In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.     

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.     

Rapid changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat estrous cycle

Distributions of inhibin alpha-, beta A-, and beta B-subunits in different ovarian compartments were studied in cycling female rats by in situ hybridization with complementary RNA probes and using immunohistochemical localization with antibodies selective for each inhibin subunit. Consistent with earlier studies showing inhibin production by granulosa cells of maturing follicles, we also detected mRNAs for inhibin alpha-, beta A-, and beta B-subunits in granulosa cells of these follicles. However, based on immunohistochemistry and in situ hybridization, we found that inhibin alpha- is not only expressed in granulosa cells of mature follicles but in follicles at all stages of maturation, including primary to tertiary follicles. A number of primordial follicles also contained alpha mRNA and immunodetectable alpha-subunit. Interestingly, theca interna and interstitial gland cells contained inhibin alpha mRNA and alpha-subunit. Low levels of inhibin alpha immunoreactivity as well as specific hybridization to the complementary inhibin alpha mRNA probe were observed in newly formed luteal tissue. beta-Subunits, on the other hand, were detected exclusively in granulosa cells of healthy tertiary follicles. The changes in expression of inhibin alpha-, beta A-, and beta B-subunits were more pronounced during the follicular phase of the cycle: inhibin alpha reached its highest level in granulosa cells, theca interna, and interstitial gland cells a few hours after the LH/FSH surge, while at the same time the beta-subunits decreased dramatically in granulosa cells of mature follicles. Immediately before ovulation (estrus 0200 h), the alpha-subunit sharply declined in preovulatory follicles and was present mainly in granulosa cells from nonovulatory follicles at various stages of maturation. At that time, the beta A- and beta B-subunits could not be detected in preovulatory follicles but were localized mainly in small tertiary follicles (less than 300 microns). Unlike for the alpha- and beta B-subunits, beta A mRNA and immunoreactivity was present in large tertiary follicles (approximately 600 microns) immediately before ovulation. The present findings support the hypothesis that a decrease in inhibin production could be responsible for the secondary FSH surge observed early on estrus. This could be initiated by a change in the ratios of activin-inhibin production by decreasing first, the levels of beta-subunits, second, the levels of alpha-subunit, and third, by a resurgence of activin A produced mainly by granulosa cells from large tertiary follicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.     

Gene expression in abnormal ovarian structures of ewes homozygous for the inverdale prolificacy gene

Animals heterozygous (I+) for the Inverdale prolificacy gene (FecX(I)) have an increased ovulation rate whereas those homozygous (II) for FecX(I) are infertile with "streak" ovaries and follicular development arrested at the primary (type 2 follicle) stage. The streak ovaries also contain small oocyte-free nodules with granulosa-like cells and often tumor-like structures. It has been hypothesized that these abnormal structures are of granulosa cell origin, and the aim of this study was to determine whether genes normally expressed in granulosa cells are also expressed in the nodules and tumor-like structures. The mRNAs encoding c-kit and its ligand stem cell factor (SCF), FSH receptor (FSH-R), follistatin, alpha-inhibin subunit, and the beta(A)- and beta(B)-activin/inhibin subunits were localized in ovaries of ewes with 0 (++), 1 (I+), or 2 (II) copies of the FecX(I) gene (n = 4-9 animals per genotype per gene) using in situ hybridization. Ontogeny of expression of all mRNAs examined was similar between ++ and I+ ewes. Expression of c-kit mRNA was observed in the oocyte of all follicular types present in ++, I+, and II ewes. Moreover, granulosa cells of type 2 (II) and type 2 and larger follicles (++, I+) expressed SCF mRNA. The mRNAs encoding FSH-R, follistatin, alpha-inhibin subunit, and beta(B)-activin/inhibin subunit were identified in type 3 and larger follicles of ++ and I+ ewes but not in follicles of II ewes that were only at the type 1, 1a, or 2 stages of development. However, the cells within the oocyte-free nodules of II ewes expressed all of these genes. The mRNAs encoding c-kit and beta(A)-activin/inhibin subunit were not observed in granulosa cells until antrum formation (type 5 follicles) or in the nodules of II ewes. Tumors from 4 ewes were obtained and classified as cystic, semisolid, or solid structures containing granulosa-like cells or as solid structures containing predominately fibroblast- and luteal-like cells. Often, two tumors were present on the same ovary. Tumors containing granulosa-like cells (n = 3-4 per gene) expressed the mRNAs encoding alpha-inhibin subunit, beta(A)-, and beta(B)-activin/inhibin subunits, follistatin, and the FSH-R but did not contain detectable amounts of mRNA for c-kit or SCF. Tumors composed predominately of fibroblast- and luteal-like cells expressed very low levels of SCF mRNA; of the other mRNAs examined, none were detected. Also, none of the genes examined were found to be expressed by the surface epithelium, theca externa, fibroblast, or vascular cells within the ovary of animals of any genotype. These findings are consistent with the hypothesis that the somatic cells in oocyte-free nodules and tumor-like tissue in II ewes originate from the granulosa cells of the small follicles.     

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.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Periovulatory changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in hormonally induced immature female rats

Immature female rats were treated with PMSG and human CG to induce ovulation. Sequential treatment with these hormones allowed us to investigate variations in the production of inhibin subunits shortly before ovulation and during the induced luteal phase. Using this model, we found that expression patterns for the alpha-, beta A-, and beta B-subunits were similar to those observed in mature cycling animals: administration of PMSG (to mimic the gonadotropin surge) led to a sharp increase in the expression of all three subunits in large preovulatory follicles whereas injection with human CG (to induce ovulation) caused a decrease in the levels of the respective mRNAs. In contrast to mature females, shortly before ovulation, levels of inhibin alpha-subunit mRNA were low in small antral follicles (approximately 350 microns). In addition, at that time, inhibin beta A- and beta B-subunits mRNAs were present in several large follicles (greater than 500 microns). More than 2 days after ovulation, inhibin beta A- and beta B-subunit mRNAs could not be detected in small antral size follicles (approximately 350 microns) of hormonally induced females. On the other hand, hybridization signals for the inhibin alpha-subunit were observed in some small antral and preantral size follicles, while signals were very low or undetectable in a large number of atretic follicles. Using this synchronized ovulation model, hybridization patterns for inhibin beta A-subunit mRNA was observed in interstitial cells, 8-10 h after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activin B: precursor sequences, genomic structure and in vitro activities

We report here the complete amino acid sequence of the human inhibin beta B-subunit as deduced from the sequence of cDNA and genomic clones. The primary translation product of the beta B mRNA predicts a protein of 407 amino acids, containing a prepro region of 292 amino acids separated by basic amino acids from the mature C-terminal 115 amino acids. Mammalian tissue culture cells transfected with a beta B-subunit expression plasmid secreted an activin B homodimer of approximately 22K mol wt. Coexpression of the beta A- and beta B-subunit mRNAs resulted in the secretion of the three forms of activin, A, AB, and B. Purified activin B was shown to elicit FSH release in an in vitro pituitary assay and trigger the accumulation of hemoglobin in K562 cells. The potency of activin B in both of these assays (ED50 approximately 2 ng/ml) was indistinguishable from that observed for activin A.     

Cellular localization of inhibin mRNA in the bovine ovary by in-situ hybridization

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.     

Up-regulation of alpha-inhibin expression in the fetal ovary of estrogen-suppressed baboons is associated with impaired fetal ovarian folliculogenesis

We recently demonstrated that the number of primordial follicles was significantly reduced in the ovaries of near-term baboon fetuses deprived of estrogen in utero and restored to normal in animals administered estradiol. Although the baboon fetal ovary expressed estrogen receptors alpha and beta, the mechanism(s) of estrogen action remains to be determined. It is well established that inhibin and activins function as autocrine/paracrine factors that impact adult ovarian function. However, our understanding of the expression of these factors in the primate fetal ovary is incomplete. Therefore, we determined the expression of alpha-inhibin, activin beta(A), activin beta(B), and activin receptors in fetal ovaries obtained at mid and late gestation from untreated baboons and at late gestation from animals in which fetal estrogen levels were reduced by >95% by maternal administration of the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 and estradiol benzoate. Immunocytochemical expression of alpha-inhibin was minimal to nondetectable in fetal ovaries from untreated baboons. In contrast, in baboons depleted of estrogen, alpha-inhibin was abundantly expressed in pregranulosa cells of interfollicular nests and granulosa cells of primordial follicles. Thus, the number (mean +/- SEM) per 0.08 mm2 of fetal ovarian cells expressing alpha-inhibin, determined by image analysis, was similar at mid and late gestation and increased approximately 8-fold (P < 0.01) near term in baboons treated with CGS 20267 and was restored (P < 0.01) to normal in baboons treated with CGS 20267 plus estradiol. Activin beta(A) was detected in oocytes and pregranulosa cells at midgestation and in oocytes and granulosa cells of primordial follicles at late gestation. Activin beta(B) was also expressed in pregranulosa cells and granulosa cells at mid and late gestation, respectively, but was not detected in oocytes. Neither the pattern nor the apparent level of expression of activin beta(A) or beta(B) were altered in fetal ovaries of baboons treated with CGS 20267 or CGS 20267 and estrogen. Activin receptors IA, IB, IIA, and IIB were detected by Western blot analysis in fetal ovaries at mid and late gestation, and expression was not altered by treatment with CGS 20267 or CGS 20267 and estrogen. Activin receptors IB and IIA were localized to oocytes and pregranulosa cells at midgestation and to granulosa cells and oocytes of primordial follicles at late gestation. Thus, the decrease in the number of follicles in the primate fetal ovary of baboons deprived of estrogen in utero was associated with increased expression of alpha-inhibin. Therefore, we propose that estrogen regulates fetal ovarian follicular development by controlling alpha-inhibin expression and, thus, the intraovarian inhibin:activin ratio.     

Alterations in peripheral concentrations of inhibin A in cattle studied using a time-resolved immunofluorometric assay: relationship with estradiol and follicle-stimulating hormone in various reproductive conditions

The aims of this study were to develop a sensitive and specific assay for bovine inhibin A using europium and to investigate the endocrine role of inhibin A in various reproductive conditions by characterizing the relationship between profiles of inhibin A, FSH, and estradiol and follicle growth during the postpartum period, during the intact estrous cycle, and in cows with follicular cysts. The time-resolved immunofluorometric assay (Tr-IFMA) for bovine inhibin A, using purified polyclonal antibodies to alpha and beta(A) subunits, was specific for bovine inhibin A and did not cross-react with bovine activin A, activin AB, activin B, pro-alphaC or human recombinant inhibin B. The detection limit of the IFMA was 3.3 pg/ml expressed in terms of bovine 32-kDa inhibin A. Dose-response curves of plasma samples obtained from intact and FSH-stimulated cows and cystic cows were parallel to the standard without any preassay processing of samples. Plasma inhibin A levels increased (P < 0.01) concomitant with emergence of nonovulatory or ovulatory follicular waves during the postpartum period. In cystic cows, plasma inhibin A was sustained at high levels for a longer period, associated with growth of persistent dominant follicles. The highest levels of inhibin A were noted during the growth phase of normal and persistent dominant follicles; however, inhibin A levels declined (P < 0.01) as these dominant follicles ceased to grow or ovulated. An inverse relationship between patterns of plasma inhibin A and FSH existed during each follicular wave in the three physiologic conditions. Increases in plasma inhibin A levels were associated with increases in plasma estradiol levels during most follicular waves; however, there was no increase in plasma estradiol level and no relationship between patterns of estradiol and FSH during follicular waves observed during the early postpartum period or midluteal phase of the estrous cycle. In conclusion, the Tr-IFMA does not require pretreatment of samples and can be used for precise measurement of bovine inhibin A without interference with free inhibin alpha subunits. Inhibin A, produced primarily during growth of the dominant follicle, functions as a negative feedback regulator for FSH secretion throughout the postpartum period and the estrous cycle, whereas estradiol appears to have a minor role in regulation of FSH compared with inhibin A, especially during the early postpartum period and midluteal phase of the estrous cycle. The results also indicate that a persistent dominant follicle sustains inhibin A production for a longer period than the dominant follicle emerging in the estrous cycle and establishes long-term dominance by suppressing emergence of a new follicular wave.     

Local regulation of primate granulosa cell aromatase activity

Granulosa cells produce inhibin and activin, proteins implicated in the local regulation of preovulatory follicular development. To assess interactions among FSH, LH, inhibin and activin on primate granulosa cell aromatase activity, we studied primary granulosa cell cultures from the ovaries of the common marmoset (Callithrix jacchus), a monkey with an ovarian cycle similar in length to the human cycle. The distinctive action of activin was augmentation of gonadotropin-responsive aromatase activity throughout antral follicular development. FSH-stimulated aromatase activity in granulosa cells from immature follicles was augmented many fold by picomolar amounts of activin. In cell cultures from preovulatory follicles, the presence of activin stimulated basal aromatase activity in the absence of gonadotropin, as well as augmenting the action of LH. Thus, locally produced activin has the potential to modulate aromatase activity in developing ovarian follicles. By contrast, inhibin or inhibin -subunit purified from bovine follicular fluid had minimal effects on aromatase activity. The only significant effect was slight suppression of FSH-inducible aromatase activity in granulosa cells from immature follicles at an inhibin concentration of 100 ng/ml. The finding that inhibin has a negligible effect on aromatase activity in granulosa cells from mature follicles suggests that it is unlikely to exert a physiologically significant influence on aromatase activity in vivo. However, evidence from other studies suggests that inhibin might affect aromatization indirectly through acting locally to modulate thecal androgen (aromatase substrate) production. Therefore, both inhibin and activin have the potential to contribute at different levels to paracrine and autocrine regulation of follicular oestrogen synthesis.     

Differential expression of inhibin subunits and follistatin, but not of activin receptor type II, during early murine embryonic development.

Activins are known to be potentially important regulators of early developmental processes in amphibians, birds, and mammalians. In this study we report the expression of the inhibin subunits, including those that make up activin, the activin-binding protein follistatin, and activin receptor type II in several in vitro systems that model early murine embryonic development, namely embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and their differentiated derivatives. In addition, we examine the expression pattern of these factors in different stages of the mouse embryo itself. Expression of inhibin alpha and beta A subunits is restricted to certain differentiated cell types, while beta B subunits are expressed in both differentiated and undifferentiated cells. Our results further indicate a change in the expression pattern of inhibin subunits during early development from beta B at the blastocyst stage largely to beta A in postgastrulation embryos. This is similar to the expression pattern at equivalent stages of Xenopus and chick development. Expression of the activin-binding protein follistatin is altered by the induction of differentiation of P19 EC and ES cells by several factors, including retinoic acid. In contrast to the inhibin subunits and follistatin, activin receptor levels are not influenced by differentiation in these cell types. The results of this study demonstrate that the inhibin subunits and follistatin, but not the activin receptor type II, are differentially expressed during early murine development and suggest that the different forms of activin/inhibin are involved in the regulation of different developmental processes.     

Localization of inhibins and activins in normal endocrine cells and endocrine tumors of the gut and pancreas: an immunohistochemical and in situ hybridization study.

Activins and inhibins, which belong to the TGF beta family, are composed of different combinations of alpha-, betaA-, and betaB-subunits, resulting in inhibin A (alphabetaA), inhibin B (alphabetaB), activin A (betaAbetaA), activin B (betaBbetaB), and activin AB (betaAbetaB). They regulate several cell functions, acting as paracrine/autocrine factors. Their actions, which depend on binding to specific receptors, are also modulated by follistatin. Gastroenteropancreatic (GEP) endocrine cells and endocrine tumors (ETs) produce several growth factors, but it is not well known whether they express follistatin and the various inhibin/activin subunits. We studied their expression in 65 GEP ETs using immunohistochemistry (IHC) and in situ hybridization (ISH). The alpha-subunit and follistatin were not identified in normal GEP endocrine cells and were poorly expressed in ETs. A betaA-subunit immunoreactivity (IR) was detected in A-, G-, EC-, and GIP-cells, while betaB-chain IR was present only in D-cells. The mRNAs encoding for these molecules were poorly expressed in normal tissues. BetaA- and betaB-subunits were identified in several ETs by both IHC and ISH: betaA-subunit mainly in G-cell and A-cell ETs, and betaB-subunit in D-cell, A-cell, and EC-cell ETs. Our results demonstrate a differential expression of activin/inhibin subunits among different types of GEP endocrine cells and related tumors, suggesting a role in modulation of biological functions of these normal and neoplastic endocrine cells.     

N-linked oligosaccharides direct the differential assembly and secretion of inhibin alpha- and betaA-subunit dimers

The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.