Follicle-stimulating hormone regulates low density lipoprotein metabolism by swine granulosa cells

FSH amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells in a synergistic manner. The mechanisms subserving this synergism included the following. 1) FSH increased by 2.1-fold the number of specific high affinity low density lipoprotein (LDL) receptors on granulosa cells, with no change in apparent binding affinity. 2) FSH augmented by 1.8- and 3.6-fold the maximal rates of [125I]iodo-LDL internalization and degradation, respectively, without altering half-maximally effective concentrations of LDL supporting these processes. 3) FSH increased significantly the mass (micrograms) of free and esterified cholesterol (measured by fluorometry) contained in granulosa cells. 4) FSH stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester from exogenous [3H]cholesteryl linoleate-labeled LDL and amplified [3H]progesterone secretion by granulosa cells exposed to this source of lipoprotein-borne sterol. 5) The stimulatory effects of FSH on LDL degradation and cholesterol side-chain cleavage activity were mimicked by other activators of granulosa cell cAMP production, e.g. forskolin and cholera toxin. We conclude that FSH and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via cellular mechanisms that result in significantly augmented binding, internalization, and degradation of LDL in concert with increased effectual utilization of sterol substrate in the cholesterol side-chain cleavage reaction. These observations are consistent with a key trophic role for FSH in regulating the sterol-metabolizing ability and steroidogenic differentiation of granulosa cells and indicate that such actions of FSH can be accounted for by activation of the cAMP-effector pathway.     

Insulin regulates low density lipoprotein metabolism by swine granulosa cells

Insulin synergistically amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells. The mechanisms subserving this facilitative interaction included the following: 1) insulin's synergism with LDL was profoundly attenuated by covalent modification of arginine residues in LDL by 1,2-cyclohexanedione treatment; 2) insulin increased by 2- to 6-fold the number of specific high affinity LDL receptors on granulosa cells, with no change in apparent binding affinity; 3) insulin augmented rates of [125I]iodo-LDL internalization and degradation without enhancing nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrollidone); 4) insulin increased by 2.5- to 3-fold granulosa cell content of free and esterified cholesterol (measured by fluorometry) in response to treatment with unlabeled LDL; 5) insulin stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester, and amplified [3H]progesterone secretion by granulosa cells exposed to [3H]cholesteryl linoleate-labeled LDL; and 6) insulin action was specific in that it was not mimicked by desoctapeptide insulin, epidermal growth factor, fibroblast growth factor, or relaxin. We conclude that insulin and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via specific mechanisms that depend upon 1,2,-cyclohexanedione-sensitive residues within LDL apoprotein. Insulin action results in significantly augmented binding, internalization, and degradation of LDL, which is accompanied by increased effectual delivery of cholesterol substrate into cellular sterol pools that participate in enhanced steroidogenesis.     

Insulin-like growth factor type I (somatomedin-C) stimulates high density lipoprotein (HDL) metabolism and HDL-supported progesterone biosynthesis by swine granulosa cells in vitro

Type I insulin-like growth factor (IGF-I) stimulated high density lipoprotein (HDL)-promoted progesterone production by swine granulosa cells cultured under serum-free conditions in vitro. In the presence of pure human IGF-I (50 ng/ml), the half-maximally effective concentration of swine HDL was 16 micrograms/ml (67% confidence limits; 15-17 micrograms/ml) after 2 days of exposure to this growth factor, 5.4 (2.6-9.8) micrograms/ml after 4 days, and 3.8 (1.2-4.8) micrograms/ml after 6 days. Maximal progesterone production increased approximately 10-fold in the presence of IGF-I and HDL on day 2, 125-fold on day 4, and 330-fold on day 6. The facilitative action of IGF-I on HDL-supported progesterone biosynthesis was accompanied by time-dependent stimulatory effects of IGF-I on trypsin-releasable HDL, trypsin-resistant cell-associated HDL, and degraded HDL (P less than 0.01). Moreover, incubation of swine granulosa cells with [3H]cholesteryl oleate-labeled HDL demonstrated that IGF-I exerted a time-dependent stimulatory effect on [3H]free cholesterol and [3H]cholesteryl ester accumulation in granulosa cells, and significantly augmented the secretion of [3H]progesterone (separated by two-dimensional TLC). In addition to the ability of IGF-I to amplify the cellular acquisition of radiolabeled sterol, this growth factor also increased the total mass of cellular cholesteryl ester and total cellular cholesterol as measured by microfluorometric assay (P less than 0.01). We conclude that IGF-I facilitates the effective delivery of HDL-derived sterol substrate into the steroidogenic pool of ovarian cells. Such observations offer an additional role for the differentiative actions of this somatomedin in the expression of full steroidogenic potential by granulosa-luteal cells.     

The role of cholesterol esterification in ovarian steroidogenesis: studies in cultured swine granulosa cells using a novel inhibitor of acyl coenzyme A: cholesterol acyltransferase

We have used a novel competitive inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035 [3-(decyldimethyl-silyl)N-[2-(4-methyl-phenyl)1-phenylethyl propanamide], to assess the importance of the cholesterol esterification reaction in ovarian steroidogenesis. Compound 58-035 markedly (greater than or equal to 96%) inhibited ACAT activity of swine ovarian microsomes in a dose-dependent (0.1-3.5 micrograms/ml) fashion. In addition, treatment of cultured granulosa cells with this fatty acylamide effectively (greater than or equal to 98%) suppressed hormonally stimulated cholesterol esterification, as assessed by the incorporation of [3H]oleic acid into cholesteryl ester. Accordingly, we used this inhibitor to test the role of cholesterol esterification in ovarian cells. In cultures with limited or no serum supplementation, long term (2- to 6-day) treatment of granulosa cells with compound 58-035 significantly increased basal progesterone production and amplified by 2- to 10-fold the stimulatory actions of trophic hormones, such as estradiol, FSH, estradiol combined with FSH, or insulin. The amplifying effect of ACAT inhibition on hormone-stimulated progesterone production could be mimicked by providing exogenous cholesterol substrate in the form of low density lipoprotein (LDL). Cotreatment with compound 58-035 and LDL resulted in no further augmentation of steroidogenesis. In contrast to the facilitative effects of compound 58-035 in longer term cultures, this ACAT inhibitor did not alter progesterone biosynthesis acutely (2-20 h) in swine or hamster ovarian cells. These observations suggest that there is an obligatory partitioning of some sterol into the ester pool in granulosa cells. In times of diminished availability of cholesterol, inhibition of the esterification pathway can make additional cholesterol available for use in steroid hormone biosynthesis. Thus, in the intact Graafian follicle, where LDL cholesterol delivery to granulosa cells and intracellular cholesteryl ester stores are limited, regulation of the ACAT reaction may significantly modulate rates of progesterone biosynthesis. The present results indicate that the use of a selective inhibitor of cholesterol esterification can permit one to probe the functional significance of the esterification reaction in steroidogenic cells.     

Stimulation of progesterone synthesis in luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-adenosine 3',5'-monophosphate: the effect of low density lipoprotein

Primary cultures of luteinized human granulosa cells reduced progestin secretion when taken from serum- and gonadotropin-containing medium into serum- and hormone-free medium. When added individually hCG, 8-bromo-cAMP and low density lipoprotein (LDL) stimulated progestin secretion by the cells after they spent 48 h in serum- and hormone-free medium. However, combinations of hCG or 8-bromo-cAMP and LDL were most effective in increasing steroidogenesis. The effects of hCG in enhancing steroidogenesis in the presence of LDL were first detectable after 3 h, but were most marked after 20 h of culture. hCG and 8-bromo-cAMP increased the conversion of [3H]cholesteryl linoleate, which had been incorporated into the core of LDL, into [3H]progesterone. hCG also stimulated cellular accumulation of LDL cholesterol, as assessed by incorporation of [1-14C]oleic acid into sterol esters or by measurement of total cellular cholesterol in the presence of amino-glutethimide to block steroidogenesis. In contrast to progesterone secretion, estradiol secretion was not affected by the addition of LDL in the absence or presence of 8-bromo-cAMP. We conclude that LDL cholesterol is required for maximal rates of progestin synthesis by human luteinized granulosa cells. When granulosa cells are stimulated by hCG, uptake of LDL cholesterol is promoted, and there is increased utilization of LDL cholesterol for steroid synthesis.     

Synergistic actions of estradiol and the insulin-like growth factor somatomedin-C on swine ovarian (granulosa) cells

Estradiol amplified synergistically the dose- and time-dependent stimulatory actions of human somatomedin-C on progesterone biosynthesis by cultured swine granulosa cells. This facilitative interaction was not attributable to inhibition of the catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one, but, rather, reflected time-dependent stimulation of pregnenolone synthesis measured in the presence of exogenous soluble sterol substrate for cholesterol side-chain cleavage. Moreover, treatment with somatomedin-C was accompanied by increased synthesis of two immunoprecipitable cholesterol side-chain cleavage constituents, viz. cytochrome P-450scc and adrenodoxin. The synergism between estradiol and somatomedin-C was associated with significantly greater specific binding of somatomedin-C in estrogen-treated than control cultures, with no change in apparent receptor affinity. In vitro synergism occurred at somatomedin-C concentrations estimated by sequence-specific immunoassay to be attainable in ovarian follicular fluid in vivo and was specific in that it was not mimicked by the insulin-like peptide relaxin or by epidermal growth factor or fibroblast growth factor. However, high concentrations of insulin-like growth factor II (multiplication-stimulating activity) and insulin were able to interact with estradiol in a facilitative fashion to enhance progesterone production. In addition, the estrogenic component of the synergism was specific, since it was antagonized by the selective antiestrogen LY156758 and was mimicked sparingly by the nonaromatizable androgen 5 alpha-dihydrotestosterone. We conclude that estradiol and somatomedin-C interact synergistically in a time- and dose-dependent manner to enhance the biosynthesis of pregnenolone and progesterone by swine granulosa cells. Since estradiol and somatomedins are present in significant concentrations in the antral fluids of maturing Graafian follicles, we suggest that coordinated trophic effects of estradiol and insulin-like growth factor(s) may effectively prepare granulosa cells for the high rates of progesterone biosynthesis ultimately required after ovulation.     

Bipotential actions of estrogen on progesterone biosynthesis by ovarian cells. II. Relation of estradiol's stimulatory actions to cholesterol and progestin metabolism in cultured swine granulosa cells

Although both inhibitory and stimulatory actions of estradiol on swine granulosa cells have been described, the bases for these inconsistent effects are not clear. We have tested properties of ovarian follicles and in vitro culture conditions that result in consistently stimulatory effects of estradiol on progesterone biosynthesis. Stimulatory actions of estradiol (in contrast to inhibitory effects) were critically dependent upon the density of granulosa cells in culture and the size and maturational status of the parent Graafian follicles. Granulosa cells isolated from small, rather than medium or large sized, swine follicles exhibited the greatest peak response to estradiol, although half-maximally stimulatory concentrations (ED50) of estradiol were similar (mean, 81 ng/ml). Granulosa cells from atretic follicles also secreted increased quantities of progesterone in response to estradiol, but the ED50 for estrogen stimulation was significantly higher (ED50 = 322 ng/ml estradiol) than that of comparable healthy follicles (ED50 = 109 ng/ml). This estrogen-responsive system was used to test the mechanisms subserving estrogen's trophic actions on granulosa cells. Estradiol significantly enhanced the activity of 3 beta-hydroxysteroid dehydrogenase with consequently increased production of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Estrogen also augmented functional cholesterol side-chain cleavage activity in a dose- and time-dependent fashion with a resultant increase in pregnenolone biosynthesis. Moreover, parallel observations documented concordant dose responses for the synthesis of all three major progestins by pig granulosa cells. The trophic actions of estrogen on the steroidogenic pathway were associated with enhanced hydrolysis of endogenous cholesteryl ester stores but were not significantly antagonized by inhibition of de novo cholesterol biosynthesis. We conclude that suitable follicle selection and appropriate in vitro culture conditions provide a consistently estrogen-responsive granulosa-cell system, in which estradiol modulates certain key aspects of progestin and cholesterol metabolism. These trophic actions of estrogen are likely to prepare granulosa cells for the increased rates of progesterone biosynthesis ultimately required by fully differentiated luteal cells.     

Mechanisms subserving calcium's modulation of luteinizing hormone action in isolated swine granulosa cells

We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [14C]acetate or the aromatization of testosterone to 17 beta-estradiol. The possible role of calmodulin in mediating calcium's actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21-4.88 micrograms calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID50)]: penfluridol (1 microM), trifluoroperazine (9 microM), chlorpromazine (95 microM), and trifluoperazine sulfoxide (greater than 300 microM). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID50 of 16.7 microM. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID50, 18 microM). However, even 100 microM TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH's stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [14C]acetate, or progesterone production from exogenously supplied sterol substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanisms subserving insulin's differentiating actions on progestin biosynthesis by ovarian cells: studies with cultured swine granulosa cells

To characterize the nature of insulin action on ovarian cells, an in vitro system of swine granulosa cells was developed in which 3- to 50-fold stimulation of progesterone production was observed in response to insulin under serum-free or sparsely (1%) serum-supplemented conditions. These studies demonstrate that optimal cell density is critical for the full expression of insulin action, and that the dose-dependent responses of granulosa cells to insulin are also significantly influenced by the maturational status of the parent follicle. The striking (greater than 30-fold) responses of medium-sized and healthy preovulatory follicles to insulin were not attributable to the presence or absence of atresia or to a selective inhibition of progesterone's catabolism to 20 alpha-dihydroprogesterone, since 20 alpha-dihydroprogesterone production was also markedly increased in response to insulin. The mechanisms subserving insulin action were explored further by testing the capacity of insulin to 1) increase progesterone accumulation in response to exogenously provided pregnenolone, and 2) stimulate pregnenolone biosynthesis in the presence of trilostane, an inhibitor of pregnenolone metabolism. The effects of insulin were to increase the biosynthesis of progesterone from available pregnenolone and increase the production of pregnenolone from endogenous sterol substrate. The physiological relevance of these differentiative actions of insulin is suggested by insulin's ability to significantly enhance the stimulatory effects of FSH, LH, epinephrine, prostaglandin E2, and cAMP effectors, cholera toxin and 8-bromo-cAMP. In summary, the present studies delineate culture conditions in which swine granulosa cells exhibit a high degree of responsiveness to the stimulatory actions of insulin on progestin biosynthesis. The effects of insulin are critically influenced by cell density, stage of follicle maturation, and the presence or absence of classical ovarian effector hormones with which insulin can interact synergistically. Moreover, our studies indicate that insulin exerts significant actions at several levels of progestin biosynthesis, including the production of pregnenolone, progesterone, and 20 alpha-dihydroprogesterone by granulosa cells. In view of the high concentrations of insulin-like growth factors and somatomedins attained in porcine Graafian follicles, we suggest that trophic actions of insulin or insulin-like peptides and the synergism of insulin with classical ovarian effector hormones are likely to be of physiological importance to the differentiation of granulosa cells in the developing ovarian follicle.     

Properties of low density lipoprotein binding by cultured swine granulosa cells

The properties of low density lipoprotein (LDL) binding to ovarian cells were investigated in cultured swine granulosa cells in serum-free monolayer cultures. Swine and human LDL bound with high affinity and specificity, with apparent dissociation constant (Kd) values for swine and human LDL of 1.1 and 3.6 micrograms/ml at 4 C, and 2.7 and 4.8 micrograms/ml at 37 C. In contrast, high density lipoprotein competed sparingly with [125I]iodo-LDL with an apparent half-maximally inhibitory concentration of 650 micrograms/ml. Binding of LDL was dependent upon arginine residues within the apoprotein B moiety, since covalent modification of LDL with 1,2-cyclohexanedione markedly reduced its ability to compete for binding or degradation and to support progesterone biosynthesis. This specific, high affinity saturable binding of LDL to pig granulosa cells exhibited a maximal binding capacity of 0.7 micrograms LDL protein/mg DNA, which corresponds to approximately 5500 LDL receptors per cell. The relative time course of LDL binding, internalization, and degradation by swine granulosa cells was assessed by examining trypsin-releasable (surface-bound) and trypsin-resistant (internalized) [125I]iodo-LDL. At 37 C, granulosa cells exhibited a rapid increase in surface-bound lipoprotein, followed by delayed but subsequently progressive increases in internalized and degraded LDL. LDL degradation by swine granulosa cells was a saturable, temperature- and time-dependent process, with half-maximal degradation occurring at a concentration of 16 micrograms/ml LDL. This correlates closely with the half-maximal concentration of LDL that stimulates progesterone secretion. Degradation of [125I]iodo-LDL was not attributable to bulk fluid-phase pinocytosis, since the cellular ingestion of an impermeant probe, [125I]iodo-polyvinylpyrrolidone, occurred at 1/40 the rate of lipoprotein degradation. In addition, degradation of [125I]iodo-LDL was specifically inhibited by excess unlabeled LDL, decreased by prior exposure of granulosa cells to the soluble sterol, 25-hydroxycholesterol, and antagonized by the lysosomotrophic agent, chloroquine. Moreover, in separate experiments, rates of degradation of LDL were found to be significantly correlated with progesterone one production (r = +0.88, P less than 0.01). In summary, swine granulosa cells exhibit specific, high affinity, saturable, and low capacity receptors for homologous and heterologous LDL. These LDL recognition sites on ovarian cells depend upon cyclohexanedione-sensitive arginine residues with the apoprotein B moiety for effective binding, internalization, and functional (steroidogenic) responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human chorionic gonadotropin and 8-bromo-adenosine 3',5'-monophosphate stimulate [125I]low density lipoprotein uptake and metabolism by luteinized human granulosa cells in culture

In nonsteroidogenic cells, cellular cholesterol requirements and sterol availability determine low density lipoprotein (LDL) receptor expression and LDL metabolism. We wished to learn if hCG and cAMP increase LDL metabolism by cultured luteinized human granulosa cells and whether this increase is dependent on enhanced metabolism of cellular cholesterol stores to steroid. Granulosa cells were cultured for 48 h in medium containing 20% human male serum and then for 48 h in serum- and hormone-free medium. The cells then received either fresh medium (no additions) or one of the following treatments: 500 mIU hCG/ml, 1.5 mM 8-bromo-cAMP, 100 micrograms aminoglutethimide (AG)/ml to inhibit cholesterol metabolism to steroid hormones, hCG plus AG, or 8-bromo-cAMP plus AG. After 6-48 h of exposure to tropic agents, specific metabolism of [125I]LDL was determined. hCG and 8-bromo-cAMP significantly increased (P less than 0.05) the amount of [125I]LDL bound (2.2-fold), internalized (2.3-fold), and degraded (2.9-fold) by the luteinized granulosa cells. The apparent Km values for LDL degradation in control and hCG-treated cells were similar (2.0 and 2.6 micrograms/ml, respectively). As little as 10 mIU hCG/ml stimulated LDL metabolism in a time-dependent fashion: a stimulatory effect was detected within 6 h of exposure to hCG and was greater after 24 h. AG attenuated but did not prevent the hCG- or 8-bromo-cAMP-stimulated increase in both LDL uptake and metabolism, although it completely inhibited the steroidogenic response. AG alone had no significant effect on [125I] LDL metabolism. We conclude that hCG and cAMP increase LDL metabolism by luteinized human granulosa cells. These effects are apparently not simply a consequence of enhanced cellular cholesterol metabolism to steroids.     

The source of cholesterol for progesterone synthesis in cultured preovulatory human granulosa cells

There are three possible sources of cholesterol for immediate use in progesterone production by preovulatory human granulosa cells: follicular fluid high-density lipoprotein, de novo synthesis of cholesterol, and performed intracellular cholesteryl ester stores. In the present study these three alternatives were investigated. First, an in vitro model was established that mimics the preovulatory environment, including short-term cultures and use of autologous follicular fluid in the culture medium, instead of serum. Using this model it was found that the presence of high-density lipoprotein from follicular fluid in the culture medium did not affect the synthesis of progesterone by the granulosa cells. Next, addition of inhibitors of de novo sterol synthesis, like low-density lipoprotein, 25-OH cholesterol and compactin to the culture medium, did not reduce [14C]acetate incorporation into sterols and steroids by the cells. The sterol synthesis was accordingly interpreted to be at a low and therefore uninhibitable level. Finally, the content of free and esterified cholesterol in freshly isolated granulosa cells was found to be 50 +/- 7 and 52 +/- 13 pmol/mg cell protein, respectively. We suggest that neither follicular high-density lipoprotein nor endogenous synthesis is the immediate cholesterol source for the progesterone production in preovulatory human granulosa cells. However, granulosa cells have a large store of cholesteryl esters that may provide free cholesterol for the preovulatory progesterone production.     

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.     

Insulin-like growth factor type I increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells

Insulin-like growth factor type I (IGF-I) is an important intraovarian peptide that stimulates granulosa cell steroidogenesis during follicular development. The cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) that converts cholesterol to pregnenolone is the rate-limiting step in progesterone biosynthesis. Since treatment of primary cultures of immature porcine granulosa cells with IGF-I will increase progesterone production as well as the synthesis of immunoprecipitable P450scc enzyme, we examined possible molecular mechanisms subserving these inductive effects of IGF-I. To this end, cultures of porcine granulosa cells were maintained in serum-free medium with or without IGF-I under various treatment paradigms. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a 32P-labeled 1-kilobase porcine cDNA clone. Northern blot autoradiogram densitometry data were normalized with a constitutively expressed 1.2-kilobase chicken glyceraldehyde-3-phosphate dehydrogenase cDNA clone. Steroidogenesis was monitored by measuring concomitant progesterone accumulation in the culture medium. Treatment with pure recombinant human IGF-I (100 ng/ml) significantly increased P450scc mRNA concentrations after 18 h, and maximal stimulation (10- to 20-fold) occurred by 48 h for both P450scc mRNA and progesterone accumulation. The IGF-I dose-response curve studied at 48 h showed a significant increase in P450scc mRNA levels at a minimal IGF-I concentration of 1 ng/ml (although progesterone production was not increased). Treatment with equimolar concentrations of epidermal growth factor, IGF-I, or insulin significantly increased P450scc mRNA concentrations, whereas fibroblast growth factor did not. To examine possible mechanisms underlying stimulation of P450scc by IGF-I, immature granulosa cells were treated with aminoglutethimide (a P450scc enzyme inhibitor), low density lipoprotein (to increase cholesterol delivery to granulosa cells), or estradiol in the presence or absence of IGF-I. Aminoglutethimide had no effect, alone or with IGF-I, on P450scc mRNA concentrations, but suppressed progesterone production. Low density lipoprotein alone also did not stimulate P450scc mRNA levels and only slightly increased progesterone accumulation, but acted synergistically with IGF-I to augment P450scc mRNA concentrations and progesterone accumulation. Estradiol alone did not stimulate P450scc mRNA concentrations, but did significantly increase progesterone production. Estradiol cotreatment with IGF-I synergistically enhanced progesterone production, but did not alter IGF-I-stimulated P450scc mRNA concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of estradiol as a biological amplifier of the actions of follicle-stimulating hormone: in vitro studies in swine granulosa cells

We describe an in vitro system of swine granulosa cells which remain responsive to estradiol (E2) and FSH. In this system, we examined mechanisms by which E2 amplifies the stimulatory actions of FSH. E2 stimulated progesterone production in a dose-dependent manner and enhanced the tropic effects of FSH. The facilitative interaction between E2 and FSH could not be accounted for by mitogenic effects, by a leftward shift in the dose-response curves for FSH or E2, or by catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. E3 also enhanced stimulatory actions of 8-bromo-cAMP, choleratoxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Lipoproteins were required for maximal interactive effects between E2 and FSH. However, estrogen significantly increased the effects of FSH even in serum- and lipoprotein-free medium. In addition, when de novo cholesterol biosynthesis by granulosa cells was suppressed by Compactin, the actions of E2 and/or FSH were not impeded. In contrast, E2 alone and FSH alone significantly augmented progesterone production in response to the oxygenated sterol 5-cholesten-3 beta, 25-diol, which is an effective substrate for cholesterol side-chain cleavage. In the presence of 5-cholesten-3 beta, 25-diol, the magnitude of the synergism between E2 and FSH was also increased markedly. We conclude that E2 augments the stimulatory actions of FSH or cAMP on steroidogenesis. This facilitative interaction is amplified by exogenous lipoproteins and does not seem to depend upon endogenous cholesterol biosynthesis. Studies with 5-cholesten-3 beta, 25-diol suggest that estrogen and FSH exert significant stimulatory effects at the level of cholesterol side-chain cleavage. These observations reveal important interactions of E2 and fSH in preparing granulosa cells for the high rates of steroidogenesis they ultimately express in the luteinized state.     

2-hydroxyestradiol enhanced progesterone production by porcine granulosa cells: dependence on de novo cholesterol synthesis and stimulation of cholesterol side-chain cleavage activity and cytochrome P450scc messenger ribonucleic acid levels

2-Hydroxyestradiol (2-OH-E2) stimulates progestin secretion by granulosa cells, but the intracellular locus of the stimulatory effect has not been clarified. The objectives of the present studies were to 1) determine the role of de novo sterol synthesis in the effect of 2-OH-E2 on progestin biosynthesis, and 2) examine the effects of 2-OH-E2 on cholesterol side-chain cleavage (SCC) activity and the level of messenger RNA (mRNA) for P450scc. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase with lovastatin (5 micrograms/ml) or mevinolin (5 micrograms/ml) reduced FSH- and 2-OH-E2-stimulated (but not E2-stimulated) progesterone production. Mevalonate (20 mM) enhanced basal progesterone production and reversed the inhibitory effect of lovastatin but did not affect progesterone biosynthesis in the presence of 2-OH-E2. As an index of the activity of cholesterol SCC enzyme, granulosa cells were exposed to 25-hydroxycholesterol (10 micrograms/ml) for 24 h and progesterone secretion monitored. Conversion of 25-hydroxycholesterol into progesterone was stimulated 2- to 3-fold by maximally effective concentrations of 2-OH-E2, E2, and FSH. 2-OH-E2 and/or E2 further enhanced 25-hydroxycholesterol conversion in the presence of FSH, LH, and epinephrine. Aminoglutethimide, an inhibitor of SCC, reduced 2-OH-E2- and 2-OH-E2 plus FSH-stimulated progesterone production by 97% and 95%, respectively. 2-OH-E2 also increased basal (by 2 to 3-fold) and FSH-stimulated (to 3.5-fold of FSH-treated controls) levels of mRNA for cytochrome P450scc. Collectively, our studies support the hypothesis that 2-OH-E2-enhanced progesterone biosynthesis by porcine granulosa cells is dependent on de novo cholesterol synthesis and is associated with increased levels of the mRNA encoding cytochrome P-450scc, which leads to increases in basal and gonadotropin-induced SCC activity.     

Ovarian actions of tumor necrosis factor-alpha (TNF alpha): pleiotropic effects of TNF alpha on differentiated functions of untransformed swine granulosa cells.

We have examined interactions between tumor necrosis factor-alpha (TNF alpha), a product of the immune system, and ovarian cells using serum-free monolayer cultures of untransformed swine granulosa cells. Recombinant human TNF alpha, a potent cytoactive product of activated macrophages, bound specifically and with high affinity to intact granulosa cells. Binding sites had an apparent Kd of 0.17 nM (95% confidence interval, 0.065-0.31), and a binding capacity of 80 nmol/micrograms DNA (95% confidence interval, 52-110). The binding capacity of granulosa cells for TNF alpha (but not the binding affinity) was increased approximately 2-fold by treatment with FSH and insulin. The biological effects of TNF alpha on pig granulosa cells were expressed after 48 and 96 h in culture. At the latter time, TNF alpha significantly suppressed insulin- and insulin- plus FSH-stimulated progesterone accumulation, with respective ID50 values of 0.08 +/- 0.008 and 0.06 +/- 0.014 nM, but did not affect basal progesterone accumulation or DNA content. TNF alpha also significantly attenuated the stimulatory effect of combined treatment with FSH and insulin on cAMP generation during 48-96 h of culture. TNF alpha inhibited the stimulatory effects of forskolin, cholera toxin, and the cAMP analog 8-bromo-cAMP on progesterone accumulation, indicating multiple sites of action of this immune modulator. Inhibition of progestin biosynthesis was observed even in the presence of 25-hydroxycholesterol, a soluble oxygenated sterol substrate for the cholesterol side-chain cleavage reaction, and was accompanied by decreased concentrations of specific cellular mRNA encoding cholesterol side-chain cleavage enzyme. There were no changes in the amounts of a constitutively expressed enzyme, phosphoglyceraldehyde dehydrogenase. Inhibitory actions of TNF alpha were specific to de novo steroid hormone biosynthesis, since nanomolar concentrations of this cytokine stimulated accumulation of prostaglandin E2 and prostaglandin F2 alpha basally and during treatment with FSH, cholera toxin, or 8-bromo-cAMP. In contrast, prostaglandin accumulation was not enhanced by interferon-gamma or interleukin-2. In summary, untransformed porcine granulosa cells exhibit specific, high affinity, low capacity saturable binding sites for TNF alpha, and the number of such binding sites can be regulated by combined treatment with insulin and FSH. Granulosa cells are susceptible to the inhibitory actions of TNF alpha on FSH- and insulin-supported progesterone biosynthesis and cAMP accumulation. One important locus of TNF alpha action is blockade of hormonally stimulated increases in specific mRNA encoding the cholesterol side-chain cleavage cytochrome P450 enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin-like growth factor-binding protein (IGF-BP) inhibition of granulosa cell function: effect on cyclic adenosine 3',5'-monophosphate, deoxyribonucleic acid synthesis, and comparison with the effect of an IGF-I antibody.

The effects of insulin-like growth factor binding proteins (IGF-BPs) purified from porcine follicular fluid on granulosa cell function were examined using serum-free cultures of rat granulosa cells obtained from immature, diethylstilbestrol-treated rats. Both the so-called GH-dependent (IGF-BP3) and non-GH-dependent (IGF-BP2) proteins dose dependently inhibited granulosa cell estradiol and progesterone production with IC50s of 4.1-7.6 nM for IGF-BP3 and 12.6-12.9 nM for IGF-BP2, the actual value depending upon the steroid being measured. A specific antiserum directed against IGF-I also dose dependently suppressed both estradiol and progesterone production, although the effect on the latter was more marked. Experiments using cells that were primed with FSH to induce functional LH receptors showed that the inhibitory action of IGF-BP3 was specific to FSH. However, both IGF-BP3 and IGF-BP2 were capable of inhibiting both forskolin- and cholera toxin-stimulated steroidogenesis, confirming that neither compound was competing with FSH for binding to its receptor. Neither IGF-BP had any effect on either basal or FSH-stimulated cAMP production, while exogenously added IGF-I was stimulatory in this respect. However, both IGF-BPs inhibited FSH-stimulated [3H]thymidine uptake by the granulosa cells, while IGF-I had no effect on this parameter, suggestive of an IGF-independent effect on granulosa cell proliferation. Our data suggest that IGF-BPs have a multifaceted mode of action on granulosa cell function, and may therefore be an important regulator of follicular growth and differentiation.     

Insulin-like growth factor-I as an amplifier of follicle-stimulating hormone action: studies on mechanism(s) and site(s) of action in cultured rat granulosa cells

The ovarian granulosa cell has recently been shown to be a site of insulin-like growth factor-I (IGF-I) production, reception, and action. In large measure, IGF-I action (in the rat) appears contingent upon its ability to synergize with FSH, a major promoter of granulosa cell differentiation. It is the objective of the in vitro studies reported herein to elucidate the cellular mechanism(s) whereby IGF-I amplifies FSH hormonal action, placing special emphasis on the potential role of the putative intracellular second messenger cAMP in this regard. Basal FSH binding (115.7 +/- 2.1 fmol/mg cell protein) to rat granulosa cells cultured under serum-free conditions remained unchanged after 72 h of treatment with IGF-I (50 ng/ml) by itself (107.1 +/- 1.0 fmol/mg cell protein). In contrast, treatment with FSH (20 ng/ml) resulted in a significant (P less than 0.05) decrease in FSH binding capacity (but not affinity) relative to controls in either the absence or presence of IGF-I. Whereas treatment with FSH resulted in a substantial increase in forskolin-stimulatable adenylate cyclase activity (10 +/- 1.7% conversion of [3H] ATP to [3H]cAMP), concurrent treatment with IGF-I resulted in 2.2-fold enhancement of FSH action. This IGF-I effect proved dose dependent with an apparent median effective dose of 3.6 +/- 0.8 ng/ml, a concentration in keeping with its granulosa cell receptor binding affinity. Significantly, however, IGF-I proved capable of enhancing Bt2cAMP-stimulated progesterone accumulation suggesting that IGF-I may be also acting at site(s) distal to cAMP generation. Taken together, these and previous studies indicate that nanomolar concentrations of exogenously added IGF-I may be interacting with the FSH transduction signal at multiple cellular site(s) to effect amplification of FSH action.