Human chorionic gonadotropin and prolactin modulation of early luteal function and luteinizing hormone receptor-binding activity in cultured human granulosa-luteal cells

These studies were undertaken to explore the roles of both hCG and PRL in the modulation of early luteal function in the human. Human granulosa-luteal cells isolated during cycles stimulated by human menopausal gonadotropin hCG were obtained at the time of follicle aspiration and cultured to determine the effects of hCG and PRL on both progesterone and hCG receptor binding. Progesterone production by hCG-stimulated granulosa-luteal cells was increased 3.5-fold over unstimulated levels after 120 h, with maximal stimulation at hCG concentrations greater than 1 IU/ml. [125I]hCG binding to granulosa luteal cells was increased 3-fold in cells cultured with hCG (10 IU/ml) after both 48 h (P less than 0.03) and 96 h (P less than 0.02) in culture. hCG (1 IU/ml) stimulated a significant increase in progesterone production above basal levels after 72 h of culture, which continued to increase until 96 h of culture; 20 alpha-dihydroprogesterone (20 alpha-OH progesterone) production also was increased by hCG (1 IU/ml) at 72 h of culture, but unlike progesterone production, showed no further increase. In both the presence and absence of hCG, granulosa-luteal cells cultured with PRL (100 ng/ml) produced significantly more 20 alpha-OH progesterone (P less than 0.04 and P less than 0.02, respectively) after several days than cells cultured without PRL. In addition, progesterone production in the presence of hCG (10 IU/ml) decreased significantly (P less than 0.04) as 20 alpha-OH progesterone levels increased. Equivalent amounts of [125I]hCG were bound by human granulosa-luteal cells cultured with and without PRL (100 ng/ml). These results show that cultured human granulosa-luteal cells are responsive to hCG, with parallel increases in both progesterone production and [125I]hCG receptor binding. The presence of PRL (100 ng/ml) had no effect on [125I]hCG binding. In both the presence and absence of hCG, PRL resulted in an increase in 20 alpha-OH progesterone production and, in the presence of hCG (10 IU/ml), a decrease in progesterone production after several days in culture.     

Ovulation induction with human menopausal gonadotropin compared to human urinary follicle-stimulating hormone results in a significant shift in follicular fluid androgen levels without discernible differences in granulosa-luteal cell function

Follicular fluid estradiol, progesterone, testosterone, and androstenedione levels were compared in 2 groups of spontaneously ovulatory women undergoing ovulation induction with human menopausal gonadotropin (hMG; which contains equal amounts of LH and FSH) or human urinary FSH (huFSH). The results were correlated with the ratios of embryo cleavage and pregnancy. Although significantly more FSH [1268 +/- 38 (+/- SEM) vs. 953 +/- 38 IU; P less than 0.05] was required for equivalent hyperstimulation in hMG compared to huFSH cycles, the number of oocytes retrieved and fertilized and the number of embryos transferred were similar for the 2 ovulation induction protocols. Forty-two follicles from 21 women stimulated with hMG and 38 follicles from 15 women stimulated with huFSH were examined and found to be representative of the total cohort of aspirated follicles. Follicular fluid estradiol and progesterone levels were similar, but hMG-stimulated follicles contained significantly more testosterone [7.83 +/- 0.52 (+/- SEM) vs. 6.30 +/- 0.42 ng/ml; P less than 0.03] and less androstenedione (24.4 +/- 3.6 vs. 37.8 +/- 5.0 ng/ml; P less than 0.03) than did huFSH-stimulated follicles. Embryonic cleavage rates were similar for all fertilized oocytes from both hMG- and huFSH-stimulated cycles, although pregnancy rates were significantly higher in huFSH cycles (40% vs. 9.5%; P less than 0.05). In addition, aromatase activity, progesterone production, and [125I]hCG-binding activity were compared in granulosa-luteal cells isolated from some of these women. Cells from 21 follicles from 9 women stimulated with hMG and 24 follicles from 9 women stimulated with huFSH were studied. There were no significant differences in aromatase activity, progesterone production, or [125I]hCG binding. Thus, the presence or absence of exogenous LH during ovulation induction with FSH has little direct effect on granulosaluteal cell function. However, the presence of LH during ovulation induction with FSH does appear to alter thecal androgen metabolism, resulting in higher testosterone and lower androstenedione levels in follicular fluid. Such a shift in androgen milieu may impair oocyte development and successful implantation.     

Effect of estradiol and progesterone on myometrial LH/hCG receptors in pigs.

High affinity luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors have been identified in porcine, rabbit and rat uteri and immunocytochemically demonstrated in the human uterus. We have now assessed the effect of estradiol and progesterone on the capacity and affinity of LH/hCG binding sites in crude membrane fractions of porcine myometria. Nineteen cross-bred gilts were ovariectomized at 6-7 months of age. Five weeks later, the experiment was conducted and gilts were given estradiol benzoate 2 mg (N = 5), progesterone 50 mg (N = 4) and 2 mg of estradiol benzoate plus 50 mg of progesterone in 2 ml of corn oil (N = 6), im for five consecutive days. Controls (N = 4) received 2 ml of the vehicle. Gilts were hysterectomized 24 h after the last injection. Blood samples for assays of LH, estradiol and progesterone were collected 1 h before hysterectomy. The numbers and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. The results indicate that treatment with estradiol benzoate increases (p less than 0.01) the number of LH/hCG binding sites compared with gilts receiving corn oil. Progesterone treatment caused elevation in the number of LH/hCG receptors (p less than 0.05), when compared with estradiol alone (2.9 +/- 0.3 vs 1.2 +/- 0.1 fmol/mg protein, respectively). Combined administration of estradiol and progesterone increased receptor capacity to 2.7 +/- 0.4. Steroid treatment did not alter the affinity (Ka) of [125I]hCG binding to receptors and it varied from 1.8 +/- 0.8 to 2.9 +/- 0.2 x 10(11) l/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internalization and degradation of human chorionic gonadotropin in ovine luteal cells: effects of inhibition of protein synthesis

Studies were undertaken to investigate the possibility that receptors for LH in monolayer cultures of enzymatically dissociated ovine luteal cells are recycled. Cultured cells (3-10 X 10(5) total steroidogenic cells/dish) were incubated with or without cycloheximide (CHX; 10(-4) M) and 2 X 10(6) cpm [125I]iodo-hCG in the presence or absence of 30 micrograms nonradioactively labeled hCG for 0, 12, 24, 36, or 48 h. At each time point, the amounts of radioactivity bound to the cells (bound [125I]iodo-hCG), located intracellularly (intracellular [125I]iodo-hCG), and degraded and returned to the medium as [125I]monoiodotyrosine (degraded [125I]iodo-hCG) were determined. The number of receptors for LH was determined by Scatchard analysis. Cell viability was also monitored by: 1) trypan blue dye exclusion, 2) the ability of the cells to synthesize protein, and 3) basal and hCG-stimulated secretion of progesterone. More than 90% of the cells remained viable after 48 h of culture, and CHX had no effect on cell viability. Protein synthesis in CHX-treated cells was inhibited by more than 90%. Basal and hCG-stimulated secretion of progesterone were also inhibited by CHX. Treatment with CHX increased the amounts of membrane-bound and internalized [125I]iodo-hCG and decreased the amounts of [125I]iodo-hCG that were degraded. When the quantities of radioactivity in these three fractions (plasma membrane-bound, internalized, and degraded) were added together to obtain a value for the total amount of [125I]iodo-hCG that had been bound to receptor during the 48-h time course (total receptor-associated [125I]iodo-hCG), the value for control cells was not significantly different from the value for CHX-treated cells. Furthermore, the total receptor-associated [125I]iodo-hCG was approximately 2-fold greater than the amount of [125I]iodo-hCG required to saturate receptors at time zero. These data indicate that synthesis of new receptors is not required for the continued binding, internalization, and degradation of [125I]iodo-hCG. Further, the data are compatible with the hypothesis that receptors for LH are recycled or that a portion of the total receptor population is in an unavailable form when the cells are intact but are available for binding after homogenization of the cells.     

Lymphokines, including interleukin-2, alter gonadotropin-stimulated progesterone production and proliferation of human granulosa-luteal cells in vitro

The effects of human interleukin-1 (IL-1) and IL-2 on human granulosa-luteal cell progesterone production were examined with or without hCG stimulation in vitro. Human granulosa-luteal cells were recovered from follicular fluid obtained from women undergoing in vitro fertilization procedures and cultured for up to 7 days before supernatant progesterone level measurement. Lymphokine-rich conditioned medium was prepared from mitogen-stimulated human peripheral blood leukocytes (HPL-CM). The influence of HPL-CM on both granulosa-luteal cell progesterone production and cell growth was inhibitory. In contrast, supernatants of the IL-2-producing cell line MLA-144 (MLA-CM) stimulated both basal progesterone secretion and cell proliferation. Human recombinant IL-2 (from 0.1-100 IU) alone did not change progesterone levels, compared to control values, after 24 h of cell culture. However, 1, 10, and 100 IU IL-2 significantly inhibited progesterone secretion from cells stimulated by 5 IU hCG (P less than 0.01). The enhanced progesterone levels stimulated by forskolin were also significantly inhibited by 10 IU IL-2 (P = 0.01). This effect was not mediated through decreased cAMP, since the forskolin-enhanced cAMP level was not influenced by IL-2, IL-1, with or without hCG, did not show any effect on progesterone production during either 24 or 48 h of cell culture. It is concluded that 1) human recombinant IL-2 significantly inhibits progesterone production stimulated by hCG in human granulosa-luteal cells; 2) IL-2 also had a marked inhibitory effect on forskolin-induced progesterone release, but did not influence the increased cAMP level stimulated by forskolin; 3) the inhibitory influence of IL-2 on progesterone synthesis may be down-stream in the signal transduction pathway from cAMP activation; and 4) HPL-CM and MLA-CM produced inhibitory and stimulatory effects, respectively, on both basal and hCG-stimulated progesterone levels as well as on granulosa-luteal cell proliferation. These activities cannot be completely attributed to IL-2, and other mediators of leukocyte origin may, therefore, exist.     

Gonadotropin receptor occupancy and stimulation of cAMP and testosterone production by purified Leydig cells: critical dependence on cell concentration

Rat testicular interstitial cells have been separated by discontinuous/continuous gradient of Percoll, yielding four cell fractions. The light cells in fraction I bound luteinizing hormone/human chorionic gonadotropin (LH/hCG) with high affinity but were not steroidogenic in response to hormone. Fraction II consisted mainly of germ cells. Although fraction III contained Leydig cells, this fraction was contaminated with germ cells and was less responsive to hormone as compared to the Leydig cells in fraction IV. The Leydig cells in fraction IV produced cAMP and testosterone in response to hormone action in a manner which was critically dependent upon cell concentration. The production of cyclic adenosine monophosphate (cAMP) in the presence of saturating concentrations of hCG (2.4 X 10(-10) M) was linear as a function of cell concentration up to 7.0 X 10(6) cells/1.25 ml and thereafter, a slight inhibition (26%) was seen at 10 X 10(6) cells/1.25 ml. The average value for cAMP production by hCG was 133.8 +/- 8.5 pmol cAMP/2 X 10(6) cells. The production of testosterone was biphasic, increasing linearly up to 5 X 10(6) cells/1.25 ml and decreasing thereafter. Two million cells, in the presence of 2.4 X 10(-10) M hCG, produced an average of 24.2 +/- 1.7 ng of testosterone in reaction volumes ranging from 1 to 2 ml whereas the same number of cells only produced 5.1 +/- 0.6 ng of testosterone in 250 microliters. The binding of 125I-labeled hCG to the same batch of cells increased with increasing cell concentrations as expected but under the conditions of maximal steroidogenesis at low cell concentrations (1.25, 2.0, and 2.5 X 10(6) cells/1.25 ml), it was barely detectable. Thus, we conclude that there is an inverse relationship between the parameters of binding and biological response in purified Leydig cells.     

Interleukin-2 is a potent inhibitor of Leydig cell steroidogenesis

Interstitial tissue of the testis consists of Leydig cells, macrophages, lymphocytes, plasma cells, mast cells and fibroblasts. Previously we have reported that interleukin-1 (IL-1) inhibits Leydig cell androgen production. In the present study, the effect of IL-2 was investigated. Leydig cells (10(5) cells/ml) from adult Sprague-Dawley rats were cultured with or without IL-2 for 24 h. After medium changes, human CG (hCG), 8-bromo-cAMP, or forskolin was added with or without IL-2. Cultures were continued for an additional 24 h, and testosterone and cAMP levels were measured. IL-2 up to 100 U/ml had no effect on basal testosterone production. hCG-stimulated testosterone formation was inhibited in a dose-dependent manner by the addition of IL-2. IL-2 in a concentration of 100 U/ml decreased hCG-induced testosterone formation from 49.6 +/- 3.6 ng/ml (mean +/- SE) to 8.5 +/- 4.2 ng/ml. The hCG dose-response curve was shifted to the right by the addition of IL-2. Maximal testosterone production in response to hCG was reduced 40% in the presence of IL-2 (50 U/ml) without alteration of median effective dose (ED50). IL-2 also inhibited hCG-induced cAMP formation and 8-bromo cAMP- and forskolin-stimulated testosterone production. However, IL-2 did not alter the binding of [125I]hCG to purified Leydig cells. Furthermore, IL-2 significantly inhibited the conversion of 20-OH-cholesterol, 22-OH-cholesterol, pregnenolone, progesterone, 17 alpha-hydroxypregnenolone, and 17 alpha-hydroxyprogesterone to testosterone but did not alter the conversion of dehydroepiandrosterone and androstenedione to testosterone. Our results suggest that a T cell growth factor, IL-2, is a potent inhibitor of steroidogenesis. IL-2 may play a paracrine role in modulating Leydig cell function.     

Mechanism of thyroid stimulation by human chorionic gonadotropin in sera of normal pregnant women.

To elucidate the mechanism of thyrotropic activity of human chorionic gonadotropin in sera of normal pregnant women, we examined the effect of blockade of the TSH receptor on the serum-induced cAMP accumulation and the effect of hCG on the TSH binding to FRTL-5 cells. In the presence of crude immunoglobulin fractions in sera from patients with primary hypothyroidism, cAMP accumulation induced by both crude and purified hCG, and normal pregnant women serum were significantly inhibited compared with that in the presence of normal IgGs. The mode of inhibition of these IgGs on the cAMP accumulation was similar for TSH and hCG when analysed by Lineweaver-Burk plots. Moreover, binding of [125I]bTSH to the TSH receptor in porcine thyroid cell membrane was apparently inhibited by adding 4 x 10(6) IU/l of purified hCG. Binding studies of TSH in FRTL-5 cells also indicated the dose-dependent displacements of [125I]TSH by hCG. Although half-maximal inhibitory concentration of hCG was about 20 times as high as that of TSH on a molar basis, displacement of [125I]TSH was observed at a concentration of hCG of 10(5)IU/l or more, which could be a physiological concentration of hCG in sera of normal pregnant women. These results suggest that thyrotropic activity of hCG in sera of normal pregnant women is, at least in a part, mediated by TSH receptors.     

Direct inhibitory effect of gonadotropin-releasing hormone upon luteal luteinizing hormone receptor and steroidogenesis in hypophysectomized rats

The effects of gonadotropin-releasing hormone (GnRH) and its potent agonist [des-Gly10, D-Leu6-N alpha Me) Leu7, Pro9,NHEt-GnRH (GnRH-A)] on ovarian luteal functions maintained by PRL were studied in vivo and in vitro. Hypophysectomized, diethylstilbestrol-treated female rats were primed with FSH for 2 days, followed by an ovulating dose of LH or hCG. Two days later, ovarian luteal functions were maintained by daily injections of 250 microgram PRL for 3 days. PRL treatment increased the serum progesterone level from 13.0 +/- 0.5 to 298 +/- 24 ng/ml and increased the ovarian hCG-binding capacity from 5.8 +/- 1.3 to 584 +/- 86 ng bound hCG/ovary. In contrast, concomitant treatment with GnRH or GnRH-A resulted in dose-dependent decreases in the PRL-induced increase of serum progesterone and ovarian LH/hCG receptor content. GnRH at 100 microgram/day caused a 60% decrease in serum progesterone and an 80% decrease in ovarian LH receptor content, whereas GnRH-A was effective at a 1-microgram dose level. Neither GnRH nor GnRH-A affected the binding affinity (Kd) of ovarian LH receptor. The direct inhibitory effects of GnRH and GnRH-A upon granulosa-luteal cell function were also tested in vitro. FSH treatment for 2 days induced functional LH and PRL receptors in cultured PRL, increased (by approximately 3-fold) progesterone production by these granulosa-luteal cells, whereas concomitant treatment with GnRH-A inhibited progesterone production in a dose-dependent manner. Thus, these studes demonstrated that GnRH and GnRH-A exert direct inhibition on ovarian luteal functions by decreasing LH receptor and progesterone production in vivo as well as inhibiting progesterone production by cultured granulosa-luteal cells in vitro.     

Regulation of steroidogenesis in cultured porcine theca cells by growth factors

Growth factors [insulin-like growth factors (IGF-I, IGF-II), transforming growth factor-beta (TGF beta), epidermal growth factors (EGF)], found in the ovary and known to alter granulosal function, were assessed for their ability to modulate porcine thecal steroidogenesis. Theca cells from large porcine follicles (8-10 mm) were plated (5 x 10(5) cells/ml.well) in serum-free M199, treated with increasing doses of growth factors: IGF-1 (0.1-50 ng/ml), IGF-II (0.5-200 ng/ml), EGF (0.021-100 ng/ml), TGF beta (0.001-40 ng/ml), or insulin (0.01-50 micrograms/ml), with or without human CG [(hCG); 20 ng/ml], and incubated for 72 h. Levels of steroids in media were determined by RIA. Insulin increased (P less than 0.05) basal and gonadotropin-induced secretion of androstenedione, progesterone, estradiol, and testosterone. IGF-I increased (P less than 0.05) the basal and hCG-induced secretion of progesterone and androstenedione at the highest doses, but did not affect basal secretion of estradiol or testosterone. IGF-II, at the highest doses, increased (P less than 0.05) thecal steroidogenesis, but only after administration of hCG. In contrast, TGF beta increased (P less than 0.05) basal and gonadotrophin-induced secretion of estradiol but inhibited thecal secretion of progesterone, androstenedione, and testosterone. EGF did not alter thecal secretion of progesterone, androstenedione, or testosterone but significantly (P less than 0.05) inhibited basal and hCG-stimulated secretion of estradiol. In conclusion, insulin IGF-I, IGF-II, EGF, and TGF beta can modulate steroidogenesis in porcine theca cells.     

Regulation of luteal cell lipoprotein receptors, sterol contents, and steroidogenesis by estradiol in the pregnant rat

Although estradiol has been found to possess receptors in the luteal cell and to stimulate progesterone synthesis, its mechanism of action in the corpus luteum remains completely unknown. To determine whether estradiol modulates cellular uptake of lipoprotein substrate and intracellular cholesterol utilization, pregnant rats were hypophysectomized and hysterectomized on day 12 to reduce the luteal content of estradiol. They were treated with either 100 micrograms estradiol daily or with a 1-cm capsule filled with testosterone, which maintained luteal estradiol at levels found in intact pregnant rats. Blood was obtained 24, 48, and 72 h later for progesterone and cholesterol measurement. At 72 h, rats were killed, and corpora lutea (CL) were isolated for measurement of cholesteryl ester, free cholesterol, and [125I]iodo high density lipoprotein [( 125I]iodo-HDL)- and [125I]iodo-hCG-binding activities. In vivo treatment with estradiol or testosterone increased serum progesterone concentrations from 35 +/- 7 ng/ml in vehicle-treated rats to 128 +/- 21 and 118 +/- 16, respectively, and luteal weight from 2.1 +/- 0.2 mg/CL to 3.9 +/- 0.3 and 4.0 +/- 0.3 within 72 h. However, steroid treatment did not induce a change in luteal cell number, since the content of DNA per CL remained similar in all groups. It also did not modify levels of serum cholesterol. [125I]Iodo-HDL binding in luteal cells increased from 3.2 +/- 0.3 pg/cell in vehicle-treated rats to 9.9 +/- 0.8 and 7.9 +/- 0.6 after estradiol or testosterone treatment, while the luteal cell content of cholesteryl ester declined from 12.5 +/- 2.0 to 7.7 +/- 0.5 and 8.4 +/- 0.9 micrograms/CL, respectively. Thus, estradiol or testosterone increases luteal cell size but not cell number, depletes cholesteryl ester, and enhances HDL receptor content and progesterone synthesis. These results suggest that one possible mechanism by which estradiol and testosterone stimulate luteal cell steroidogenesis is by increasing the delivery of cholesterol substrate through a receptor-mediated process and by enhancing cholesterol utilization.     

Estrogen regulates low density lipoprotein metabolism by cultured swine granulosa cells

To test estrogen's possible regulation of lipoprotein metabolism by granulosa cells, swine granulosa cells were cultured under serum-free conditions in the presence or absence of estradiol. Treatment with estradiol significantly enhanced high affinity, saturable, [125I]iodo-low density lipoprotein (LDL) binding with a median 2.85-fold (range 2.3- to 5.6-fold, n = six experiments) increase in the calculated number of LDL receptors and no change in the apparent dissociation constant (Kd) for LDL binding (Kd = 3.4 +/- 0.92 micrograms/ml in control and 4.0 +/- 0.87 micrograms/ml human LDL in estradiol-treated cultures). Estradiol also significantly increased [125I]iodo-LDL internalization by granulosa cells and augmented the maximal rate of LDL degradation by 2.0 to 2.5-fold without altering the apparent Michaelis-Menten constant (Km) for this process. Estrogen's dose-dependent enhancement of [125I]iodo-LDL binding, internalization, and degradation could be observed at minimum estradiol concentrations of approximately 100 ng/ml and was accompanied by increased progesterone secretion by granulosa cells. Further studies indicated that estrogen's stimulation of LDL internalization and degradation was not simply attributable to increased rates of nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrrolidone) or increased steroidogenesis per se (tested by blocking cholesterol side-chain cleavage with aminoglutethimide). We conclude that estradiol amplifies LDL binding by swine granulosa cells by increasing the number of high affinity, saturable LDL receptors with no alteration in their apparent affinity. Moreover, estrogen action is accompanied by enhanced rates of progesterone production in the presence of LDL, and increased rates of LDL internalization and degradation, which could not be accounted for simply by accelerated nonspecific bulk fluid-phase pinocytosis. We suggest that the significant facilitative actions of estradiol on lipoprotein binding and metabolism are likely to assist in preparing granulosa cells for the increased rates of progesterone biosynthesis ultimately required in functional corpora lutea.     

In vitro heteroregulation of LH receptors by prolactin and FSH in rat granulosa cells

The purpose of the present study was to further characterize the regulation of LH/hCG receptors by FSH in granulosa cells and test the hypothesis that the LH/hCG receptor levels are heteroregulated by PRL. Granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2-4 days in defined medium containing androstenedione (10(-7) M) and/or FSH and PRL, after which [125I]iodo-hCG binding to the granulosa cells was measured. When granulosa cells were cultured for 2 days (days 0-2) with increasing concentrations of FSH (0.1-100 ng/ml), there was a dose related increase in [125I]iode-hCG binding from a control value of 1.05 +/- 0.2 fmoles/10(6) cells to a maximum of 20 +/- 1.8 fmoles/10(6) cells. The miminum, half-maximum (ED50) and maximum doses of FSH were 0.3, 0.5 and 3 ng/ml, respectively. At concentrations of FSH greater than 3 ng/ml there was a progressive decrease in [125I]-iodo-hCG binding to a low value of 6.1 +/- 1 fmoles/10(6) cells at 100 ng/ml of FSH. No changes in [125I]iodo-hCG binding were observed in response to PRL (1 microgram/ml) during the day 0-2 incubation. When granulosa cells were stimulated for 2 days with 20 ng/ml of FSH, washed, and then recultured for another 2 days (days 2-4) with FSH, the LH/hCG receptor content remained high (F leads to F = 17.4 +/- 2.8 fmoles/10(6) cells). In contrast, when FSH-primed cells were recultured for 2 days without FSH, the [125I]iodo-hCG binding decreased sharply to near control levels (F leads to C = 2.5 +/- 0.2 fmoles/10(6) cells). This marked loss of LH/hCG receptors was largely prevented when FSH primed cells were recultured with PRL (F leads to P = 10.3 +/- 1.5 fmoles/10(6) cells). This stimulatory effect of PRL on [125I]iodo-hCG binding was dose-dependent: minimum, ED50, and maximum doses of PRL were 0.2, 0.5 and 1 microgram/ml, respectively. Scatchard-plot analysis revealed that although the dissociation constant (Kd) of the LH/hCG receptors stimulated by FSH and PRL were of similar high affinity (approximately 8 x 10(-11) M), the maximum binding (Bmax) values in the PRL-treated cells were less. Addition of 10(-7) estradiol together with the PRL did not cause a further increase in Bmax values above that observed with PRL alone.     

125I]hCG binding to bovine thecal tissue from healthy and atretic antral follicles

[125I]hCG binding to thecal tissue from healthy bovine follicles was examined and compared to [125I]hCG binding to other bovine ovarian tissues. [125I]hCG bound specifically to theca interna but not to theca externa. Binding to theca interna was a time- and temperature-dependent process, the rate of association obeying second-order kinetics with calculated rate constants of 1.97 +/- 0.13 X 10(5) and 0.85 +/- 0.04 X 10(5) 1 M-1 sec-1 at 37 and 22 degrees C, respectively. The dissociation of [125I]hCG from theca interna was a slow biphasic process with only 40% of specifically bound [125I]hCG being liberated after 8 h at 37 degrees C. Unlabelled hCG and LH, but not FSH, prolactin, GH, TSH or GnRH, inhibited [125I]hCG binding to theca interna. The specific binding of [125I]hCG to theca interna was saturable and equilibrium binding data produced a linear plot when fitted to the Woolf equation. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) calculated from Woolf plots were 0.21 +/- 0.02 nM (mean +/- SEM) and 34 +/- 4 fmoles/mg protein, respectively. Constants for [125I]hCG binding to granulosa cells and luteal tissue, respectively, were 0.29 +/- 0.02 and 0.31 +/- 0.04 nM for the Kd values and 32 +/- 6 and 116 +/- 13 fmoles/mg protein for the Bmax values. [125I]hCG binding constants for small (less than 8 mm dia.) and large (greater than or equal to 8 mm dia.) follicles (healthy or atretic) were not significantly different. In addition, there was no difference in the [125I]hCG binding constants of healthy and atretic follicles (large or small).(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of gonadotropins and testosterone in progesterone production by human ovarian granulosa cells

Granulosa and theca cells obtained from patients were isolated and cultivated in a chemically defined medium containing gonadotropins and/or testosterone. Progesterone secretion by granulosa cells was consistently stimulated (2-40-fold) in all 5 patients by the addition of follicle-stimulating hormone (FSH, 0.25 micrograms/ml). In the presence of testosterone (0.5 micro M) alone, progesterone production was stimulated (2-8-fold) in 4 out of the 5 patients and cells of one patient showed a greater response to testosterone than to FSH alone. In 2 of the 5 patients, it was also noted that FSH and testosterone acted in a synergistic manner to stimulate the production of progesterone by granulosa cells. On the other hand, human chorionic gonadotropin (hCG, 1.0 IU/ml) alone failed to exert any significant effect. None of the treatments examined altered the production of progesterone by theca cells. These results suggest a role for FSH and testosterone in regulating progesterone biosynthesis by granulosa cells of the human ovary during follicular development.     

Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory Graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin

Human granulosa-luteal cells were harvested from preovulatory Graafian follicles at the time of oocyte retrieval for in vitro fertilization after induction of follicle maturation by sequential injections of menopausal gonadotropins and hCG. Such highly differentiated granulosa cells produced large quantities of progesterone basally (6.8 pg/cell X 2 days) in monolayer culture. Human LH significantly increased progesterone biosynthesis after 6, 12, 48, 96, or 144 h in culture, with a maximal increase of 8- to 20-fold occurring at 96 h. The stimulatory effect of LH could be observed under serum-free conditions and was maximal in the presence of 4% serum. Human granulosa-luteal cells also exhibited significant stimulatory responses to hCG, prostaglandin E2, or the cAMP effectors 8-bromo cAMP, choleratoxin, or forskolin in serum-free incubations. Concentrations of 17 beta-estradiol that are attained physiologically in ovarian follicles in vivo markedly suppressed basal and LH (or cAMP)-stimulated progesterone production in vitro (maximal suppression, greater than 90%). The nonaromatizable androgen 5 alpha-dihydrotestosterone also inhibited progesterone production, but by no more than 45-50% even at supraphysiological concentrations. Estradiol's blockade of progesterone synthesis was associated with a corresponding increase in pregnenolone accumulation. The present studies indicate that human granulosa-luteal cells isolated from preovulatory follicles induced with exogenous gonadotropins and hCG secrete large quantities of progesterone in vitro. Such cells retain stimulatory responses to human LH, hCG, prostaglandin E2, and classical cAMP effectors in serum-free incubations. Moreover, physiological concentrations of 17 beta-estradiol suppress progesterone production, probably by inhibiting cellular conversion of pregnenolone to progesterone. Thus, the present in vitro system permits an investigation of hormone action in well differentiated, human granulosa-luteal cells isolated from preovulatory Graafian follicles that have a defined endocrine history of prior gonadotropin exposure in vivo.     

Independence of steroidogenic capacity and luteinizing hormone receptor induction in developing granulosa cells.

The relationship between FSH-induced acquisition of LH/hCG receptors and the steroidogenic capacity of granulosa cells from estrogen-primed hypophysectomized rat ovaries has been examined. Granulosa cells harvested from the immature preantral follicles of animals not treated with FSH (controls) displayed negligible specific human [125I]iodo-hCG binding and produced only minimal amounts of progesterone during 48 h of culture in vitro. Addition of highly purified hFSH or prostaglandin-E2 (PGE2) to the culture medium elicited substantial increases in progesterone production which were not accompanied by measurable increases in [125I]iodo-hCG binding. Treatment with oFSH in vivo for 24 h led to the initiation of antrum formation in many follicles and was accompanied by an 8-10-fold increase in hCG binding by freshly isolated granulosa cells. Basal, hFSH-, and PGE2-stimulated progesterone production during culture was also greater than controls. In contrast, cells from animals receiving oFSH in vivo for only 12 h showed no increase in hCG binding either before or after culture, yet basal and stimulated progesterone production in vitro was significantly greater than controls, indicating that the initiation of steroidogenesis was antecedent to LH/hCG receptor induction. Only those cells obtained after the 24-h in vivo treatment with oFSH produced elevated amounts of progesterone when incubated in the presence of hCG, thereby showing that the observed increases in [125I]iodo-hCG binding reflected the induction of functionally active LH/hCG receptors. Pharmacological stimulation of steroidogenesis by cell suspensions with N,O'-dibutyryl cAMP resulted in consistently high levels of progesterone production irrespective of previous treatment with FSH in vivo. This uniform expression of in vitro steroidogenic capacity occurred in the complete absence of measurable increases in LH/hCG receptors, suggesting that these two fundamental developmental processes are independent phenomena which may be under separate regulation in vivo.     

Inhibition by estradiol-17 beta of porcine thecal androgen production in vitro.

Thecal preparations from medium-sized procine ovarian follicles (3.5-5 mm diameter) were incubated for 4 h in a chemically defined medium in the presence or absence of highly purified luteinizing hormone (LH) and/or estradiol-17 beta (estradiol). LH (1 microgram/ml) stimulated the thecal production of testosterone (T) and dihydrotestosterone (DHT) by 2- to 3-fold. Although estradiol (10 microgram/ml) alone had only a slight but non-significant inhibitory effect on basal testosterone production, it significantly inhibited the production of both T and DHT as well as decreasing the DHT/T ratio in a dose-related manner in the presence of LH. Production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone by the thecal cells was stimulated 50-to 200-fold and 2.5-fold, respectively, by LH. Estradiol had no significant effect on thecal cAMP and progesterone production in the presence or absence of the gonadotropin. These findings are consistent with the concept that estradiol produced by granulosa cells following hormonal stimulation may serve as a local negative feedback mechanism to control thecal androgen production.     

Effects of 2-hydroxyestradiol on the number of granulosa cell beta-adrenergic receptors

Recent studies have shown that 2-hydroxyestradiol (2-OH-E2) synthesized locally from estradiol (E2) stimulates progesterone production by granulosa cells (GC) and enhances the action of other trophic hormones. A particularly strong synergistic interaction between 2-OH-E2 and beta-adrenergic agonists was observed. Therefore, the present studies were undertaken to determine if this synergism was due to a 2-OH-E2-stimulated increase in the number of beta-adrenergic binding sites in GC. Binding of the 125I-labeled beta-adrenergic ligand iodocyanopindolol ([125I]iodo-CYP) to GC was evaluated and was found to be saturable with time and ligand concentration, and of high affinity (Kd = 29 +/- 8 pM; maximum binding = 0.55 +/- 0.02 fmol/10(6) cells). Rank order potency for agonist/antagonist binding was consistent with a beta-adrenergic receptor: propranolol greater than isoproterenol greater than norepinephrine. 2-OH-E2 did not compete for the [125I]iodo-CYP-binding sites. Incubation of cultured porcine GC with 2-OH-E2 caused a time- and dose-dependent increase in the number of specific [125I]iodo-CYP-binding sites. Averaged over eight separate experiments, 4-day treatment with 4 micrograms/ml 2-OH-E2 increased the number of [125I]iodo-CYP-binding sites 3.1 +/- 0.9-fold above the control value (P less than 0.05). A similar 4-day treatment with 2 micrograms/ml E2 or 200 ng/ml LH or FSH was without effect on [125I]iodo-CYP binding (P greater than 0.05) despite stimulation of progesterone secretion to a degree similar to that seen with 2-OH-E2. These results support the hypothesis that 2-OH-E2 produced by GC may enhance progesterone production stimulated by catecholamines, in part by increasing the numbers of beta-adrenergic binding sites on GC.     

Divergent responses by human and mouse thyroids to human chorionic gonadotropin in vitro

hCG is a known stimulator of mouse thyroid in vivo. Studies were therefore performed to ascertain whether the thyroid-stimulating activity of hCG in the mouse could also be demonstrated by the in vitro techniques that had failed to show any activity of hCG in the human thyroid. When labeled with 125I and incubated at 22 degrees C in 20 mM Tris-0.5% bovine serum albumin (Tris-BSA), pH 7.45, with increasing concentrations (70-300 micrograms protein/ml) of a mouse thyroid fraction, a purified hCG preparation [( 125I]hCG) showed 5-12% specific binding. In contrast, its binding to a human thyroid particulate fraction, over the same range of protein concentrations, did not exceed 1%. When similar studies were performed at 37 degrees C in 10 mM Tris-50 mM NaCl-0.5% BSA, pH 7.45, [125I]hCG showed no detectable binding either to the human or the mouse thyroid fractions. At concentrations ranging from 1 to 20 mIU/ml (0.9-18 X 10(-9) M), bTSH stimulated cAMP release from human thyroid slices into the medium in a dose-dependent manner. In contrast, hCG concentrations from 10(3) to 10(4) IU/ml (2-20 X 10(-6) M) were without effect on cAMP release. bTSH, at concentrations of 4.5 and 9.0 mIU/ml (4 and 8 X 10(-9)M), stimulated cAMP release from the mouse thyroid, producing in the medium approximately 11- and 28-fold increases in cAMP concentration. hCG also stimulated cAMP release from the mouse thyroid, the increases being approximately 2.3- and 1.8-fold, in the presence of 2270 and 4540 IU/ml (4.5 and 9.0 X 10(-6) M), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)