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Biomedical subjects

G F Erickson

Publications and source records attributed to G F Erickson.

At least 55 records · Page 3Linked to original sources
PubMed1989

Transforming growth factor-beta promotes differentiation of ovarian thecal-interstitial cells but inhibits androgen production.

Evidence that transforming growth factor-beta (TGF beta) is produced by thecal-interstitial cells (TIC) has suggested the hypothesis that TGF beta may be an autocrine regulator of TIC function. The purpose of these studies is to begin to test this hypothesis. In the present experiments we tested the effects of TGF beta on steroid production by TIC isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When TIC (10(4) viable cells/well) were cultured in serum-free medium (0.2 ml in 96-well plates), low amounts of androsterone (less than 4 ng/ml) were produced at 2, 4, and 6 days. TGF beta (0.01-100 ng/ml) did not change basal androsterone production. Treatment with LH (50 ng/ml) stimulated a 100-fold increase in androsterone at 2 days and 60-fold increases at 4 and 6 days. Concomitant treatment with TGF beta (10 ng/ml) caused a 65% inhibition (ED50 = 2.3 +/- 0.7 ng/ml) of androsterone production at each time period. Analysis of key steroid metabolites demonstrated that androsterone and androstenedione were inhibited equally, while progesterone was significantly increased (ED50 = 1.2 +/- 0.2 ng/ml). Time-course studies revealed that TGF beta alone did not alter progesterone production at 2 days, but markedly increased progesterone (10-fold) above control levels at 4 and 6 days. Dose-response experiments showed that TGF beta did not alter the sensitivity of the TIC to LH stimulation, indicating that LH activation of the intracellular signaling pathway was not blocked by TGF beta. Treatment with insulin-like growth factor-I (IGF-I) together with LH caused a synergistic increase in androsterone production. The synergistic stimulation of LH action by IGF-I could be blocked by TGF beta. Interestingly, TIC were more sensitive to TGF beta in the presence of IGF-I (ED50 = 0.18 +/- 0.04 ng/ml). In contrast, TGF beta enhanced progesterone production only at the highest dose of TGF beta (10 ng/ml). To further elucidate the mechanism of TGF beta action, the effects of TGF beta on the TIC content of 17 alpha-hydroxylase/C17-20 lyase (P450(17)alpha) and cholesterol side-chain cleavage (P450scc) were analyzed by immunoblotting. TGF beta alone or in combination with LH stimulated an increase in P450scc content, but did not alter P450(17 alpha content. These results lead us to conclude that 1) the TIC are targets for TGF beta; 2) IGF-I increases the sensitivity of TIC to TGF beta action; and 3) TGF beta acts directly on TIC to stimulate progesterone while inhibiting androgen production.

Androgens↗
PubMed1989

Insulin-like growth factor-I regulates aromatase activity in human granulosa and granulosa luteal cells.

The purpose of this research was to test the hypothesis that insulin-like growth factor-I (IGF-I) regulates estradiol (E2) synthesis in human granulosa and granulosa luteal cells. Cells from individual follicles from spontaneous and human menopausal gonadotropin/CG-stimulated cycles were cultured in serum-free medium containing androstenedione, IGF-I, FSH, and/or CG. At 2, 4, and 6 days, E2 in the medium was measured by RIA. In the granulosa experiments, control cells produced basal levels of E2 at 2 days, and the levels increased with increasing follicle size. Treatment with FSH stimulated E2 production (on the average, 5-fold), and the effect was dose dependent (ED50 = 5 ng/mL or 16 mIU/mL). Incubation with IGF-I alone caused increases in E2 production comparable to those caused by FSH, and the IGF-I effect was dose dependent (ED50 = 8 ng/mL). In most cases, coincubation with FSH and IGF-I augmented E2 levels more than either hormone alone, and at 4 and 6 days the interaction was synergistic. The data from dose-response experiments suggested that the basis of the synergy between FSH and IGF-I was a marked potentiation by either hormone (approximately 10-fold) in the potency of the complementary hormone to stimulate E2 production. In the experiments with granulosa luteal cells from spontaneous and in vitro fertilization preovulatory follicles, the controls synthesized very high levels of E2 spontaneously at 2 days; however, E2 production declined 700% at 4 days, and no E2 was produced by control cells at 6 days. Treatment with FSH, CG, or IGF-I did not cause a significant increase in the high basal levels of E2 at 2 days. During subsequent culture, however, all three hormones stimulated E2 production at 4 and 6 days, but the increases were modest and not sustained. In contrast, coincubation of granulosa luteal cells with FSH plus IGF-I or CG plus IGF-I dramatically enhanced E2 production at 4 and 6 days (on the average, 4-fold), and the effects were sustained throughout the culture period. (ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1988

An improved method for primary culture of ovarian androgen-producing cells in serum-free medium: effect of lipoproteins, insulin, and insulinlike growth factor-I.

Although luteinizing hormone (LH) alone stimulates ovarian interstitial cells cultured in serum-free medium to synthesize large amounts of androgens, there seem to be additional factors in vivo that modulate the time course and magnitude of the cellular responses to LH. In an attempt to develop a more nearly physiologic cell culture model, lipoproteins, insulin, and insulinlike growth factor-I (IGF-I) were added to the serum-free medium. The effects of these modifications on androgen biosynthesis by dispersed cells from ovaries of hypophysectomized immature rats cultured in 96-well tissue culture plates were examined. A saturating dose of LH stimulated a 25-fold increase in androsterone synthesis at 2 d, which decreased at 4 and 6 d. Addition of human high density (hHDL) or human low density lipoprotein (hLDL) caused a 2.5-fold increase in LH-stimulated androsterone synthesis. Cells were approximately twice as sensitive to hHDL (ED50 = 5.5 +/- 0.5 micrograms cholesterol/ml) compared to hLDL (ED50 = 9.1 +/- 1.1 micrograms cholesterol/ml). Surprisingly, rat HDL caused only a 40% increase in LH-stimulated androsterone synthesis. When insulin alone was added to cells cultured with a saturating dose of LH, there was a 2.8-fold increase in androsterone synthesis. Addition of hHDL and insulin together caused a synergistic increase in LH-stimulated androsterone synthesis. In contrast to hHDL, which did not change the time course of LH-stimulated androsterone production, insulin prolonged maximal LH-stimulated androsterone synthesis at 4 and 6 d. Inasmuch as the ED50 for insulin action (1.3 +/- 0.1 micrograms/ml) was supraphysiologic, the effects of IGF-I on LH-stimulated androgen synthesis were examined. IGF-I mimicked all of the effects of insulin, but at a physiologic concentration (ED50 = 2.5 +/- 0.3 ng/ml). Ovarian cells cultured in serum-free medium supplemented with hHDL and insulin or IGF-I exhibit responses that closely approximate the physiologic responses observed in vivo. These results suggest that lipoproteins and IGF-I are important physiologic stimulators of ovarian theca-interstitial cell androgen biosynthesis which, when added to the serum-free medium, make the cellular responses in this in vitro model more nearly approximate the responses in vivo.

Androgens↗
PubMed1988

A monoclonal antibody recognizes a 39 kDa protein expressed in atretic granulosa cells.

A major problem in ovarian physiology is the lack of conveniently quantifiable markers of atresia. Towards this end, we identified a monoclonal antibody (anti-OA-2) that selectively recognizes granulosa cells in atretic follicles. When cryostat sections of rat ovaries were incubated with anti-OA-2, granulosa cells in atretic follicles showed intense immunofluorescent labeling. In contrast, no anti-OA-2 immunoreactivity was observed in the granulosa of the healthy follicles. The amount of anti-OA-2 binding was significantly enhanced when atresia was stimulated by treatment with human chorionic gonadotropin, testosterone, or estrogen withdrawal. The results of immunoprecipitation and Western blot analyses indicated that the OA-2 antigen is a 39 kDa protein which is actively synthesized by the granulosa during atresia. The 39 kDa protein is localized at or near the inner surface of the plasma membrane. We conclude that the anti-OA-2 monoclonal will prove useful as a convenient analytical tool to study the regulation of granulosa atresia.

Animals↗
PubMed1988

Luteinizing hormone-independent luteinization and ovulation in the hypophysectomized rat: a possible role for the oocyte?

Immature hypophysectomized, estrogen-treated rats were used to study the regulation of luteinization. Particular attention was focused on the potential role of the oocyte in this process. Rats were injected for 2 days with follicle-stimulating hormone (FSH) to stimulate follicular development. Within 48 h following FSH treatment, many follicles became luteinized, as determined by morphometric analysis. This luteinization occurred in the absence of detectable levels of luteinizing hormone (LH). The number of follicles undergoing luteinization was dependent on the FSH dose. In addition, ovulation occurred in some of the animals receiving the highest doses of FSH (3-micrograms or 5-micrograms injections). The majority of follicles undergoing luteinization or ovulation were greater than 400 microns in diameter. Luteinized follicles exhibited positive reactivity for cholesterol side-chain cleavage enzyme, 3 beta-hydroxysteroid dehydrogenase, lipid, and alkaline phosphatase, which was similar to that found in corpora lutea of the cycle. Serum progesterone (P0) and 20 alpha-hydroxypregn-4-en-one levels were elevated in animals with luteinized follicles, especially in those animals that also underwent ovulation. Morphological evaluation of oocytes showed that the majority of luteinized follicles contained a degenerating oocyte. Oocyte degeneration was highly correlated (r = 0.94) to luteinization. These results demonstrate that luteinization and ovulation can occur in the FSH/estrogen-primed hypophysectomized rats in the absence of detectable serum LH. Furthermore, LH-independent luteinization was strongly correlated to degenerative changes in the oocyte. These results provide new evidence to support the concept that the oocyte may be an intraovarian regulator of luteinization.

Animals↗
PubMed1988

Evidence for protein kinase C regulation of ovarian theca-interstitial cell androgen biosynthesis.

The hypothesis that protein kinase C may be an important regulator of ovarian theca-interstitial cell steroidogenesis was tested by using phorbol-12-myristate-13-acetate (PMA) and phorbol-12, 13-dibutyrate (PDB) to directly stimulate protein kinase C activity. Collagenase-dispersed cells (4 x 10(5) viable cells/dish) form ovaries of hypophysectomized immature rats were cultured in serum-free medium in the presence and absence of 0-100 ng/ml of luteinizing hormone (LH), PMA (0-100 nM), and/or PDB (0-100 nM). Treatment with 100 ng/ml LH stimulated androsterone production 100-fold at Day 4 of culture. The presence of 100 nM PMA or PDB had no effect on basal androsterone production; however, treatment with increasing concentrations of PMA or PDB (0-100 nM) caused a dose-related inhibition (maximum 70%) of LH-stimulated androsterone synthesis (ID50 = 1.8 nM and 2.4 nM, respectively). PMA and PDB did not significantly alter DNA, protein, or cell viability, indicating that their inhibitory effects were not due to changes in cell number or viability. Cells treated with LH and 100 nM 4 alpha-phorbol didecanoate (4 alpha-PDD; a phorbol ester that does not activate protein kinase C) failed to show significant decreases in androsterone production. Time-course studies revealed that when PMA treatment was delayed until Day 2 or 4 of culture, dramatic inhibitory effects on LH-stimulated androsterone production were still observed. These results suggest that the biological activity of protein kinase C is retained after the cells have expressed their differentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1988

Purification of ovarian theca-interstitial cells by density gradient centrifugation.

The study of the biochemical mechanisms regulating the differentiation of the ovarian androgen-producing cells has been difficult because of the lack of a method for isolating the theca-interstitial cells (TIC) from the granulosa and other cell types. We report here a simple, rapid and reproducible method for obtaining large numbers of highly enriched TIC using density gradient centrifugation. When dispersed cells from ovaries of hypophysectomized immature rats were centrifuged in a Percoll gradient (20-70%), the cells were separated into five incompletely resolved bands. Of these, band V contained the androgen-producing cells. The band V cells produced 3.2- and 3.9-fold more cAMP and androgen, respectively, than the whole ovarian cells and contained 3.8- and 3.5-fold more 125I-hCG binding and 3 beta-HSDH labeled cells, respectively. These data indicated that the TIC were 65% pure. In order to improve the degree of purification, a discontinuous density procedure was used. When the ovarian cells were centrifuged in d = 1.055 g/ml Percoll, the purified TIC produced 4.7- and 5.9-fold more cAMP and androgen than the whole ovarian cells and they contained 5.3-fold more 125I-hCG binding. The TIC were cleanly separated from the granulosa cells and histochemical staining for 3 beta-HSDH activity revealed that the TIC were 93.0 +/- 1.3% pure. This method provides, for the first time, the opportunity to study TIC differentiation under defined conditions without granulosa cell contamination.

Animals↗
PubMed1987

Evidence for widespread atresia in the hypophysectomized estrogen-treated rat.

The question considered in this study concerns to what extent the ovaries of the immature hypophysectomized diethylestilbestrol-treated rat are populated by atretic follicles. Histological studies of ovaries on postoperative days 4-7 reveal the presence of numerous follicles showing signs of ill health. The granulosa cells constituting these follicles are heterogeneous and exist in three distinct zones. The cells in zone 1 are affixed to the basal lamina and form a tightly packed sheet of cuboidal epithelium one to two cells thick. The cells constituting zone 2 are located just medial to zone 1 and appear as a mass of loosely aggregated cells which show intense necrosis. Zone 3 is composed of the prospective cumulus-oocyte complex which appears completely normal. Results of histochemical studies show heterogeneity in the granulosa cells with respect to the presence and location of markers specific for atresia. In atretic follicles, the granulosa cells populating zones 1 and 2 contain lipid droplets and esterase activity, but are negative for the lysosomal enzyme acid phosphatase. The exact reverse is true for zone 3, where the cumulus express intense acid phosphatase activity but are negative for lipid and esterase activities. The granulosa cells in healthy follicles are totally negative for these histochemical markers. As a healthy preantral follicle grows, there is a dramatic increase in the mitotic index of the granulosa cells; however, in all atretic follicles, granulosa cell division is decreased by more than 90%. Further experiments using monoclonal antibodies as probes confirm the presence of atretic follicles and demonstrate that the structure-function changes in zones 1 and 2 are associated with striking changes in the antigenic properties of the granulosa cells. Morphometric studies indicate there are a total of 235 +/- 29 preantral follicles/ovary in the hypophysectomized diethylstilbestrol-treated rat. On postoperative day 4, 37 +/- 1.2% of the preantral follicles are atretic; this number increases linearly with time posthypophysectomy, reaching nearly 50% on postoperative day 7. Examination of follicles at different stages of development reveals that atresia is rare in the very early stages, e.g. follicles less than 200 microns in diameter; however, in the population of larger preantral follicles (those 200-400 microns in diameter), atresia is common, with nearly 80% being atretic on postoperative day 7. Collectively, these findings provide convincing evidence of widespread atresia in the ovaries of the hypophysectomized diethylstilbestrol-treated rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Norepinephrine amplifies human chorionic gonadotropin-stimulated androgen biosynthesis by ovarian theca-interstitial cells.

Ovarian theca-interstitial cells, when cultured in serum-free medium, secreted androgens in response to hCG stimulation. This production was dependent on time (maximum production attained after 96 h) and dose (half-maximal effective dose of hCG, 9 ng/ml). When the sympathomimetics norepinephrine, epinephrine, and isoproterenol were added to the medium, androgen production in response to hCG was enhanced by 100-300%. The ability of the catecholamines to stimulate androgen production was dependent on the continuous presence of hCG. Treatment with catecholamines alone did not induce theca-interstitial cells to produce androgens. Catecholamine stimulation of steroid hormone metabolism was selective for intermediates in the delta 4-pathway, with greatest increases occurring in the production of androstenedione and testosterone. It was found that the effect of the catecholamines on androgen production was dependent on both beta 1-and beta 2-adrenergic receptors. The acquisition of catecholamine responsiveness was specific to hCG; if theca-interstitial cells were induced to differentiate with either prostaglandin E2 or cholera toxin, then isoproterenol did not enhance androgen synthesis. The catecholamine-induced increases in androgen production were not due to a granulosa cell contribution of steroid. The interstitial cells are the only steroid-producing cells in the ovary that are directly innervated by norepinephrine-containing fibers of the sympathetic nervous system. Our finding of catecholamine-augmented androgen production provides a direct link between the autonomic nervous system and regulation of ovarian steroid synthesis.

Androgens↗
PubMed1985

A monoclonal antibody to a mammary cell line recognizes two distinct subtypes of ovarian granulosa cells.

When screening supernatant fluids from hybridoma clones, Dulbecco and co-workers found that a mouse monoclonal antibody generated against a mammary tumor cell line showed rather striking high binding to rat oocytes and granulosa cells. In this study we have specifically investigated the reactivity of the monoclonal antibody (designated anti-OA-1) with granulosa cells during the differentiation process. This was accomplished using the two-step indirect immunocytochemical technique. When a primordial follicle is recruited to initiate growth, intense immunoreactivity appears in the surface membrane of the granulosa cells. As a follicle proceeds through the preantral stages, the plasma membrane of the granulosa cells is strongly positive for anti-OA-1 reactivity, and the granulosa appear as a homogeneous population. However, once Graafian follicle development is initiated, a major shift in anti-OA-1 immunoreactivity occurs among the granulosa cells. As the antrum expands, 75% of the granulosa in the mural region (those nearest the basal lamina) elongate and become negative for anti-OA-1. This is in contrast to the periantral and cumulus granulosa, which remain rounded and show strong anti-OA-1 reactivity up to the preovulatory stage. The disappearance of anti-OA-1 reactivity in the subpopulation of mural cells is specifically initiated by FSH and occurs very rapidly after a 12-h lag phase. After the loss of anti-OA-1 reactivity, the elongated mural granulosa cells express their terminal differentiated state by acquiring LH/hCG receptor, 3 beta-hydroxysteroid dehydrogenase activity and cytoplasmic lipid inclusions. By contrast, the periantral and cumulus granulosa, which remain positive for anti-OA-1, do not express these differentiated functions; however, they do differentiate ultrastructurally, indicating that they respond to the FSH signal. These results strongly suggest that a monoclonal antibody recognizes a major surface differentiation antigen in the granulosa cell. This antigen is under hormonal control and is inversely linked to expression of the terminal differentiation program in the granulosa cells. We anticipate that the monoclonal antibody will be a valuable probe to aide in the analysis of structure/function relationships in subpopulations of granulosa cells.

Animals↗
PubMed1985

Gonadotropin-releasing hormone induces classical meiotic maturation in subpopulations of atretic preantral follicles.

GnRH has been shown to induce premature meiotic maturation in preantral follicles of the immature estrogen-primed hypophysectomized rat. As these animals are free of circulating gonadotropins and contain large numbers of full-grown oocytes in preantral follicles, we have investigated this model to determine its usefulness in studying meiotic maturation. We show that a maximum dose of the agonist D-Trp6,Pro9,Net-LRF (GnRH-a) induces approximately 25% of full grown oocytes to resume meiosis within a 12-h period. This response is dose dependent (ED50 = 0.24 microgram/rat) and specific for GnRH-a. GnRH-a stimulates germinal vesicle breakdown and first polar body formation within 2 and 8 h, respectively. More than 75% of those oocytes that initiate meiotic maturation reach metaphase II by 15 h. This effect of GnRH parallels the time course of physiological meiotic maturation triggered by LH as well as that of oocytes maturing spontaneously in vitro. Oocytes in primordial and primary follicles do not respond to GnRH. The majority of affected follicles are small tertiary follicles (200-400 micron in diameter) and show signs of atresia. This atresia is not caused by GnRH-a and does not, in itself, result in meiotic maturation, but appears to confer susceptibility to GnRH-a-induced meiotic maturation. Our studies indicate that this animal model will be useful to elucidate further the mechanisms and requirements for meiotic maturation. It will also facilitate investigation of the role of atresia in the GnRH response of tertiary follicles and the issue of follicle heterogeneity within these animals.

Animals↗
PubMed1984

Studies on the effect of gonadotropin-releasing hormone and its agonist on human luteal steroidogenesis in vitro.

The possible direct effect of gonadotropin-releasing hormone (GnRH) and a potent GnRH agonist [( imBzl )-D- His6 -Pro9-NEt]-GnRH on basal and human chorionic gonadotropin (hCG)-stimulated progesterone, androstenedione, and estradiol production by cultured human luteal cells was examined. Luteal cells from the early or midluteal phase of the menstrual cycle responded to hCG stimulation with two to fivefold increases in steroid production in both short-term (4 hours) and long-term (up to 144 hours) culture in chemically defined medium without serum. After 48 hours in this system, levels of androstenedione and estradiol were very low, and progesterone was the predominant steroid produced. The addition of GnRH or a potent GnRH agonist to the medium had no effect on either basal or hCG-stimulated steroid secretion. When luteal cells were cultured longer (for up to 10 days) in the presence of serum, GnRH agonist caused no significant alteration of either basal or hCG-stimulated progesterone production. Collectively, these results support the conclusion that GnRH and its potent agonist do not act directly on human corpora luteal cells to modulate steroidogenesis.

Androstenedione↗
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↗
PubMed1983

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells.

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.

Animals↗