Expression of aldo-keto reductase 1C23 in the equine corpus luteum in different luteal phases.

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3ß-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

Cytochalasin-stimulated steroidogenesis from high density lipoproteins.

The cytochalasins stimulate steroid secretion of Y-1 adrenal tumor cells two-to threefold. The order of potencies is cytochalasin E is greater than D is greater than B, but the maximum response is the the same and always less than with ACTH. Like that with ACTH, the stimulation has a rapid onset, is easily reversible, is inhibited by cucloheximide and aminoglutethimide, and occurs at a stage before pregnenolone. Although the cytochalasin, like ACTH, produce cell rounding, it is shown that this morphological change is not necessarily coupled to steridogenesis. Unlike ACTH, cytochalasin B does not measurably increase cellular levels of cAMP at concentrations that lead to maximal steroidogenesis. The cytochalasin B-induced stimulation of steroidogenesis, unlike the short-term ACTH effect, fails to occur in the absence of serum. This lack of response can be corrected by even low concentrations of human high density lipoproteins (HDL) but not by low density lipoproteins (LDL). We, therefore, propose that cytochalasin B enhances the availability of cholesterol bound to HDL for steroidogenesis by Y-1 adrenal cells.

A role for calmodulin in the regulation of steroidogenesis.

TWO APPROACHES WERE USED TO STUDY THE POSSIBLE ROLE OF CALMODULIN IN THE REGULATION OF STEROID SYNTHESIS BY MOUSE ADRENAL TUMOR CELLS: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents.

Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.

We have recently succeeded in immortalizing rat granulosa cells by co-transfection with SV-40 DNA and the Ha-ras oncogene. These cells lost their response to gonadotropins, but expressed the cytochrome P450scc mitochondrial system enzymes and produced progesterone and 20 alpha-hydroxy-4-pregnan-3-one (20 alpha-OH-P) upon cAMP stimulation (Suh, B. S., and A. Amsterdam. 1990. Endocrinology. 127:2489-2500; Hanukoglu, I., B. S. Suh, S. Himmelhoch, and A. Amsterdam. 1990. J. Cell Biol. 111:1973-1981). In an attempt to restore the steroidogenic response to gonadotropins in immortalized cells, lutropin/choriogonadotropin (LH/CG-R) receptor expression plasmid was prepared by introducing the complete coding region of LH receptor cDNA (McFarland, K. C., R. Sprengel, H. S. Phillips, M. Köhler, N. Rosemblit, K. Nikolics, D. L. Segaloff, and P. H. Seeburg. 1989. Science (Wash. DC). 245:494-499) into a SV-40 early promoter based eucaryotic expression vector. Granulosa cells from preovulatory follicles were transfected with this LH receptor expression plasmid, together with SV-40 DNA and the Ha-ras oncogene. Cell lines obtained after this triple transfection accumulated cAMP in a dose-dependent manner in response to hCG. Moreover, they produced progesterone and 20 alpha-OH-P upon hCG stimulation with an ED50 of 125 pM and 75 pM, respectively, which is within the physiological range. Concomitantly with hCG induced differentiation, inhibition of cell proliferation was evident following stimulation with hormone concentrations as low as 40 pM. The number of hCG receptor sites per cell after numerous passages and several freezing and thawing cycles was 1.9 x 10(4), they showed a Kd of 180 pM. Stimulation with hCG induced pronounced morphological and biochemical changes in these cells including formation of mitochondrial located adrenodoxin, a marker enzyme for enhanced steroidogenesis. These findings make possible the expression in immortalized granulosa cells, of selectively mutated receptor molecules which preserve their steroidogenic potential, thereby opening the way to analysis of structure-function relationships of the receptor molecule.

Inhibition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1) by the ionophore A23187. Role of protein biosynthesis.

Addition of the ionophore A23187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 . 10(-7)M). Inhibition is rapid in onset and is not overcome by addition of external Ca2+. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effect is not overcome by high concentrations of Ca2+. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steroid synthesis after entry of cholesterol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca2+.

The non-benzodiazepine anxiolytic drug etifoxine causes a rapid, receptor-independent stimulation of neurosteroid biosynthesis.

Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.

Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses.

Several clonal lines of cultured Leydig tumor cells have been established and characterized in terms of gonadotropin receptors and steroid production. Although freshly isolated cells derived from the M5480P tumor have functional hCG receptors, only two of the five clonal lines established were shown to bind significant quantities of hCG. In these clones, steroid production can be stimulated to the same extent by hCG, cholera toxin, and 8-Br-cAMP. The other three clones bind a small amount of hCG and respond to the hormone with a marginal increase in steroidogenesis. Steroid production, however, is significantly stimulated by cholera toxin or 8-Br-cAMP. A comparison of the steroids produced by freshly isolated cells and two of the clones revealed some changes in the steroidogenic pathway. The most obvious change is an increase in the ability of the cultured cells to synthesize 20 alpha-dihydroprogesterone (20 alpha-hydroxypregn-4-en-3-one). These clonal lines may provide a suitable model system for the study of gonadotropin actions and regulation of the expression of differentiated functions of Leydig cells.

Pregnancy in young and aged rats. I. Ovarian progesterone and 20 alpha-hydroxypregn-4-en-3-one formation.

The conversions of [3H]pregnenolone (P5) to [3H]progesterone (P4) and [3H]20 alpha-hydroxypregn-4-en-3-one (20-OHP4) were determined in vitro in ovaries of 3 (young)- and 11 (aged)-month-old rats throughout pregnancy. Aged females exhibited an increased incidence of embryonic-fetal resorption beginning at midgestation. The mean +/- SE numbers of live and dead fetuses in aged females between day 11 of gestation and term were 8.6 +/- 0.5 and 2.3 +/- 0.3, respectively, compared to 12.0 live fetuses in young females. In young animals, ovarian conversion of P5 to P4 approximated 30% on days 1-3 of gestation, increased (P less than 0.001) to a plateau of about 45% on days 6-11, then declined to 23.5% by day 15. In aged females, ovarian formation of P4 from P5 showed no significant progressive rise or fall throughout pregnancy. The increment in P4 formation observed on days 6-11 in young rats, therefore, did not occur in aged females, and the percent conversion of P5 to P4 was 40% lower (P less than 0.05 to P less than 0.005) in aged rats during this interval. Ovarian P4 formation was similarly low in aged females that did or did not exhibit embryonic death on days 9-11. From day 12 of gestation until term, the percent conversion of P5 to P4 was similar in all young and aged animals. Conversion of P5 to 20-OHP4 in young rats increased (P less than 0.005) steadily to a peak of 21.9 +/- 5.8% (mean +/- SE) at midgestation, then declined thereafter. 20-OHP4 formation was about 50% greater in aged than in young females on days 2-6, but values were similar thereafter. The mean ratio of conversion of P5 to P4 to that of P5 to 20-OHP4, therefore, was 50% lower (P less than 0.025) in aged (1.8 +/- 0.2) than in young (3.3 +/- 0.4) rats between days 6 and 11 of gestation. The subnormal amount of P4 recovered after ovarian incubation in aged females apparently reflected subnormal P4 formation and/or enhanced P4 catabolism to steroid metabolites. The present results, therefore, demonstrate intrinsic changes in ovarian function during aged rat pregnancy. The onset of fetal demise in aged females, however, does not appear to be related to this alteration in ovarian steroidogenesis.

Microtubules and the gonadotropic regulation of granulosa cell steroidogenesis.

The involvement of microtubules in the gonadotropic regulation of granulosa cell steroidogenesis was assessed at the preantral (E2-cells) and antral (PMS-cells) stages of follicular development. The influence of agents that alter microtubule-tubulin equilibrium on basal and FSH-stimulated progesterone production was determined in vitro and compared with that on microtubule integrity and organization using immunofluorescence. Basal and FSH-stimulated progesterone production was approximately 2-fold higher in PMS-cells than in E2 cells. Colchicine and nocodazole, two agents that depolymerize microtubules, significantly stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one production in PMS-cells. Although progesterone production by E2-cells was increased by nocodazole, the amount produced was considerably less than that produced by PMS-cells. FSH-stimulated progesterone biosynthesis was reduced by colchicine and nocodazole in both cell types. Taxol, an agent that stabilizes microtubules, markedly reduced FSH-stimulated progesterone production in both E2- and PMS-cells, but failed to exert a comparable effect on basal steroid production. A close association existed between the concentrations of colchicine, nocodazole, and taxol that altered basal and/or FSH-stimulated steroidogenesis and those that affected microtubule organization and/or distribution. Whereas granulosa cells appeared flattened with numerous cytoplasmic processes after 24 h of culture in medium alone, they were almost spherical and devoid of projections after culture with these agents. FSH-stimulated cells also occupied less area than controls, although cytoplasmic processes were present. These findings indicate an involvement of microtubules in the regulation of granulosa cell steroidogenesis. It is proposed that one of their roles is to facilitate the movement of cholesterol from lipid droplets to mitochondria, possibly by bringing these cellular inclusions closer together.

Biosynthesis of the neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha hp), a specific inhibitor of FSH release.

The gonadal steroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) is a neuroactive steroid with anxiolytic and analgesic actions. In addition, 3 alpha HP has been shown to inhibit GnRH activity on gonadotropes and selectively suppress FSH release from pituitary cells, without an effect on LH. The enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) has been presumed to be the enzyme responsible for the conversion of progesterone to 3 alpha HP, but this has never been confirmed in vitro or in vivo. We have now determined the mechanism of 3 alpha HP synthesis in vivo using specific enzyme inhibitors and in vitro using recombinant proteins. Incubation of [(3)H]progesterone with purified recombinant rat and human 3 alpha HSD isoforms showed that both the rat 3 alpha HSD and the human type 2(brain) 3 alpha HSD converted progesterone to 3 alpha HP. Age-dependent 3 alpha HP production was demonstrated in pituitary and cortex. Incubation of both tissues with indomethacin, a known 3 alpha HSD inhibitor, decreased the conversion of progesterone to 3 alpha HP by at least 70%, indicating that 3 alpha HSD was responsible for this conversion. As human type 2 3 alpha HSD is expressed in a region-specific fashion in the brain, 3 alpha HP may only be made in specific regions of the brain. Furthermore, the data suggest that the pituitary has the capacity for 3 alpha HP production, which may provide an additional mechanism for regulation of GnRH action.

The influence of exogenous free cholesterol on steroid synthesis in cultured adrenal cells.

Previous studies showed that Y-1 cells in which the concentration of cholesterol in plasma membranes (expressed relative to phospholipid as a molar ratio, C/P) was raised by incubation with cholesterol produced more 20 alpha-dihydroprogesterone than cells with normal or low plasma membrane C/P values. This difference was not associated with any difference in the production of cAMP. The cells respond to (Bu)2cAMP in the order high greater than normal greater than low C/P. High C/P is associated with higher levels of cholesterol in the cytosol and higher concentrations of this steroid in the inner mitochondrial membrane. The rate of transport of cholesterol to the inner membrane differed in the three groups of cells: high greater than normal greater than low C/P. Production of pregnenolone by mitoplasts was no different in the three groups, and the same applies to the production of pregnenolone by mitochondria from cells incubated with and those incubated without (Bu)2cAMP. 25-Hydroxycholesterol increased the production of 20 alpha-dihydroprogesterone by cells showing normal or low C/P values, but not those with high values. It is concluded that the transport of cholesterol to the inner mitochondrial membrane is regulated by at least two processes, namely the supply of substrate (cholesterol), which can saturate this transport mechanism, and some mechanism(s) that is stimulated by cAMP and is capable of stimulating transport above maximal levels produced by high concentrations of cholesterol alone. The second mechanism requires the synthesis of a new protein(s).

The influence of plasma membrane cholesterol on the response of adrenal cells to adrenocorticotropin.

The concentration of cholesterol in plasma membranes of Y-1 cells was altered between values of the molar ratio of cholesterol to phospholipids (C/P) of 0.6:1.6 to study the influence of plasma membrane cholesterol on the response to ACTH. Increasing concentrations of membrane cholesterol (C/P) were associated with an increase in the number of ACTH receptors and, hence, production of cAMP and steroids without affecting the nature of binding (Kd) of ACTH to its receptor. Binding studies revealed spare ACTH receptors at all concentrations of membrane cholesterol. However, production of cAMP and steroids by Y-1 cells were tightly coupled. Studies with cholera toxin showed that extreme changes in C/P were without influence on the activity of Ns or that of the cyclase itself. It was also shown that production of steroids in the absence of ACTH and that in response to cholera toxin involve some effect of membrane cholesterol not dependent on cAMP.

Androgens augment vasoactive intestinal peptide- and growth hormone-releasing hormone-stimulated progestin production by rat granulosa cells.

Vasoactive intestinal peptide (VIP) has been shown to stimulate steroid production by cultured rat granulosa cells independently of FSH. In the present study, we have examined the modulatory effects of various steroids on this response. Rat granulosa cells cultured for 2 days with only VIP showed small but significant increases in progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) production. Concomitant treatment with either a synthetic estrogen (diethylstilbestrol), a synthetic progestin (R5020), or cortisol did not augment VIP-stimulated progesterone production; however, the latter two steroids slightly, but significantly, augmented VIP-stimulated 20 alpha-OH-P production. In contrast, concomitant treatment with a synthetic androgen (R1881) dramatically augmented both VIP-stimulated progesterone and 20 alpha-OH-P production. These effects were dose dependent for both VIP and R1881 and could be blocked by the androgen antagonist cyproterone acetate. Time course studies revealed that progesterone content of the culture media rapidly increased over the first 24 h of culture then remained fairly constant for the next 48 h; 20 alpha-OH-P content, on the other hand, was low for the first 12 h of culture and steadily increased thereafter. Dose-response analysis for R1881 revealed an ED50 of approximately 2 x 10(-8) M for the synthetic androgen, and comparison with other naturally occurring androgens provided the rank order of potency R1881 > androstenedione > testosterone = dihydrotestosterone. Additional studies with another member of the VIP peptide family, GH-releasing hormone, showed dose-dependent stimulation of progesterone and 20 alpha-OH-P production by this peptide. These effects were also augmented by R1881 but not by diethylstilbestrol, R5020, or cortisol. These studies demonstrate that androgens, but not estrogens, progestins, or glucocorticoids, augment VIP- and GH-releasing hormone-stimulated progestin production by cultured rat granulosa cells.

Inhibition of cortiocosteroidogenesis by delta-9-tetrahydrocannabinol.

ACTH, cholera toxin, cyclic AMP but not pregnenolone-induced steroidogenesis in Y-1 functional mouse adrenal tumor cells was significantly inhibited by delta-9-tetrahydrocannabinol, cannabidiol, and cannabinol. The inhibition of steroidogenesis could not be correlated with a general depression in cell function or viability. The data suggest that cannabinoids inhibit corticosteroidogenesis at a site between the synthesis of cAMP and of pregnenolone.

Leptin modulates the glucocorticoid-induced ovarian steroidogenesis.

Leptin regulates food intake and other activities through its hypothalamic receptor. Leptin receptors are also found in other organs, including the ovary. Direct effects of leptin in ovarian steroid production were studied in primary rat granulosa cells and in rat and human granulosa cell lines. Leptin (0.6-18 nM) suppressed ovarian steroid synthesis costimulated by FSH and dexamethasone. Production of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one was inhibited by leptin. This inhibition was due at least in part to reduced expression of adrenodoxin, a component of the P450scc system enzyme. Costimulation of progesterone production by forskolin and dexamethasone was also inhibited by leptin, whereas the forskolin-induced cAMP production was not affected. We find that leptin induces c-Jun expression and attenuates the transcriptional activity of the glucocorticoid receptor (GR) in granulosa cells. Elevation of c-Jun expression by other means, e.g. 12-O tetradecanoyl-phorbol-13-acetate or transfecting with a c-Jun expression vector, abolished the transcriptional activity of the GR. A leptin-induced elevation of c-Jun modulates the transcriptional activity of the GR, possibly leading to the observed attenuation of steroidogenesis. It was recently shown that glucocorticoids stimulate leptin expression in vivo, which in turn, inhibits cortisol synthesis. A direct action of leptin on the ovary is an additional element of a regulatory network that maintains the homeostasis of steroid production.

Metabolism of high density lipoproteins reconstituted with [3H]cholesteryl ester and [14C]cholesterol in the rat, with special reference to the ovary.

In order to study the metabolism of high density lipoprotein (HDL)-carried sterol in the rat, human HDL was reconstituted with [14C]cholesterol and [3H]cholesteryl ester. After iv injection into immature PMSG-human CG primed rats pretreated with 4-aminopyrazolopyrimidine and aminoglutethimide, there was time-dependent accumulation of 3H and 14C in various organs which reached a maximum by 15-90 min. On a milligram wet weight basis, uptake of 3H and 14C was greatest in the adrenals, next in ovaries, followed by the liver, with little uptake by kidneys and spleen. On an organ basis, accumulation was greatest by the liver. At 15-45 min post injection, 60% of the 3H in the ovary was in free sterol, indicating hydrolysis of the accumulated cholesteryl esters, whereas 95% of the 3H in serum remained in sterol esters associated with HDL. Coadministration of excess unlabeled HDL, but not human low density lipoprotein, reduced accumulation of radioactivity by the ovaries and adrenals by 60%, indicating a specific and saturable uptake process. Granulosa cells cultured in lipoprotein-deficient medium with reconstituted HDL formed 3H- and 14C-labeled 20 alpha-hydroxypregn-4-en-3-one. Over a 24-h period, utilization of both [14C]cholesterol and [3H]cholesteryl ester was linear, but rates of utilization of the two sterol moieties were not parallel. There was preferential uptake and utilization of free sterol. A dose-response study demonstrated a Michaelis-Menten constant (Km) of 40-60 micrograms sterol/ml for both free and esterified cholesterol. Lysosomotropic agents (chloroquine and NH4Cl) had no effect on utilization of either free or esterified cholesterol for steroidogenesis but reduced degradation of 125I-labeled low density lipoprotein apoprotein. These findings lend further support to the concept of a distinct HDL pathway in steroidogenic cells of the rat, which involves 1) preferential uptake and utilization of free cholesterol from HDL and 2) does not require lysosomal activity.

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.

Estrogen regulation of steroidogenesis in rabbit luteal cells.

Estradiol is a potent modifier of gonadotropin-stimulated steroidogenesis. By the seventh day after ovulation, estradiol is the only agent required for the stimulation of progesterone synthesis by corpora lutea of superovulated pseudo-pregnant rabbits. To learn which control points in steroidogenesis are susceptible to regulation by estradiol alone, we have studied the production of pregnenolone, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one by corpora lutea of estradiol-stimulated and estradiol-deprived pseudopregnant rabbits. In previous investigations, we learned that estradiol deprivation in vivo, on day 9 of pseudopregnancy, causes an abrupt cessation of progesterone and 20 alpha-hydroxy-4-pregnen-3-one production, which is associated with accumulation of cholesterol and cholesteryl ester in the luteal tissue. We now report that production of pregnenolone, measured as its concentration in serum, also decreases abruptly by 84% within 48 h when the estradiol stimulus is removed on day 9 of pseudopregnancy. In addition, short term incubations of luteal tissue demonstrate that corpora lutea from estradiol-deprived rabbits do not use stores of luteal intracellular cholesterol for production of pregnenolone and progestin. These findings suggest that upon estradiol deprivation, rabbit luteal cells lose their capacity for using stored cholesteryl ester, or cholesterol synthesized de novo, for the production of pregnenolone and progestins. We, therefore, tested the hypothesis that a blockade of steroidogenesis caused by estrogen deprivation occurs at the point of cytochrome P-450 cholesterol side-chain cleavage (P-450scc), a principal rate-limiting step in the conversion of cholesterol to hormonal steroid products. To this end, we assayed the P-450scc activity in mitochondria-rich fractions of corpora lutea from rabbits that were deprived of estradiol for 24 and 48 h beginning on day 9 after induction of superovulation. Surprisingly, withdrawal of the estradiol stimulus did not cause loss of luteal P-450scc activity, measured as the amount of aminoglutethimide-inhibitable conversion of 25-hydroxycholesterol to pregnenolone by mitochondria-rich preparations. From these results, we infer that the luteotropic action of estradiol is probably not effected at P-450scc in the rabbit corpus luteum, but, presumably, occurs at control points that regulate the availability of stored cholesterol and/or its movement to or within the mitochondria for conversion to pregnenolone.

Effects of human chorionic gonadotropin in the rabbit corpus luteum: loss of estrogen receptor and decreased steroidogenic response to estradiol.

The effects of hCG on luteal estrogen receptor and steroidogenesis were examined in 9-day-pseudopregnant rabbits. Twenty-four hours after the injection of ovulatory dosages of hCG (10-100 IU), a dose-related loss of luteal estrogen receptor and in vitro progesterone production was observed. The declining progesterone production was not due to the increased conversion of progesterone to the major metabolite, 20 alpha-dihydroprogesterone. The loss of steroidogenesis induced by hCG was not permanent and could be reversed by estradiol treatment started within 24 h of hCG injection. In those animals with a higher luteal estrogen receptor content at the start of the estrogen treatment, the steroidogenic response was greater. These findings indicate that hCG-induced loss of steroidogenesis is associated with the loss of luteal estrogen receptor. The degree of restoration of progesterone synthesis induced by a 24-h period of estradiol treatment may be related to the content of unoccupied cytoplasmic estrogen receptor in the corpus luteum.

Estrogen augmentation of gonadotropin-stimulated progestin biosynthesis in cultured rat granulosa cells.

The influence of estrogens on gonadotropin-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) was examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of FSH in the presence or absence of either diethylstilbestrol (DES) or estradiol. FSH treatment increased progestin production in a dose-dependent manner, whereas treatment with estrogens alone were ineffective. In contrast, concomitant addition of either DES or estradiol augmented FSH-stimulated production of progesterone and 20 alpha-OH-P. Increasing concentrations of estradiol (10(-10) - 10(-7) M) augmented the stimulatory effect of FSH (30 ng/ml) on progesterone production in a dose-dependent manner with ED50 values of approximately 3 X 10(-9) M. The facilitatory action of estradiol was time-related, becoming significant after 36 h of treatment. Granulosa cells were also cultured for 2 days with FSH to induce functional LH receptors. The FSH-primed cells were treated for an additional 3 days with increasing concentrations of LH (0.3-30 ng/ml) in the absence or presence of DES (10(-7) M). LH stimulated progesterone and 20 alpha-OH-P production in a dose-dependent manner, whereas concomitant addition of DES further enhanced LH-induced progestin biosynthesis. (Bu)2cAMP also increased progesterone and 20 alpha-OH-P production by the granulosa cells; however, concurrent addition of DES did not augment the actions of (Bu)2cAMP. The effect of estrogens on gonadotropin-stimulated cAMP accumulation was also examined. FSH treatment dose-dependently increased cAMP accumulation, whereas concomitant treatment with estradiol further increased the FSH action. Similarly, LH treatment also stimulated cAMP accumulation in FSH-primed cells, whereas concurrent addition of DES further augmented LH action. Thus, the stimulatory effect of estrogens upon gonadotropin-stimulated progestin production may be related to the augmentation of cAMP biosynthesis. The present observations suggest that intraovarian estrogens may act locally to enhance the sensitivity of granulosa cells to FSH and LH, thereby increasing the biosynthesis of progestins and cAMP by the granulosa cells.