Progesterone Accumulation Research Articles

Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of Rana pipiens.

Pituitary homogenates (FPH) provoke a cascade of responses in the amphibian ovarian follicle, culminating in progesterone biosynthesis and oocyte maturation (GVBD). Calcium may play an important role as an intracellular second messenger in regulating these physiological responses. Experiments were carried out on cultured, isolated follicles of Rana pipiens to assess the effects of varying extracellular calcium on follicular progesterone accumulation and oocyte maturation. In hormonally unstimulated follicles, an increase in extracellular Ca2+ alone produced a significant increase in progesterone in methanol extracts of follicles after 4 hours of culture, and in some cases also provoked oocyte maturation assessed after 24 hours of culture. In no case did elevated Ca2+ alone stimulate maximal progesterone accumulation as compared with FPH-stimulated follicles, although the time-course of accumulation was similar. The calcium ionophore, A-23187, similarly increased progesterone accumulation in a dose-dependent manner when introduced in amphibian Ringer's (1.35 mM Ca2+), but inhibited progesterone elevation caused by increasing calcium concentrations in the culture media and FPH stimulation. Depleting free calcium from the culture medium with graded doses of the chelator EGTA decreased FPH-induced progesterone accumulation and inhibited FPH- and progesterone-induced GVBD. The calcium channel blocker, verapamil, also inhibited FPH-induced progesterone accumulation and GVDB in a dose-dependent manner, while having no effect on progesterone-induced meiotic resumption. These data strongly implicate intracellular calcium levels regulating progesterone production by ovarian follicle cells and subsequent oocyte maturation.

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians ( Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian ( Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0–3 μ M), cAMP (0–3 m M), or dibutyryl cAMP (dbcAMP, 0–2.5 m M) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 m M) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 m M) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0–3 μ M) and IBMX (0-0.09 m M) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 m M) and IBMX (0.09 m M). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.

Protein synthesis involvement in regulating pituitary-induced progesterone levels in ovarian follicles of Rana pipiens.

Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production and/or accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated. Intrafollicular progesterone produced by FPH is considered to mediate oocyte maturation; thus, cycloheximide may interfere with production and/or action of progesterone. Simultaneous treatment of FPH-stimulated follicles with cycloheximide inhibited FPH-induced progesterone accumulation (measured by RIA) and the accompanying-GVBD in a dose-dependent fashion. Inhibitory effects of cycloheximide on either FPH-induced progesterone production or GVBD were not reversed when follicles were washed and returned to fresh medium devoid of FPH and cycloheximide. However, subsequent restimulation of washed follicles with FPH resulted in increased progesterone levels and oocyte maturation. The extent of reversibility, in terms of GVBD and progesterone production, after FPH restimulation varied between animals. Pretreatment of follicles with cycloheximide for 6 hours, without FPH, had little or no effect on progesterone production when follicles were washed and treated with FPH. Delayed addition of cycloheximide to follicles following FPH stimulation blocked further progesterone accumulation as indicated by measurement of intrafollicular progesterone at the time of cycloheximide addition and at the end of the incubation period. The results indicate that cycloheximide rapidly inhibits progesterone production and that continuous protein synthesis is required for progesterone accumulation. Furthermore, protein synthesis does not appear to be required for progesterone metabolism since intrafollicular progesterone declined with prolonged culture even in the presence of cycloheximide. The nature of protein(s) involved in follicular progesterone production remains to be elucidated. FPH mediation of oocyte maturation within ovarian follicles appears to depend upon protein synthesis in somatic follicle cells, which is required for progesterone production, and in the oocyte, to mediate the response to the steroid trigger.

Androgen modulation of follicle-stimulating hormone-induced granulosa cell steroidogenesis in the primate ovary.

The role of androgen in regulating FSH-induced steroidogenesis in primates was investigated in granulosa cell cultures from reproductively suppressed (acyclic) marmoset monkeys. Progesterone accumulation and induction of aromatase activity were measured during a 48-h culture of granulosa cells (isolated from 0.5-1.0 mm diameter follicles) in medium 199 containing human (h) FSH and/or various sex steroids. Steroidogenesis in control cultures was minimal, but the presence of hFSH (0.3-100 ng/ml) caused dose-dependent stimulation. Maximal responses (mean +/- SE) were observed with 30 ng/ml of hFSH (aromatase, 1.0 +/- 0.2 pmol estradiol/10(3) cells X 3 h; progesterone, 4.5 +/- 0.8 pmol/10(3) cells X 48 h) and were 100 times basal values. The presence of testosterone (10(-6)M) during the 48-h culture enhanced the responses to hFSH two- to six-fold over the range 0.3-3.0 ng hFSH/ml. In the presence of a submaximal stimulatory dose of hFSH (3 ng/ml), the effects of testosterone on granulosa cell steroidogenesis were dose-related. Maximum responses were obtained with doses of testosterone between 10(-8) and 10(-7)M. Similar dose-dependent effects were found with 5 alpha-dihydrotestosterone (a non-aromatizable androgen), but not with estradiol, suggesting specific androgen synergism with FSH. Maximal aromatase activity induced after in vitro treatment with hFSH approached that in granulosa cells freshly isolated from a preovulatory follicle of a cyclic animal. These results demonstrate steroid modulation in vitro of FSH-responsive function, similar to that observed in rodent granulosa cells. Therefore, androgen may play a local role in the regulation of FSH-stimulated granulosa cell function during follicular development in primates.

Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.

The mechanisms by which phorbol ester inhibits LH stimulation of progesterone production in rat granulosa cells.

Tumor-promoting phorbol esters are believed to affect cell functions by activating a Ca+2-and lipid-dependent protein kinase (protein kinase C). 12-0-Tetradecanoyl phorbol-13-acetate (TPA) inhibits LH stimulation of progesterone (P) accumulation in rat granulosa cells. To determine the mechanisms by which TPA inhibits LH stimulation of P accumulation, TPA regulation of various ovarian steroidogenic enzymes was investigated. Cells were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h. TPA decreased the P responses to LH, cholera toxin, and (Bu)2cAMP by 20%, 24%, and 28%, respectively. One locus of inhibition of LH action, therefore, was after cAMP. TPA decreased LH-stimulated 3-beta-hydroxysteroid dehydrogenase activity. Furthermore, TPA stimulated 20-alpha-hydroxysteroid dehydrogenase activity. The combination of TPA and A23187 inhibited LH-stimulated P accumulation to the same extent as TPA alone. These data suggest that TPA-induced decreases in LH-stimulated P production result from both the inhibition of P biosynthesis and the stimulation of P breakdown.

Mechanisms of Action of Gonadotropin Releasing Hormone on Rat Granulosa Cells

To elucidate the mechanisms of stimulatory actions of GnRH on rat granulosa cells (GC), we have compared the actions of a GnRH agonist with those of a tumor-promoting phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA) and Ca+2 ionophore, A23187. GC were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG. Following prelabeling with 3[H]arachidonic acid (AA), the cells were incubated with the test substances for 10 min and AA release determined. A GnRH agonist, [D-Ala6, des-Gly-NH2(10)] GnRH ethylamide (GnRHa; 10 ng/ml) increased AA release 175% compared to the control value. AA release in the presence of GnRHa was larger than that due to 1 microM A23187 or 40 nM TPA alone. A23187 or TPA increased GnRHa-stimulated AA release further. GC were incubated with the test substances for longer time periods, i.e., up to 5 h. GnRHa caused a 4-fold increase in prostaglandin (PG) synthase activity at 5 h. GnRHa increased PGE accumulation to the same extent as TPA, but only increased PG synthase activity about half as much. In combination with TPA, GnRHa had no influence on TPA-stimulated PG synthase activity, but increased PGE accumulation to levels comparable to those with A23187 plus TPA. GnRHa caused a 2.5 fold increase in progesterone (P) accumulation, which was the same as TPA. P accumulation in the presence of GnRHa was affected by neither A23187 nor TPA. These data indicate that the combination of TPA and A23187 can substitute for GnRH action on PGE and P accumulation in rat GC.

Mechanisms subserving the bipotential actions of estrogen on ovarian cells: Studies with a selective anti-estrogen, LY156758, and the sparingly metabolizable estrogen agonist, moxestrol

Estrogen exerts biphasic effects on progesterone biosynthesis by swine granulosa cells, such that initial transient inhibition is followed by delayed but sustained stimulation. We have tested the functional role of the estradiol receptor in these biphasic responses by utilizing the highly selective estrogen-receptor antagonist, LY156758, and the synthetic estrogen agonist, moxestrol. The acute inhibitory action of estradiol was mimicked in a dose-dependent action by moxestrol (half-maximally inhibitory dose: 54.3 ± 25 ng/ml), but was not antagonized by LY156758. Rather, the antiestrogen alone significantly suppressed basal progesterone synthesis, and accentuated the suppressive effect of submaximally inhibitory doses of estradiol. Inhibition was accompanied by increased pregnenolone accumulation, with a consequently augmented ratio of pregnenolone to progesterone. Moreover, in cell-free sonieates of granulosa cells, LY156758 directly inhibited 3β-hydroxysteroid dehydrogenase activity in a dose-dependent fashion, with half-maximal inhibition expressed at a drug concentration of 2.44 ± 0.31 μg/ml compared with 85 ± 19 ng/ml for estradiol. In addition, the combination of LY156758 and submaximally inhibitory doses of estradiol resulted in further suppression of 3β-hydroxysteroid dehydrogenase activity. The sustained stimulatory phase of estrogen action was also mimicked by moxestrol in a dose-dependent fashion. However, in contrast to its acute inhibitory effects, longer-term treatment with LY156758 slightly enhanced basal progesterone accumulation, and effectively antagonized estradiol's stimulatory actions. In summary, our results with moxestrol demonstrate that both the inhibitory and the stimulatory actions of estradiol are effectively mimicked by this synthetic estrogen agonist. Results with the selective anti-estrogen LY156758 indicate a small degree of intrinsic estrogen agonist activity (approx 4% that of estradiol), which is reflected by its acute and direct inhibition of 3β-hydroxysteroid dehydrogenase activity. However, under longer-term conditions in which estradiol's stimulation of progesterone production is expressed, LY156758 significantly antagonizes estradiol's trophic actions. Accordingly, we suggest that the acute suppressive effects of estradiol on progesterone production are mediated predominantly by direct inhibition of 3β-hydroxysteroid dehydrogenase activity, while delayed stimulatory effects are transduced via estrogen-receptor mechanisms. We conclude that the disparate effects of estrogen on granulosa-cell progesterone biosynthesis are dissociable under in vitro conditions and expressed via distinct and separable mechanisms.

Differentiation of rat ovarian thecal cells: evidence for functional luteinization.

To determine if thecal cells of rat preovulatory (PO) follicles become functionally luteinized, theca from small antral (SA) and PO follicles were isolated before and 8 h after iv injection of an ovulatory dose (10 IU) of hCG. Thecal explants were cultured for 30 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium containing 1% fetal calf serum (FCS) with or without 5 ng/ml ovine LH or 10 microM forskolin. Whereas theca from SA, hCG-treated SA, and PO follicles were dependent on LH or forskolin to maintain progesterone (greater than 10 ng/ml) and androstenedione (greater than 10 ng/ml) accumulation, luteinizing theca (hCG-treated PO) accumulated more than 10 ng/ml progesterone and more than 2 ng/ml androstenedione with or without LH or forskolin for 30 days. Granulosa cells were isolated from these same follicles and cultured under similar conditions, including 10 ng/ml testosterone and 25 ng/ml ovine FSH. Only granulosa cells isolated from luteinizing follicles (hCG-treated PO) maintained progesterone (greater than 20 ng/ml) and estradiol (10 ng/ml) accumulation with or without FSH or forskolin for 30 days. Basal concentrations of cAMP were 5 to 10-fold higher in thecal and granulosa cells from luteinizing follicles than in these tissues isolated from SA or PO follicles. We conclude that thecal cells as well as granulosa cells of rat PO follicles respond to the LH/hCG surge by becoming functionally luteinized, less dependent on LH, and capable of maintaining an increased accumulation of basal cAMP. Furthermore, the data suggest that one luteinizing thecal explant produces a similar amount of progesterone as one follicle equivalent of luteinizing granulosa cells. Thus, luteinized theca have the potential of contributing significantly to progesterone secretion by the mature rat corpus luteum.

Short term androgen production by rat ovarian follicles and long term steroidogenesis by thecal explants in culture.

To characterize the aromatizable and 5 alpha-reduced androgens produced by developing ovarian follicles, small antral (SA) and preovulatory (PO) follicles, theca and granulosa cells were incubated for 4 h with or without 8-bromo-cAMP and androstenedione. In addition, thecal explants were cultured for 10 days with or without ovine LH (oLH) to determine if the hormone-induced changes in androgen synthesis by developing follicles could be mimicked in vitro. Short term incubations of SA and PO follicles, theca and granulosa cells in medium alone resulted in limited accumulation of androgen [testosterone, 5 alpha-androstan-17 beta-ol-3-one (DHT), 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha diol), and androsterone], as determined by RIA. In the presence of 8-bromo-cAMP, PO follicles produced large quantities of testosterone (3 ng), DHT (1 ng), 3 alpha diol (15 ng), and androsterone (14 ng), while SA follicles accumulated much less androgen (0.69, 0.05, 1.23, and 1.3 ng, respectively). In the presence of androstenedione and 8-bromo-cAMP, both SA and PO follicles and theca produced large amounts of aromatizable and 5 alpha-reduced androgens. SA and PO granulosa cells required the presence of the substrate androstenedione to produce androgens, primarily testosterone and 3 alpha diol. Therefore, progesterone, androstenedione, and 5 alpha-reduced androgens were used to monitor LH action on thecal cell function in culture. Small antral theca cultured in basic culture medium alone (containing 10% fetal calf serum) displayed an increased ability to accumulate androstenedione by day 6, approximately 3 times that observed on day 2. However, a 5-fold further increase in androstenedione accumulation was observed by day 6 for SA theca cultured in the presence of oLH. Maintenance of progesterone accumulation by SA theca throughout the culture period also was dependent on the presence of LH. In contrast, androstenedione accumulation by PO theca required the presence of LH in the culture medium, while progesterone accumulation in these cultures did not. Little or no 5 alpha-reduced androgen accumulated in the media of SA and PO theca cultured in basic culture medium alone. However, SA and PO theca cultured with oLH accumulated approximately 1 ng androsterone by day 10. We conclude that 1) SA and PO follicles, theca and granulosa cells possess the enzymes required to produce large amounts of 3 alpha diol and androsterone; 2) low concentrations of oLH are required to stimulate SA thecal steroidogenesis and to maintain PO thecal androstenedione accumulation in culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotrophic control of steroidogenesis in human granulosa-lutein cells.

Granulosa-lutein cells were harvested from periovulatory follicles in human ovaries and cultured for up to 6 days, equivalent to almost half of a normal luteal phase. The average rate of basal progesterone accumulation in the culture medium was constant at approximately 36 nmol progesterone/10(6) cells/day. Oestradiol accumulation was too low to measure in the absence of precursor androgen. Basal aromatase activity (measured as oestradiol formed in 3 h from 10(-6) M exogenous testosterone) was high (average 1.15 nmol oestradiol/10(6) cells/3 h) at the time of cell isolation (Day 0) but fell by greater than 90% on Day 1. By Day 2 the activity had partly recovered and averaged 62% of the Day 0 value, rising to 70% on Day 6. This loss and recovery of aromatase activity was independent of the addition of gonadotrophic hormones to the culture medium. However, dose-related increases in aromatase activity occurred in the presence of highly pure human pituitary LH (0.1-30 ng/ml). The increase was observed on Day 4 and was maximal on Day 6 (average 3-fold increase over control) in the presence of LH concentrations greater than or equal to 1.0 ng/ml. LH also caused dose-related increases in progesterone accumulation by Day 4 with maximal stimulation on Day 6 (average 3-fold increase over control) at greater than or equal to 10.0 ng/ml. Dose-related stimulation of aromatase activity by human pituitary FSH also occurred but maximal stimulation required the presence of 300 ng FSH/ml and progesterone accumulation was hardly affected at this dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prostaglandins E 2 and F 2α on progesterone metabolism by rat granulosa cells

Rat granulosa cells were cultured with or without PGE 2 and/or PGF 2α. Accumulation of endogenous progesterone and 20α-hydroxy-4-pregnen-3-one was determined. Additionally, [4- 14C]progesterone metabolism was assessed. PGE 2 increased progesterone accumulation, in part, by decreasing progesterone catabolism to 20α-reduced progestins. In contrast, PGF 2α stimulated 20α-hydroxysteroid dehydrogenase activity, thus increasing progesterone catabolism. Combined treatment with PGE 2 and PGF 2α augmented progesterone accumulation to levels above controls but below those attained with PGE 2 alone. These data indicate that PGE 2 and PGF 2α exert opposite effects on progesterone production and catabolism and that the ratio of PGE 2 to PGF 2α in the local granulosa cell milieu may be of importance in determining overall progesterone output.

Synergistic interactions of somatomedin-C with adenosine 3',5'-cyclic monophosphate-dependent granulosa cell agonists.

Recent studies have demonstrated the ability of somatomedin-C (Sm-C) to synergize with follicle-stimulating hormone (FSH) in the activation of cultured rat granulosa cell progesterone biosynthesis as well as the induction of luteinizing hormone (LH) receptors. Neither effect could be attributed to Sm-C-enhanced granulosa cell survival or replication, but could be accounted for, in part, by increased adenosine 3',5'-cyclic monophosphate (cAMP) generation. The present study was undertaken to determine if the synergistic property of Sm-C is FSH-selective and hence limited in relevance to follicular maturation, as well as to clarify further the role of cAMP in Sm-C-amplified agonist action. To this end, the ability of Sm-C to modulate the hormonal action of a series of physiologic as well as pharmacologic granulosa cell agonists was examined in vitro using cultured granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats. Concurrent treatment with highly purified Sm-C (50 ng/ml) resulted in marked increases over controls in the LH-stimulated [1 ng human chorionic gonadotropin (hCG)]-and beta 2-adrenergic-stimulated (10(-6) M terbutaline) accumulation of cAMP (3.8- and 2.6-fold, respectively and progesterone (3.2- and 7.4-fold, respectively). Similarly, concurrent treatment with Sm-C also augmented the vasoactive intestinal peptidergic stimulation of granulosa cell cAMP generation (4.1-fold) and progesterone biosynthesis (2.1-fold). In contrast, Sm-C was incapable of enhancing progesterone accumulation in response to stimulation with rat prolactin, a cAMP-independent granulosa cell agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Purines, prostaglandins and peptides--nature and cellular mechanisms of action of local assist and assassin agents in the ovary.

The evidence for a paracrine, progonadotropic role of adenosine in ovarian cells is summarized along with a capsule review of the origin and mechanisms of release and action of adenosine in other tissues. Briefly, adenosine markedly amplified rat and human luteal cell cyclic AMP and progesterone accumulation in the presence, but not the absence, of LH. The site of action of adenosine was found to be intracellular, linked to its phosphorylation, which resulted in increased levels of ATP. In rat luteal cells, adenosine blocked the acute antigonadotropic (luteolytic) action of PGF2 alpha. In the follicle, adenosine release from granulosal cells appeared to be stimulated by FSH. Adenosine and a nonmetabolized adenosine analog, augmented FSH-dependent inhibition of oocyte maturation in the presence or absence of an adenosine transport inhibitor. Inhibition of oocyte maturation by adenosine thus appears to be mediated by extracellular purinergic receptors. Paracrine, antigonadotropic agents also appear to regulate ovarian function. For example, GnRH elicits antigonadotropic activity in rat granulosal and luteal cells. We describe a novel, GnRH-like, ovarian hormone (GLOH) which may be the physiological ligand whose action GnRH mimics in rat ovarian cells. This protein was shown to be distinctly different from GnRH and a variety of other cyclic and noncyclic peptides. PGF2 alpha is a well known leutolytic agent and a summary of the antigonadotropic mechanism of PGF2 alpha action in rat luteal cells is presented. In these cells, the action of GnRH (or possibly the GnRH-like protein) and PGF2 alpha are mediated by separate membrane receptors but they appeared to share the same intracellular second messenger. Evidence for a role of products of phosphoinositol as a mediator of these antigonadotropic agents is summarized. We suggest that the ultimate mediator of antigonadotropic agents is Ca2+ which is released in the luteal cell in response to the intracellular mediator of antigonadotropic agents. For example, pharmacological agents which increase intracellular levels of Ca2+, mimicked the antigonadotropic action of GnRH and PGF2 alpha in rat luteal cells. Also, Ca2+ directly inhibited LH-sensitive adenylate cyclase activity in isolated luteal membranes, a paradigm in which GnRH and PGF2 alpha were inactive. The mechanism of Ca2+ action appeared to be linked to interference with GTP activation of adenylate cyclase. However, removal of extracellular Ca2+ did not abrogate the action of either GnRH or PGF2 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone secretion by primary cultures of rat luteal cells.

Primary cultures were prepared from ovaries of immature rats that had been superovulated. The dispersed luteal cells attached to growth surfaces, formed monolayers and secreted progesterone. Progesterone accumulation in the medium was most pronounced in the first week of culturing. Removal of serum from the medium resulted in a progressive decline in progesterone concentration in the culture medium which reached basal levels by 5 hours. In medium without serum, the addition of hCG, FSH or prolactin stimulated an increase in progesterone secretion within 1 hour. Also cholera toxin stimulated a significant increase in progesterone levels in the medium. Prior exposure of cultures to estradiol for 3 days did not augment the response to hCG and inhibited the ability of cholera toxin to stimulate progesterone secretion. These results indicate that the steroidogenic function of rat luteal cells can be studied in culture and that a number of hormones rapidly stimulate the secretion of progesterone from these cells.

Absence of LHRH effect on testosterone production by Leydig cells from fetal mouse testis

The effect of LHRH and one of its agonist (des-gly 10 ( d-Ala 6)-LHRH-ethylamide) on the functional activity (testosterone and progesterone production) of purified fetal mouse Leydig cells was examined in short-term primary culture and under dynamic conditions. The continuous presence of increasing concentrations of LHRH (10 −10 to 10 −6 M) for 3 days was unable to affect the hCG-stimulated testosterone production on any day of culture. Stimulated testosterone production progressively decreased from day 1 to day 3 of culture ( P < 0.001). Progesterone accumulation increased in both basal and hCG stimulated conditions during the same period ( P < 0.001) and was not altered by the presence of LHRH at all three concentrations tested. There was no effect of LHRH pretreatment either on the basal production or on the acute hCG stimulation studied during a subsequent 6 h incubation. Exposure of cells to hCG for 120 min enhanced testosterone accumulation. No change in kinetic characteristics was observed when LHRH (10 −6 M) was continuously present in the medium. These results show that LHRH does not have any detectable effect on the fetal population of Leydig cell in the mouse.

Prolactin effects on basal and gonadotrophin-stimulated progesterone accumulation by PMSG-primed mouse ovaries in vitro.

To examine the effects of prolactin (Prl) and human chorionic gonadotrophin (hCG) on progesterone production by murine ovarian explants, immature female mice were injected with 4 IU pregnant mare's serum gonadotrophin (PMSG) to induce follicular maturation. After 24 or 40 h mice were killed, ovaries removed, cut into fragments and maintained as explants for 24 h in the presence or absence of ovine or human Prl (25-2500 ng/ml). None of these doses of Prl affected basal progesterone accumulation into media over 24 h. To determine if Prl could modify the capacity of ovarian explants to respond to gonadotrophin, ovaries were incubated with 25 IU/ml hCG for 3 h after an initial 24 h incubation period with or without Prl. Prl had no effect on basal progesterone accumulation but significantly enhanced hCG-stimulated progesterone accumulation during the 3 h incubation period. We conclude that Prl does not inhibit but may enhance progesterone secretion by pre-ovulatory follicles in the mouse.

Use of antisera to follicle-stimulating hormone (FSH) to detect non-FSH factors in human serum which modulate rat granulosa cell steroidogenesis.

We measured the ability of serum from women to stimulate steroidogenesis in cultured granulosa cells. Serum promoted estradiol and progesterone synthesis in proportion to its FHS content measured by RIA [i.e. serum from postmenopausal women (PM) greater than serum from the midcycle at the time of the gonadotropin surge (MC) greater than serum from the first day of the menstrual cycle (D1) greater than serum from a hypophysectomized woman (AP)]. The FSH activity of these sera was reduced but not eliminated when we included excess antisera to ovine or human FSH in the culture medium (i.e. PM greater than MC greater than D1 greater than AP). These antisera completely neutralized the actions of ovine FSH, human FSH, and menopausal gonadotropin (Pergonal) added to serum. In contrast to the stimulation seen with 5% or lower concentrations of serum in the culture medium, we observed that 10-20% serum inhibited FSH-induced androgen aromatization and progesterone accumulation. The degree of stimulation or inhibition of steroidogenesis depended on the number of granulosa cells added to each culture. High initial cell concentrations inhibited the ability of the cells to respond to either serum or PMSG. In addition to factors which stimulate or inhibit FSH-induced steroidogenesis, human serum contains factors distinct from FSH which cause the cells to flatten and adhere more tightly to the culture dishes. Although progesterone synthesis was increased in cells which had flattened on the surface of the culture dishes, this phenomenon was not a prerequisite for serum-induced steroidogenesis. We conclude that serum contains factors immunologically distinct from FSH, possibly of pituitary origin, which induce granulosa cell steroidogenesis. In addition, serum contains inhibitory substances which block hormone-induced steroidogenesis and which tend to obscure the stimulatory effects of FSH. Detection of both factors depends in part on the number of granulosa cells used to innoculate the cell cultures.

Somatomedin-C Synergizes with Follicle-Stimulating Hormone in the Acquisition of Progestin Biosynthetic Capacity by Cultured Rat Granulosa Cells*

We have recently shown that nanomolar concentrations of somatomedin-C (Sm-C), are capable of enhancing the FSH-mediated (but not basal) accumulation of progesterone (Po) by cultured rat granulosa cells. To further characterize this direct cytodifferentiative effect of Sm-C, granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats were cultured under serum-free conditions for up to 96 h. Concurrent treatment with highly purified Sm-C (50 ng/ml) produced 10.2- and 3.6-fold increments in the FSH (20 ng/ml)-stimulated accumulation of Po and 20 alpha-hydroxy-4-pregnen-3-one, respectively. Sm-C-augmented Po biosynthesis was dose- and time dependent, but was independent of the FSH dose employed. Significantly, this effect of Sm-C could not be accounted for by enhancement of cellular viability or plating efficiency, nor by an increase in the number of cells, or their DNA synthesis. Furthermore, specific inhibition of DNA synthesis with cytosine-1-beta-D-arabinofuranoside was without significant effect on the ability of SM-C to enhance FSH-supported Po biosynthesis. Insulin, like Sm-C, also synergized with FSH in the induction of Po biosynthesis. However, insulin [ED50 = 19.2 +/- 1.6 (SE) micrograms/ml] was approximately 4800-fold less potent than Sm-C [ED50 = 4.0 +/- 0.3 (SE) ng/ml] in this regard, and exerted little or no effect at concentrations presumed to saturate the putative high affinity granulosa cell insulin receptor. Although maximal stimulatory doses of Sm-C (75 ng/ml) or insulin (100 micrograms/ml) produced comparable increments in FSH-supported Po biosynthesis, combined treatment with maximal doses of both peptides did not prove additive. Pertinently, the direct cytodifferentiative effect of Sm-C is exerted at (nanomolar) concentrations compatible with its receptor-binding affinity as observed in all other cell types studied. Thus, Sm-C is not likely to be acting through the putative high affinity insulin receptor but rather through its own high affinity recognition sites. Similarly, the cytodifferentiative action of high dose insulin may reflect the consequences of its cross-interaction with the putative Sm-C, rather than the insulin receptor. These findings are in keeping with the suggestion that the granulosa cell may be the site of Sm-C reception and action and that Sm-C of intraovarian or circulatory origin may participate in the differentiation, as well as replication, of the developing granulosa cell.

Intrafollicular action of estrogen in regulating pituitary-induced ovarian progesterone synthesis and oocyte maturation in Rana pipiens: Temporal relationship and locus of action

Previous studies demonstrated that estrogen inhibited frog pituitary homogenate (FPH) and progesterone-induced oocyte maturation. In order to determine whether estrogen interfered with intrafollicular progesterone synthesis, experiments were designed to study the effect of exogenous estrogen (estradiol-17β) on FPH-induced progesterone production in amphibian ( Rana pipiens) ovarian follicles cultured in vitro. Intrafollicular progesterone concentrations were monitored directly using radioimmunoassay and the occurrence of germinal vesicle breakdown (GVBD) was used as a biological indicator of the action of steroids on oocyte maturation. FPH elicited a rapid and dramatic increase in follicular progesterone concentration (1000–3000 pg per follicle) which preceded germinal vesicle breakdown. Addition of estrogen to the culture medium inhibited FPH-induced progesterone production and the accompanying GVBD in a dose-dependent fashion. The presence of estrogen did not enhance the degradation of preloaded progesterone, suggesting estrogen impeded progesterone synthesis rather than enhanced progesterone metabolism. The temporal relationship between estrogen and FPH interaction was assessed by varying the relative time of hormone addition, after which intrafollicular progesterone concentration and GVBD were monitored. Progesterone production and GVBD were drastically inhibited when estrogen was added before or simultaneously with FPH. However, when addition of estrogen was delayed until after FPH simulation, a progressive loss of the inhibitory effect of the steroid on progesterone accumulation and GVBD was observed. Thus, the estrogensensitive phase was confined to the early portion of FPH stimulation. Continuous presence of estrogen in the culture system was not required to inhibit FPH-induced events. A short exposure (15 min) of follicles to estrogen was sufficient to inhibit oocyte maturation, whereas progesterone synthesis was not significantly affected. With longer exposure, however, FPH-induced progesterone production was impeded. Washing estrogen-treated follicles did not reverse the inhibitory effect of estrogen, however the follicles remained responsive to exogenous progesterone stimulation and exhibited GVBD. Results suggest that the inhibitory effects of estrogen on FPH action and progesterone production were not reversible under the in vitro culture conditions. To determine whether specific follicular components were involved in estrogen inhibition, progesterone production was assessed following selective removal of different follicle components by microdissection prior to being treated with FPH and estrogen. Removal of the follicular epithelium, theca layer, or the cytoplasm (yolk) of the oocyte did not interfere with estrogen inhibition of progesterone production. The data suggest that the locus of action of estrogen, within the ovarian follicle, is directed against the follicle cells and hence these cells may play a pivotal role in regulating the progesterone synthesis and oocyte meiotic maturation. Intrafollicular estrogen levels may thus serve as a physiological regulator of follicular responsiveness to gonadotrophic hormones.