopulations in endometrial curettings (EC) from patients who had intra-uterine, ectopic, molar and, particularly, probable intra-uterine pregnancies. We also compared its expression with those of betaHCG, HPL and CAM 5.2. The four groups of EC investigated included: Group 1, 15 patients with intra-uterine pregnancies (IUP); Group 2, 15 patients with tubal pregnancies (TP); Group 3, 15 patients with hydatidiform moles (HM); and Group 4, 20 patients with purported history of intra-uterine pregnancies (PIUP). Positive and negative control cases were from Groups 1 and 3 and Group 2, respectively. The test cases were from Group 4. Immunohistochemistry was performed on each case testing for expression of inhibin alpha, betaHCG, HPL and CAM 5.2. Trophoblastic populations, which included syncytiotrophoblast (ST), cytotrophoblast (CT) and intermediate trophoblast (IT), were absent in all 15 negative control cases (Group 2). The 30 positive control cases (Groups 1 and 3) revealed the following: (a) ST, CT and IT were identified in all cases and were positive for CAM 5.2, (b) inhibin alpha, betaHCG and HPL (except one case) were reactive for all cases with ST, but not CT, and (c) IT positivity for betaHCG, HPL and inhibin alpha was 67, 80-93 and 100%, respectively. From the 20 test cases (Group 4), the findings were: (a) CT was absent in all cases, (b) scattered ST cells, which were identified only in 10 cases, were positive for all antibodies, (c) scattered IT cells were present in 17 cases and showed 100% CAM 5.2 positivity, and (d) IT positivity for betaHCG, inhibin alpha and HPL was 58.8% (10/17), 76.5% (13/17) and 82.4% (14/17), respectively. Background staining was observed in 22 of 65 cases (33.8%) stained with betaHCG and HPL; half of these cases came from Group 3. Inhibin alpha and CAM 5.2 staining did not show this problem. We suggest that inhibin alpha is a useful antibody in diagnosing IUP and HM and in documenting intra-uterine gestations in cases with PIUP because it is a sensitive marker in immunolabelling IT and ST. Combined application of inhibin alpha and CAM 5.2 might be more useful than betaHCG and HPL because the latter showed background staining in one third of the cases.

Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis.

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.

Localization and secretion of inhibins in the equine fetal ovaries.

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.

Localization of inhibin alpha-, betaA- and betaB-subunits and aromatase in ovarian follicles with granulosa theca cell tumor (GTCT) in 6 mares.

To clarify the morphological and immunohistochemical characteristics in mares with granulosa theca cell tumor (GTCT), the localization of inhibin subunits (alpha, betaA, betaB) and aromatase in the granulosa cell layers and theca layers in the ovarian follicles were determined by immunohistochemical staining. The follicles were obtained from the ovaries of 6 mares with GTCT and 4 normal mares as controls. Immunohistochemically, inhibin alpha-subunit was localized in the granulosa cells of all follicles showing different sizes in all GTCT cases and betaA- subunit was localized in two GTCT cases in all sized follicles. But inhibin betaB- subunit and aromatase were not localized in GTCT cases. On the other hand, inhibin alpha-, betaA-, and betaB-subunits and aromatase were localized in the large and medium sized follicles, but inhibin betaA- and betaB-subunits and aromatase were not stained in the small sized follicles in normal cases. These findings suggest that some mares with GTCT can secrete dimeric inhibin (inhibin A), but all GTCT cases cannot secrete inhibin B. By the results of aromatase staining it is clear that testosterone is not converted into estradiol due to the lack of aromatase in the GTCT follicles.

Immunohistochemical Characteristics of Adrenocortical Carcinoma: An Overview

Immunohistochemistry has played a major role in the practice of surgical pathology over the past three decades. These techniques have been of particular value in the diagnosis and classification of tumors of the adrenal cortex. The intermediate filament phenotype of adrenocortical carcinomas includes positivity for vimentin (80-100%), cytokeratin (40-50%) and neurofilament proteins (50%). Other markers that have proven useful for the diagnosis of these tumors include melan A (A103), alpha inhibin, synaptophysin, calretinin, D11 and the nuclear protein adrenal 4 binding protein. The judicious use of these markers permits the distinction of adrenocortical carcinomas from other tumor types and may also be of value in the identification of the functional characteristics of these neoplasms.