Beta A-subunit mRNA Research Articles

Different origins of two corpora lutea recovered from a pregnant African elephant (Loxodonta africana).

Elephant ovaries contain multiple corpora lutea (CLs) throughout pregnancy. Two CLs (P-1 and P-2) collected from a pregnant African elephant were used to investigate their origin and physiological state in this study. The mRNA expressions of prolactin receptor, CYP11A and inhibin betaB subunit were higher in P-2 than in P-1, while LHCGR and inhibin betaA subunit mRNA were higher in P-1 than in P-2. Protein expression of cleaved caspase-3 was detected in P-1 but not in P-2. These results suggest different origins for the two CLs in this one pregnant elephant, and we also demonstrated the production of bioactive prolactin by the elephant placenta.

Altered sex hormone concentrations and gonadal mRNA expression levels of activin signaling factors in hatchling alligators from a contaminated Florida lake

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly.

Activin βA subunit, follistatin and follistatin-like 3 are expressed in the endometrium of ovariectomized rats and regulated by estrogen replacement

Activin A is a growth factor expressed in the endometrium, where it modulates tissue remodeling and enhances decidualization. The effects of activin A are counteracted by two binding proteins, namely follistatin and follistatin-like 3 (FSTL3). We have evaluated the effects of estrogen and progestin on the endometrial expression of activin betaA subunit, follistatin and FSTL3 in ovariectomized rats. Adult female Wistar rats (n = 21) were ovariectomized and received one week later a single dose of estradiol benzoate (1.5 mg/kg body weight, i.m. injection), either alone (n = 7) or associated with depot medroxyprogesterone acetate (3 mg/kg body weight, i.m. injection, n = 7), or oil vehicle (control group, n = 7). One week later, activin betaA subunit mRNA levels had increased significantly in the uteri of rats treated with estradiol alone (7.4 fold increase over controls, P < 0.05) and to the same extent in rats receiving estradiol plus medroxyprogesterone (6.1 fold increase over controls, P < 0.05). This was accompanied by increase of betaA subunit immunostaining in estradiol and estroprogestin treated rats, which was noted only in the surface endometrial epithelium. Follistatin mRNA expression, conversely, showed a significant decrease in the groups treated with estrogen alone and estrogen plus progestin (P < 0.05), and follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprogestin-treated rats compared to controls. FSTL3 expression was similar in the 3 groups. In conclusion, the expression of activin betaA subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin.

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

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

Secretion of Inhibin and Testicular Expression of Inhibin Subunits in Male Duck Embryos and Newly Hatched Ducks

Concentrations of immunoreactive (ir-) inhibin in circulation, amniotic fluid, and testes of embryos and newly hatched ducks were determined from d 21 of incubation to d 1 of age. Plasma concentrations of FSH and LH were also determined by chicken radioimmunoassay (RIA) systems. In addition, gene expression and cellular source of inhibin were investigated by in situ hybridization and immunohistochemistry. The results showed that plasma ir-inhibin gradually declined from d 21 to d 24, followed by an increase on d 25 and remained high until d 1 after hatching. FSH in plasma was high on d 21 followed by a sharp decline toward d 25 after which FSH levels stabilized. A reverse relationship was observed between inhibin and FSH during the late stage of incubation. Embryonic testes contained high ir-inhibin levels. Testicular ir-inhibin levels were relatively high at early time points with a peak on d 23, and significantly decreased from d 23 to d 24 and stabilized thereafter. Amniotic fluid concentrations of ir-inhibin were relatively low and remained constant between d 21 and d 25. In situ hybridization demonstrated that the expression of inhibin alpha- and betaA-subunit mRNA was coexisted in the cells in the seminiferous tubules of testes on d 25. The immunoreactivity of inhibin betaA- and betaB-subunits was colocalized in the cells in the seminiferous tubules of testes on d 25. The results of dimeric inhibins determined by the ELISA method showed that inhibin B can be measured in embryonic testicular homogenate and pooled embryonic plasma. Although inhibin A was detected in testicular homogenate, it was under the detection limit in pooled embryonic plasma. In conclusion, these results indicate that cells in the seminiferous tubules of embryonic testes in ducks may secrete dimeric (bioactive) inhibins to circulation and that the FSH-inhibin feedback loop may become operational during the late stage of the incubation.

Serum and tissue expression of activin a in postmenopausal women with breast cancer.

Activins are growth factors involved in the control of cell proliferation and differentiation. Human breast tissues express immunoreactive activin subunits, and activin A is able to inhibit the replication of mammary cells in vitro. The aim of the present study was to evaluate 1) whether breast cancer expresses activin betaA subunit mRNA, 2) whether serum activin A levels are altered in postmenopausal women with breast cancer, and 3) how circulating activin A levels change after tumor removal. Four groups of women (n = 158) were enrolled for the present prospective study: two groups were composed of postmenopausal women with breast cancer (n = 74) or benign lesions (n = 15); the third was a control group composed of healthy postmenopausal women (n = 62); and the fourth group included healthy fertile women (n = 7) undergoing plastic surgery with removal of non-neoplastic mammary tissue. RT-PCR showed that betaA subunit mRNA was expressed in breast carcinoma, fibroadenoma, and normal mammary tissue, and the level of expression was higher in carcinoma than in normal tissue (P < 0.05). Dimeric activin A was detectable in homogenates of breast cancer tissue at concentrations twice as high as in non-neoplastic tissue (P < 0.01). In women with breast cancer, median serum activin A levels were significantly higher than in controls (P < 0.001). The high serum activin A levels in patients with breast cancer were not correlated with the presence of lymph node metastasis, tumor grade, or tumor diameter. After tumor excision, a significant decrease of activin A in the first and second postoperative days was observed (P < 0.01; Friedman's ANOVA). Conversely, activin A levels remained unchanged after plastic surgery in healthy women. The present results suggest that activin A is expressed and secreted in postmenopausal women with breast cancer. The pathophysiological and possible clinical implications of this finding remain to be investigated.

Inhibins in the male Göttingen miniature pig: Leydig cells are the predominant source of inhibin B.

The expression of inhibin subunits in the testes of the Göttingen miniature pig was examined by in situ hybridization and immunohistochemistry. In addition, the major forms were determined by enzyme-linked immunosorbent assay (ELISA). Strong positive immunostaining for the inhibin alpha subunit was observed in Sertoli and late-stage germ cells, but it was weak in Leydig cells. However, Leydig cells showed strong positive staining for the betaA subunit, but Sertoli cells and spermatogonia showed a weak reaction. Strong positive immunostaining for the betaB subunit was observed in Leydig cells but spermatogonia showed weak staining for it. In contrast to the staining specificity of inhibin alpha and betaA subunits, the betaB subunit did not exhibit positive staining in Sertoli cells. In situ hybridization revealed that although the a subunit mRNA signal was highly expressed in all cell types, the reaction appeared to be stronger in Sertoli cells and spermatogonia than in Leydig cells. betaA subunit mRNA expression was somewhat identical to that of the alpha subunit, however, germ cells showed a weak stain for it. A strong, positive mRNA signal for the betaB subunit was confined to Leydig cells and late-stage germ cells. ELISA results showed that concentrations of inhibin B and inhibin pro-alphaC were high in the circulation and testes. In contrast, inhibin A levels in both plasma and testes were undetectable. The present results strongly suggest that inhibin B is the major form of circulating inhibin and that Leydig cells are the predominant source of this dimeric inhibin in male Göttingen miniature pigs. Furthermore, the germ cells also appear to be an important source of circulating inhibins.

Human GnRH-secreting cultured neurons express activin betaA subunit mRNA and secrete dimeric activin A.

To evaluate the expression of activin betaA-subunit mRNA and the secretion of activin A in/from cultured GnRH-secreting neuronal cells cloned from human olfactory epithelium (FNC-B4), which showed biochemical and antigenic properties of GnRH-secreting neurons. FNC-B4 cells were cultured in basal and conditioned media. Reverse transcription-polymerase chain reaction (RTR-PCR) evaluated the expression of activin betaA-subunit mRNA. By using a specific ELISA, dimeric activin A concentrations were measured in culture media, in the absence or presence of carvone or forskolin and with different doses of progesterone, GnRH, and estradiol. RT-PCR experiments performed on total RNA isolated from FNC-B4 cells, using specific primers for the activin betaA gene, showed a 787bp DNA band corresponding to the betaA gene. FNC-B4 cells secreted activin A, and the highest accumulation in conditioned medium was achieved after 3h culture: the addition of forskolin, but not of carvone, was able to stimulate the release of activin A from cultured neuronal cells (P<0.01). When progesterone or GnRH was added, a significant accumulation of activin A was observed (P<0.01), while estradiol administration did not significantly affect activin A secretion. To date, this is the only study, in an in vitro human model reporting, that GnRH-secreting neuronal cells expressed activin betaA-subunit mRNA, and released dimeric activin A in culture medium. The expression and secretion of activin suggests that in these cells activin A might exert its action by autocrine/paracrine mechanisms.

Inhibitory effects of activin-a on osteoblast differentiation during cultures of fetal rat calvarial cells

Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts. However, the role of activin-A on osteoblasts is not clearly understood. We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones. Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay. This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture. In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis. The peak of osteocalcin mRNA was delayed. Gene expression for TGF-beta was not influenced by exogenous activin-A. The betaA subunit (activin-A) mRNA was detected during the early phase of this culture. These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.

Inhibitory effects of activin-A on osteoblast differentiation during cultures of fetal rat calvarial cells.

Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts. However, the role of activin-A on osteoblasts is not clearly understood. We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones. Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay. This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture. In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis. The peak of osteocalcin mRNA was delayed. Gene expression for TGF-beta was not influenced by exogenous activin-A. The betaA subunit (activin-A) mRNA was detected during the early phase of this culture. These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.

An essential role for ovarian inhibin in pineal gland-mediated anestrus in Syrian hamsters.

When transferred from long photoperiod (LP) to short photoperiod (SP), female Syrian hamsters exhibit depressions of follicle stimulating hormone (FSH) and follicular development, cessation of ovulation, and marked ovarian interstitial tissue hyperplasia. Pinealectomy prevents these effects of SP. The object of this study was to determine the role of inhibin in the regulation of FSH during SP-induced anestrus. Adult LSH/SsLak hamsters maintained in LP (LD 14:10 hr) were transferred to SP (LD 8:16 hr) on the day of estrus, and groups of animals killed at either 16.00 hr on proestrus or 08.00 hr on estrus during each of five consecutive 4-day estrus cycles after transfer. Groups of females that became anestrus in SP were killed either at 08.00 or 16.00 hr, 12 days after the last observed estrus discharge. Compared to LP controls, serum FSH levels on estrus increased significantly (P<0.01) during the first two cycles in SP before declining to concentrations that were significantly lower than control values (P<0.01). Serum inhibin levels increased significantly by the third, fourth, and five days of estrus in SP. Regression analysis revealed a significant inverse correlation between serum inhibin and FSH levels on estrus during SP exposure (P=0.021) but not on proestrus. Relative levels of inhibin alpha- and betaA-subunit mRNAs were lower in ovaries from SP proestrus and anestrus females killed at 16.00 hr as compared to those from proestrus LP controls; they were elevated in ovaries from SP estrus and anestrus females killed at 08.00 hr compared to those from estrus LP controls. The absence of antral follicles on estrus in the last cycles of SP and anestrus suggests that the increase in circulating inhibin and inhibin mRNAs may be derived from hyperplastic interstitium. These observations suggest that inhibin may play an essential role in suppressing FSH secretion during pineal gland-mediated anestrus in Syrian hamsters.

Second trimester levels of maternal serum total activin A and placental inhibin/activin alpha and betaA subunit messenger ribonucleic acids in Down syndrome pregnancy.

Previous data have shown that inhibin A (alpha/betaA) is increased about twofold in maternal serum samples from Down syndrome pregnancy. Our objectives were to determine whether activin A (betaA/betaA) was similarly increased in maternal serum from pregnancies affected with fetal Down syndrome, and to investigate whether increased expression of each inhibin/activin subunit occurred in placental tissue from cases of fetal Down syndrome. Maternal serum total activin A levels were measured in 20 cases of fetal Down syndrome and 100 unaffected pregnancy samples. In addition, analysis of inhibin/activin alpha and betaA subunit mRNA levels was performed in placental tissue extracts from six cases of fetal Down syndrome and six tissues with a normal karyotype. The median total activin A level in the Down syndrome cases was 0.82 MoM (multiples of the median); values did not differ significantly (P = 0.36, Mann-Whitney U analysis) from those in unaffected pregnancies. The inhibin alpha subunit/GAPDH mRNA ratio, but not that of betaA subunit/GAPDH mRNA, was significantly greater (P < 0.01, ANOVA) in placental tissue from Down syndrome than in control placental tissue. Unlike inhibin A, activin A is not significantly increased in Down syndrome relative to unaffected pregnancy. Furthermore, increased amounts of maternal serum inhibin A in Down syndrome pregnancy probably result from increased placental expression of inhibin alpha, but not betaA, subunit.

Temporal changes in inhibin subunit mRNAs during atresia of preovulatory follicles in the rat.

This study aimed to investigate the time course of disappearance of the mRNAs of the various subunits of inhibin in follicles which become atretic. An animal model was used in which atresia of preovulatory follicles could be studied in a chronological order. Injection of gonadotrophin-releasing hormone (GnRH) antagonist (20 microg) at the morning of pro-oestrus (P) blocked ovulation and the 10-12 preovulatory follicles became gradually atretic. A second injection was given the next day to prevent delayed ovulation. The rate of atresia could be delayed by simultaneous administration of a subovulatory dose of human chorionic gonadotrophin (hCG) (0.5 IU) and could be advanced by administration of a fivefold larger amount of GnRH antagonist. Functional activity of follicles becoming atretic was studied by measuring oestradiol production after incubation of individual follicles for 4 h. Follicles isolated 24 h after the first injection of GnRH antagonist (P+24) already secreted significantly less oestradiol in vitro than follicles isolated at pro-oestrus, although they were morphologically not different from pro-oestrous follicles. Follicles isolated at P+24 from hCG-treated rats secreted more oestradiol compared with follicles from rats not treated with hCG. In contrast, follicles isolated at P+24 from rats that were given a fivefold larger amount of GnRH antagonist secreted less oestradiol. Once this model was validated, temporal changes in inhibin subunit mRNAs in follicles undergoing atresia were measured by in situ hybridization and RNase protection assay. In situ hybridization showed abundant alpha- and betaA-subunit mRNA in the whole granulosa layer of preovulatory follicles at P and P+24, while betaB-subunit mRNA was restricted to the antral layer and cumulus. At P+48 the amount of alpha- and betaA-subunit mRNA had declined and was restricted to the cumulus, whereas betaB-subunit mRNA was absent. In the atretic follicles present at P+72 and P+96, mRNAs of all three inhibin subunits were absent. Administration of 0.5 IU hCG delayed the decline in the amount of alpha, betaA and betaB mRNA in preovulatory follicles at P+48. RNase protection assay of inhibin subunits in isolated follicles revealed no changes between P and P+24. However, at P+48, the mRNAs of alpha- and betaA-subunits were decreased. Expression of the mRNA of betaB-subunit declined gradually from P to P+48. The present study demonstrates that in follicles which are becoming atretic, mRNAs of alpha- and betaA-subunits decline simultaneously with the appearance of pycnotic cells in the granulosa layer, while betaB-subunit mRNA declines earlier, simultaneously with the decrease in the ability to secrete oestradiol in vitro.

Equine inhibin/activin beta A-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy.

The expression of both inhibin alpha- and inhibin/activin beta A-subunit mRNA was examined in equine uteroplacental tissues collected during pregnancy (days 90 to 300). Northern blot analysis revealed that 5 transcripts (7.0, 4.1, 3.4, 2.6, 1.5 kb) of beta A-subunit were present, and the most abundantly expressed transcript was the 1.5 kb one. Relatively high levels of the 1.5 kb transcript were seen in the second trimester of pregnancy compared to what was found in the third trimester. To identify the tissue localization of beta A-subunit mRNA, in situ hybridization was performed, and the positive signal was observed exclusively in the endometrial glands, but not in the fetal placental tissue (trophoblast) at days 150, 210, and 300 of pregnancy. On the other hand, inhibin alpha-subunit transcript could not be detected at any stage of pregnancy examined either by Northern blot analysis or in situ hybridization. Although the factor(s) regulating the gene expression of beta A-subunit in this equine tissue is currently unknown, these results suggest that activin, but not inhibin, is predominantly produced in the endometrial glands of the pregnant mare, and thus produced activin may play a paracrine or endocrine role during pregnancy in this species.

Tissue-specific expression of inhibin/activin subunit and follistatin mRNAs in mid- to late-gestational age human fetal testis and epididymis.

Inhibin/activin subunit (alpha, beta A, and beta B) immunoreactive protein localization patterns and cell type specific inhibin alpha-subunit mRNA expression have been examined in early- to midgestational age human fetal testes. The scarcity of available third trimester human fetal tissue has, however prevented a complete examination throughout the gestational period and the cell specific expression of follistatin and beta A- and beta B-subunit mRNAs are currently unknown at any gestational age. In the present study, this gap is filled and report mRNA expression patterns of inhibin/activin subunits in mid- and late-gestational age (21-33 wk) human fetal testes and testicular duct system. We also report the first examination of follistatin mRNA signals in the human fetal gonad is also resent in both tubular and interstitial cells, and beta B-subunit mRNA is expressed in seminiferous tubules, in mid- and late-gestational age human fetal testes. Inhibin/activin beta A-subunit mRNA was detected in the interstitial cells of remarkably well preserved mid (21 and 22 wk) and late (29 wk) gestational age testes, and is the only activin-system factor mRNA also expressed in tissue of the duct system of the testis (smooth muscle cells of the epididymis). Follistatin mRNA signal was equal to background levels in testicular and duct tissues at all ages examined. These cell specific expression patterns suggest prominent and possibly differential roles for the inhibins and activins, unopposed by gonadal follistatin, in the human fetal male reproductive system.

Characterization of inhibin/activin subunit, follistatin, and activin type II receptors in human ovarian cancer cell lines: a potential role in autocrine growth regulation.

Although ovarian cancer is the most common gynecological malignancy with a relatively poor 5-yr survival record, the mechanism(s) by which these tumors arise is not well understood. A role for inhibins and activins in regulating this transformation is suggested by the detection of circulating alpha or dimeric inhibin in some patients with ovarian cancer and by the alpha inhibin knockout mouse, in which development of gonadal tumors in 100% of homozygotes is associated with greatly elevated activin levels. To develop diagnostic tools with greater specificity for ovarian cancers, the present study was targeted at characterizing the biosynthetic capacity of the epithelial ovarian cancer cell lines from the American Type Culture Collection with respect to inhibin, activin, the related activin-binding protein follistatin (FS), and activin receptor type II. In addition, the functional capacity of this system was investigated by examining the ability of activin and FS to modulate cellular proliferation. All six cell lines contained abundant messenger RNA (mRNA) for activin receptor type II, but no inhibin alpha-subunit mRNA was detected in any cell line. Two cell lines contained mRNA for activin beta B-subunit (CaOV4 and SKOV3), one cell line contained beta A-subunit mRNA (SW626), and one cell line contained both (ES2); the latter also contained FS mRNA. FS mRNA was detected in another cell line (PA-1) that contained no detectable activin beta-subunit mRNA. Finally, one cell line (CaOV3) contained neither beta-subunit nor FS mRNA. Protein secretion was also examined. Consistent with the mRNA studies, the two cell lines containing FS mRNA secreted FS (PA-1 and ES2 cells), whereas three of the remaining lines secreted activin (A or B). In the cell line containing neither FS nor beta-subunit mRNA, no FS or activin could be detected. Finally, none of the cell lines secreted detectable immunoreactive inhibin. The effects of exogenous activin and FS on cellular proliferation were examined in these cell lines. No response was detected in the two cell lines that secreted FS (PA-1 and ES2). For the four cell lines not synthesizing FS, treatment with activin (1-100 ng/ml) resulted in an increase, whereas FS treatment (1-100 ng/ml) resulted in a decrease in cellular proliferation, as determined by [3H]thymidine incorporation. The response to activin correlated negatively with endogenous activin production, suggesting that autocrine activin production may be involved with cell proliferation. The differential expression and production of inhibin/activin subunits, activin receptors, and follistatin as well as the range of responses to exogenous activin among six ovarian epithelial cancer cell lines suggest that this family of hormones may be important in regulating cell proliferation in the ovary. Whether primary tumors have the same profile and the degree to which these results can be generalized to additional forms of ovarian cancer remain to be determined.

Molecular cloning of inhibin/activin beta A-subunit complementary deoxyribonucleic acid and expression of inhibin/activin alpha- and beta A-subunits in the domestic hen.

A chicken inhibin/activin beta A-subunit cDNA clone has been isolated and used to detect the beta A-subunit mRNA in the hen. A cDNA library of the hen granulosa layer was screened with a rat inhibin beta A-subunit cDNA probe, and a cDNA of 1540 bp was cloned and sequenced. The clone contains the entire open reading frame of 1272 nucleotides encoding 424 amino acids. It shows 78% nucleotide identity (85% in deduced amino acids) in the full-length coding region and 85% nucleotide identity (98% in deduced amino acids) in the 116-amino acid C-terminal mature region to the rat beta A-subunit cDNA clone. The predicted positions of one N-linked glycosylation site and all cysteine residues are fully conserved as well. Northern blot analysis showed that the major 8.4-kb inhibin/activin beta A-mRNA band was primarily expressed in the granulosa layer of the preovulatory follicles. It was also detected in much less abundance in the theca layer, thyroid gland, heart, muscle, intestine, adrenal gland, liver, lung, spinal cord, and spleen. When a chicken inhibin alpha-subunit cDNA probe was used, the 1.7-kb major band of the inhibin alpha-subunit mRNA was detected most strongly in the granulosa layer and less abundantly in the postovulatory follicle, theca layer, testis, heart, and adrenal gland. Moreover, a distinct 1.3-kb alpha-mRNA was detected in the muscle and lung. The present cloning study extends our understanding of the inhibin/activin beta A-subunit cDNA from studied species to the chicken and shows that the beta A-subunit is highly conserved during evolution. Our data indicate that the mRNAs for inhibin/activin alpha- and beta A-subunits are widely present in a variety of chicken tissues but that the granulosa layer of large preovulatory follicles is the primary site of expression. The present study also suggests that both inhibin and activin may serve as local factors to regulate cell function in chicken tissues.

Prostaglandin E2 induces inhibin alpha- and beta A-subunit mRNA and secretion of dimeric inhibin A in cultured human granulosa-luteal cells.

Prostaglandins (PG) E2 and F2 alpha are produced by the human corpus luteum in significant amounts and they are known to exert local luteotrophic and luteolytic effects respectively. The present study determined how PGE2 and PGF2 alpha are involved in the regulation of inhibin A, a granulosa cell-derived glycoprotein hormone that has an endocrine inhibitory effect on hypophyseal gonadotrophin production and a local stimulatory action on thecal cell androgen secretion. The effects of PGE2 and PGF2 alpha on inhibin alpha- and beta A-subunit mRNAs levels and on the secretion of dimeric inhibin A were studied in cultures of human granulosa-luteal cells obtained from patients undergoing an in-vitro fertilization programme. We found that PGE2 induced mRNA of inhibin alpha- and beta A-subunits in a time- and concentration dependent manner, whereas PGF2 alpha had no clear effect. In contrast to the rapidly and transiently induced beta A-subunit mRNA, alpha-subunit mRNA accumulation was stimulated with slower kinetics by PGE2 in a protein synthesis-dependent manner; this was also observed for the mRNA of the cytochrome P450scc enzyme. In the medium of cells cultured for 24 h with PGE2 (1 x 10(-6) M), 1.3-1.6-fold increase (P < 0.05) above basal values in secreted dimeric inhibin A was observed. The results suggest that PGE2 is an important inducer of inhibin A production in granulosa cells and that a local action of PGE2 may partially explain the increase in circulating inhibin A observed during the midluteal phase of the human menstrual cycle.

Regulation of inhibin/activin subunit messenger ribonucleic acids (mRNAs) by activin A and expression of activin receptor mRNAs in cultured human granulosa-luteal cells.

Recent studies have indicated that activin and inhibin may act as local regulators of cell growth and steroidogenesis in the human ovary. We studied the effect of recombinant human activin A and purified bovine inhibin A on the steady state messenger RNA (mRNA) levels of the inhibin/activin alpha-, beta A-, and beta B-subunits in cultured granulosa-luteal (GL) cells from preovulatory ovarian follicles of women undergoing in vitro fertilization. Activin A induced the expression of a 4.8-kilobase beta B-subunit mRNA transcript without affecting basal expression levels of the alpha- and beta A-subunit mRNAs. It stimulated beta B-subunit mRNA levels in a concentration- and time-dependent manner. Maximal stimulation of beta B-subunit mRNA levels was obtained with 30-100 ng/ml activin A. The level of beta B-subunit mRNAs increased significantly 8 h after stimulation, rising gradually thereafter to a maximum at 48 h. Inhibin A did not affect the mRNA levels of any inhibin/activin subunits, nor did it inhibit the effect of activin A. Recombinant human follistatin did not affect basal beta B-subunit mRNA levels, but it neutralized the effect of activin A. Although hCG induces inhibin/activin alpha- and beta A-subunit mRNA levels in human GL cells, it did not increase basal beta B-subunit levels. By contrast, it inhibited activin A-induced beta B-subunit mRNA levels. On the other hand, activin A decreased hCG-induced mRNA levels of the inhibin alpha-subunit and cytochrome P450 side-chain cleavage (P450scc) enzyme, an important rate-limiting enzyme in human GL cell progestin synthesis. Moreover, we observed by Northern blot analysis that cultured human GL cells as well as freshly isolated preovulatory granulosa cells express the specific mRNAs for all currently known serine/threonine kinase activin receptors, i.e. activin receptors I, IB, II, and IIB. Our results suggest that in GL cells, activin A may locally stimulate synthesis of the beta B-subunit in an autocrine or paracrine manner, and that in human ovary, regulation of the beta B-subunit differs from that of the alpha- and beta A-subunits.

Regulation of inhibin/activin subunit messenger ribonucleic acids (mRNAs) by activin A and expression of activin receptor mRNAs in cultured human granulosa-luteal cells

Recent studies have indicated that activin and inhibin may act as local regulators of cell growth and steroidogenesis in the human ovary. We studied the effect of recombinant human activin A and purified bovine inhibin A on the steady state messenger RNA (mRNA) levels of the inhibin/activin alpha-, beta A-, and beta B-subunits in cultured granulosa-luteal (GL) cells from preovulatory ovarian follicles of women undergoing in vitro fertilization. Activin A induced the expression of a 4.8-kilobase beta B-subunit mRNA transcript without affecting basal expression levels of the alpha- and beta A-subunit mRNAs. It stimulated beta B-subunit mRNA levels in a concentration- and time-dependent manner. Maximal stimulation of beta B-subunit mRNA levels was obtained with 30-100 ng/ml activin A. The level of beta B-subunit mRNAs increased significantly 8 h after stimulation, rising gradually thereafter to a maximum at 48 h. Inhibin A did not affect the mRNA levels of any inhibin/activin subunits, nor did it inhibit the effect of activin A. Recombinant human follistatin did not affect basal beta B-subunit mRNA levels, but it neutralized the effect of activin A. Although hCG induces inhibin/activin alpha- and beta A-subunit mRNA levels in human GL cells, it did not increase basal beta B-subunit levels. By contrast, it inhibited activin A-induced beta B-subunit mRNA levels. On the other hand, activin A decreased hCG-induced mRNA levels of the inhibin alpha-subunit and cytochrome P450 side-chain cleavage (P450scc) enzyme, an important rate-limiting enzyme in human GL cell progestin synthesis. Moreover, we observed by Northern blot analysis that cultured human GL cells as well as freshly isolated preovulatory granulosa cells express the specific mRNAs for all currently known serine/threonine kinase activin receptors, i.e. activin receptors I, IB, II, and IIB. Our results suggest that in GL cells, activin A may locally stimulate synthesis of the beta B-subunit in an autocrine or paracrine manner, and that in human ovary, regulation of the beta B-subunit differs from that of the alpha- and beta A-subunits.