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R H Schwall

Publications and source records attributed to R H Schwall.

33 records · Page 2Linked to original sources
PubMed1991

In vivo regulation of FSH synthesis by inhibin and activin.

The effect of a single sc injection of the gonadal peptide, recombinant human activin A (rhActivin A), on gonadotropin synthesis and secretion was examined in adult and immature male and female rats and the effect of recombinant human inhibin A (rhInhibin A) was examined in adult male rats. Pituitary FSH beta, LH beta and alpha messenger RNA (mRNA) levels were determined by blot hybridization. Trunk blood was collected to measure serum FSH levels. Treatment with rhInhibin A (100 micrograms/kg) resulted in a decrease in FSH beta mRNA to 2% of controls levels 6 h after injection. FSH beta mRNA levels started to rebound at 10 h, but were still significantly lower than vehicle-treated controls. Serum FSH levels were significantly reduced at 2 h and were reduced further at 6 and 10 h. There were no significant changes in alpha and LH beta mRNA levels. RhActivin A, at the highest dose (500 micrograms/kg), in immature male rats had only a modest effect (1.2- and 1.3-fold increase) on FSH beta mRNA levels and FSH secretion, respectively, at 2 h. No increase in FSH synthesis and FSH secretion was observed in adult male rats. In contrast, both immature and adult-ovariectomized E2 implanted females showed a robust response to rhActivin A. In immature females, 2 h after rhActivin A (100 and 500 micrograms/kg) administration, FSH beta mRNA levels were elevated 2.0- and 2.2-fold. At this time serum FSH was also elevated. At 6 and 10 h rhActivin A significantly reduced FSH beta mRNA levels from vehicle-treated controls. In contrast, FSH secretion was elevated at 6 h and returned to baseline at 10 h. Administration of rhActivin A (500 micrograms/kg) to adult, ovariectomized-E2 females resulted in a significant increase in FSH beta mRNA levels and FSH secretion at 2 and 6 h. There were no significant changes in alpha and LH beta mRNA levels in either males or females. Thus, these in vivo studies have shown that rhInhibin A can inhibit FSH beta mRNA levels and FSH secretion in the adult male rat. RhActivin A stimulates FSH synthesis and secretion in the immature and adult ovariectomized-E2 females, but has little or no effect in immature and adult males. Hence, there is a sexual dimorphic response to rhActivin A in vivo in the rat.

Activins↗
PubMed1991

Effect of recombinant activin on androgen synthesis in cultured human thecal cells.

The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (androstenedione and dehydroepiandrosterone) production were induced by treating the cells for 4 days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (greater than or equal to 30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activin-A, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activin-A on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary.

Activins↗
PubMed1991

The effects of activin on follicle-stimulating hormone secretion and biosynthesis in human glycoprotein hormone-producing pituitary adenomas.

The effects of activin on pituitary FSH biosynthesis have been previously characterized using primary rat pituitary cultures; however, little is known of the effects of activin on FSH biosynthesis and secretion in human pituitary tissue. Production of intact glycoprotein hormones and free subunits is increasingly recognized in pituitary tumors; however, the regulation of gonadotropins in such tumors has not been addressed. We have investigated the effects of human recombinant activin on glycoprotein hormone biosynthesis and secretion in primary cultures of 12 human glycoprotein hormone-producing pituitary adenomas and compared this with the effects of activin in normal rat anterior pituitary cells. In 33% of the human pituitary tumors studied, significant (P less than 0.05) increases in FSH beta secretion occurred in response to incubation with 20 ng/mL activin for 24 h (19-287% stimulation), without changes in the production of intact FSH. A Northern analysis performed on cells derived from one tumor indicated that FSH beta mRNA levels increased 350% after activin treatments; however, FSH secretion did not parallel the mRNA changes. None of the human glycoprotein hormone-producing tumors significantly increased FSH secretion in response to activin. To validate the biological activity of recombinant human activin-A and to confirm time and dose conditions for the human tumor cultures, we also examined its ability to stimulate FSH production in rat pituitary cultures. Activin (20 ng/mL) added to the culture medium significantly increased FSH secretion and steady state levels of FSH beta mRNA after 24 h. These data indicate that some glycoprotein hormone-producing pituitary tumors treated with purified activin have discordant responses of intact gonadotropins and free subunit responses. In contrast to responses in normal rat gonadotrophs, FSH beta biosynthetic pathways may be uncoupled from intact FSH secretion in a subset of glycoprotein hormone-producing pituitary adenomas.

Activins↗
PubMed1990

Purification and characterization of recombinant human activin B.

Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the beta subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the beta subunit of inhibin B. Activin B has an apparent pI of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Activins↗
PubMed1990

Localization of inhibin/activin subunit mRNAs within the primate ovary.

In order to gain further understanding of the physiology of inhibin and activin in the primate, the expression of inhibin/activin subunit mRNAs in the monkey ovary was examined by in situ hybridization. Granulosa cells of small antral follicles were found to express mRNA for the beta B subunit, which decreased to undetectable levels in dominant follicles. In contrast, expression of alpha and beta A subunit mRNAs was detected in granulosa cells of dominant follicles and in corpora lutea, but not in small antral follicles. These results indicate that the expression of the beta A and beta B subunits is differentially regulated during the growth and development of ovarian follicles in the monkey.

Activins↗
PubMed1989

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.

Activins↗
PubMed1988

Activin stimulates secretion of follicle-stimulating hormone from pituitary cells desensitized to gonadotropin-releasing hormone.

The secretion of follicle-stimulating hormone (FSH) by pituitary cells is stimulated by activin and gonadotropin-releasing hormone, GnRH. To examine the possible interrelationships between the intracellular actions of these secretagogues, responsiveness to activin was tested following pretreatment with 0, 0.1, or 10 nM GnRH. In cells pretreated with 0 or 0.1 nM GnRH, FSH secretion was increased approximately 2-fold during a subsequent challenge with either activin or GnRH. In contrast, in cells pretreated with 10 nM GnRH, FSH secretion became unresponsive to GnRH but could still be stimulated 2-fold by activin. These results demonstrate that activin is able to stimulate FSH secretion in cells that have undergone desensitization to GnRH.

Activins↗
PubMed1988

Selective and indirect modulation of human multipotential and erythroid hematopoietic progenitor cell proliferation by recombinant human activin and inhibin.

Activin and inhibin are biomolecules that, respectively, enhance and suppress the release of follicle-stimulating hormone from pituitary cells in vitro. Purified recombinant human (rhu) activin A and inhibin A were assessed for their effects on colony formation in vitro by human multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells. It was found that (i) rhu-activin A enhances colony formation by normal bone marrow erythroid and multipotential progenitor cells; (ii) purified rhu-inhibin A decreases activin, but not rhu-interleukin 3, rhu-granulocyte-macrophage colony-stimulating factor, or rhu-interleukin 4, enhancement of erythropoietin-stimulated colony formation by erythroid and multipotential progenitor cells; (iii) modulatory actions of rhu-activin and rhu-inhibin are mediated through monocytes and T lymphocytes within the marrow; (iv) actions are apparent in the absence or presence of serum; and (v) rhu-activin and rhu-inhibin have no effect on colony formation by granulocyte-macrophage progenitor cells. This defines an indirect mode of action and a specificity for activin and inhibin on multipotential and erythroid progenitor cells.

Activins↗
PubMed1988

Recombinant expression and characterization of human activin A.

Activin, which stimulates the secretion of FSH from anterior pituitary cells, is a dimer of the beta-subunits of inhibin. Two species of activin (A and AB) have been purified from ovarian follicular fluid and characterized. We have been able to biosynthetically produce recombinant human activin A by constructing stable cell lines expressing the mRNA for the beta A subunit of human inhibin. These cell lines secreted a 24 kilodalton beta A dimer which stimulated FSH secretion in cultured pituitary cells. The ability of this protein to stimulate FSH secretion was sensitive to reduction of disulfide bonds, exhibited a slow onset of action, and was blocked by actinomycin D. In addition, recombinant activin A stimulated hemoglobin accumulation in K562 cells. These data show that recombinant activin A has the biochemical properties and biological activities that have been ascribed to native activin A. In addition, these results provide an independent confirmation, and thereby a final proof, of the structure and function of activin.

Activins↗
PubMed1986

Changes in the distribution of sizes of ovine luteal cells during the estrous cycle.

The ovine corpus luteum is composed of two types of steroidogenic cells, which are referred to as small and large luteal cells. In this study, the size and number of steroidogenic cells were determined in corpora lutea collected on Days 4, 8, 12, and 16 of the estrous cycle. Corpora lutea were dissociated into single-cell suspensions that were stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, a marker for steroidogenic cells. The size of 3 beta-HSD-positive cells was measured with a Zeiss Videoplan Image Analyzer. On Day 4, most of the 3 beta-HSD-positive cells were less than 18 microns in diameter, the median being 11.2 microns. By Day 8, the number of 3 beta-HSD-positive cells increased 3-fold, and the median diameter increased to 12.8 microns. Although the number of 3 beta-HSD-positive cells was reduced by approximately 50% on Day 16, the median size on Days 12 and 16 was 14.6 and 16.8 microns, respectively. The ratio of large (greater than 18 microns) to small (less than 18 microns) luteal cells was 0.11 +/- 0.03 on Day 4; the ratio increased linearly to 0.67 +/- 0.09 by Day 16. This increase between Days 4 and 12 was attributable to an overall increase in the size of the cells; the increase between Days 12 and 16, however, was due to a loss of small luteal cells. When the experiment was conducted near the end of the breeding season, before animals became anestrous, the median size of the luteal cells did not change at different times of the estrous cycle but remained constant throughout. These data suggest that development of the corpus luteum is associated with an increase in the size and number of steroidogenic luteal cells, and that luteolysis is associated with a preferential loss of small luteal cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Inhibition of synthesis of luteinizing hormone (LH) receptors by a down-regulating dose of LH.

Although LH is known to cause down-regulation of the LH/human CG (hCG) receptor, the mechanisms underlying this process are not well understood. Here we have analyzed the effects of LH on the turnover of LH receptors in cultured granulosa cells. Down-regulation was stimulated in FSH-primed granulosa cells by exposure to LH for 1 h. By 24 h after LH exposure, the LH/hCG receptor content, as measured using [125I]iodo-hCG was 70% less than controls. To determine whether this loss of LH/hCG receptors was due to accelerated receptor degradation, turnover measurements were made using tunicamycin to inhibit LH/hCG receptor synthesis. Under these conditions, there was a small (16%) increase in the rate of degradation of LH/hCG receptors in LH-treated cells compared to controls. By contrast, the rate of LH/hCG receptor synthesis, estimated mathematically, was reduced by 75% in the LH-treated cells. LH treatment had no effect on the incorporation of [35S]methionine into total cellular protein. Withdrawing FSH from the culture medium had a similar effect, slightly enhancing LH/hCG receptor degradation while markedly reducing its synthesis. The role of lysosomes in the degradation of LH/hCG receptors was also investigated. The lysosomotropic amine NH4Cl failed to cause LH/hCG receptors to accumulate, contrary to what would be expected if LH/hCG receptors were degraded lysosomally. In contrast, NH4Cl inhibited the degradation of receptor-bound [125I]iodo-hCG, suggesting that the ligand and the receptor may be handled differently by the granulosa cell. Our results suggest that in the granulosa cell, LH down-regulates its own receptor by inhibiting the synthesis of LH/hCG receptors, possibly by interfering with the ability of FSH to stimulate the synthesis of LH/hCG receptors. Furthermore, they suggest that the degradation of LH/hCG receptors may occur by nonlysosomal mechanisms.

Animals↗
PubMed1983

A central role for cyclic AMP, but not progesterone, in luteinizing hormone receptor down-regulation in the granulosa cell.

Although luteinizing hormone (LH) is known to down-regulate its own receptor in several gonadal cell types, the mechanisms underlying this process are poorly understood. To elucidate these mechanisms we have examined the role of cAMP and progesterone in LH-stimulated down-regulation of the LH receptor, using cultured granulosa cells as a model. LH receptors were induced by culturing the cells with follicle-stimulating hormone for 2 days, and once induced, could be down-regulated by a brief exposure to LH. Down-regulation also occurred when cells were cultured with activators of adenylate cyclase, inhibitors of phosphodiesterase, or analogues of cAMP. Cholera toxin and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate, like LH, decreased the number of LH receptors, without affecting affinity for 125I-human chorionic gonadotropin (hCG). The extent of receptor loss after treatment with LH plus cholera toxin was no greater than that caused by LH alone. LH, hCG, and deglycosylated hCG, which binds to the LH receptor but has little bioactivity, caused down-regulation, and their relative capacity to cause down-regulation was highly correlated with their relative capacity to stimulate cAMP production. Indirect evidence suggested that maximal down-regulation requires activation of adenylate cyclase for at least 3 h. Consistent with this idea, a 3-h exposure to dibutyryl cAMP caused near-maximal down-regulation. Progesterone secretion was enhanced by all agents that caused down-regulation of the LH receptor; however, there was little correlation between progesterone secretion and down-regulation. Furthermore, maximal down-regulation occurred when progesterone secretion was inhibited greater than 99% with cyanoketone. These data indicate that cAMP, but not progesterone, plays a central role in LH receptor down-regulation in the granulosa cell and that elevation of intracellular cAMP levels for 3 h is both necessary and sufficient to trigger maximal down-regulation.

Animals↗
PubMed1983

A new in vitro model system for the study of luteinizing hormone receptor down-regulation.

The overall goal of our research is to elucidate the cellular mechanisms involved in the process of luteinizing hormone (LH)-stimulated down-regulation of the LH receptor. Towards this end, we became interested in determining whether cultured granulosa cells could be used as a physiologic model system to study this important process. In this report, we show that a very brief exposure (2 min) to physiologic concentrations of LH causes down-regulation of the LH receptor in granulosa cells cultured in serum-free medium. These data represent the first in vitro demonstration of down-regulation of the LH receptor in the granulosa cell. Furthermore, the dose dependency and kinetics of down-regulation of the LH receptor in vitro, as described here, are very similar to those reported for LH receptor down-regulation in vivo. Thus, this granulosa cell culture system is a powerful new model for studying the biochemical events involved in LH receptor down-regulation.

Animals↗