Evidence for a role for adenosine 3',5'-monophosphate in progesterone secretion by human chorion

Experiments were performed to determine whether cells from human chorion can synthesize and release progesterone. Cells were isolated from term chorion laeve by collagenase-DNAse digestion and incubated in RPMI-1640 medium. Freshly isolated cells contained 9.9 +/- 1.1 ng progesterone/10(6) cells, and released 72.0 +/- 7.1 ng/10(6) cells X 24 h in the absence of precursors. When 25-hydroxycholesterol (25HC) served as a precursor, progesterone release into the medium was concentration and time dependent from 1-20 micrograms/ml up to 8 h. When pregnenolone served as a precursor, progesterone secretion followed Michaelis-Menten kinetics (Km = 6.7 microM; maximum velocity, 1.02 nmol/10(6) cells X h). In the presence of 25HC (20 micrograms/ml), progesterone release increased significantly on exposure to cholera toxin (1 microgram/ml), methylisobutylxanthine (0.1 mM), forskolin (0.1 mM), or (Bu)2cAMP (1 mM). Cells maintained in culture released progesterone when fetal calf serum (10%) or 25HC served as precursors. These studies show that trophoblasts from fetal membranes can synthesize and release progesterone from endogenous and exogenous precurors and support the suggestion that cAMP is an important mediator in this process.     

Mevinolin (lovastatin) inhibits androstenedione production by porcine ovarian theca cells at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex

Mevinolin, putatively a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase, was used to assess the contribution of de novo synthesized cholesterol to androgen production by ovarian thecal cells in vitro. Enzymatically dispersed thecal cells from 3- to 6-mm follicles of prepubertal gilts were incubated at 150,000 cells/ml with a maximally effective dose of LH (250 ng/ml) for 24 h. Mevinolin (3-50 microM) caused dose-dependent inhibition of androstenedione production. Addition of 25-hydroxycholesterol (0.025-25 microM) failed to restore androstenedione production to levels seen in the absence of mevinolin, suggesting an additional site of action of mevinolin beyond 3-hydroxy-3-methylglutaryl coenzyme reductase. The site of this inhibitory effect was determined by measuring steroid products formed in the presence of relevant steroid precursors. Mevinolin (12 microM) inhibited the production of 17 alpha-hydroxyprogesterone from progesterone and that of androstenedione from 17 alpha-hydroxyprogesterone, while 25-hydroxycholesterol to progesterone and pregnenolone to progesterone conversions were unimpaired. That mevinolin did not affect 3 beta-hydroxysteroid dehydrogenase:delta 5-delta 4-isomerase reactions was confirmed by demonstrating that conversions of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone to progesterone, 17 alpha-hydroxyprogesterone, and androstenedione, respectively, were not affected by 12 microM mevinolin. These results indicate that mevinolin has an additional inhibitory action at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex. The degree of inhibition of androstenedione production was not decreased with increased concentrations of progesterone or 17 alpha-hydroxyprogesterone substrate, suggesting that the inhibition was not competitive in nature. As the dose of mevinolin was increased up to 50 microM, progesterone accumulation was unaffected, but pregnenolone concentrations in medium greatly increased. While the mechanism of this effect is unclear, this finding suggests that preformed intracellular cholesterol, rather than that synthesized de novo, is supplying steroidogenic substrate in these cells.     

Estrone sulfate sulfatase activity is increased during in vitro decidualization of stromal cells from human endometrium

Arylsulfatase (EC 3.1.6.1) activity in human stromal cells isolated from specimens of histologically normal proliferative endometrium was increased several-fold during culture for 8-15 days in RPMI-1640 medium plus 10% charcoal-treated fetal bovine serum in the presence of a mixture of ovarian hormones (36 nM estradiol, 1 microM medroxyprogesterone acetate, and 100 micrograms/mL relaxin). The changes in sulfatase activity, determined by measuring the rate of formation of estrone from tritiated estrone sulfate, were associated with in vitro decidualization of the stromal cells, as determined by changes in secretion of PRL into daily renewed culture medium. PRL output by the cells during the last 24 h in culture and sulfatase activity in the cells collected at the end of the culture period were related to their DNA and protein contents. Sulfatase activity in the cells cultured in the presence of the ovarian hormones was comparable to the activity found in decidual cells at term pregnancy. PRL added for 1 day to cultures of stromal cells in the absence of exogenous hormones increased sulfatase activity in the cells, probably by acting in an autocrine manner, as previously demonstrated with human decidual cells during pregnancy. These experiments also revealed a hormonal regulation of stromal cell proliferation in vitro, as estimated from measurements of both DNA and protein levels per dish. Augmentation of sulfatase activity can serve as another marker of in vitro decidualization. Physiologically, an increase in this enzymatic activity may result in a preferential estrogenic stimulation of the decidualized cells by utilization of a circulating substrate, estrone sulfate. This hypothesis could explain the preferential retention of progesterone receptors in decidual cells observed immunohistochemically during the late luteal phase of the menstrual cycle, suggestive of a shift in progestogenic actions from the epithelium to the stroma.     

Steroidogenesis by the immature rhesus monkey ovary in vitro

Primates are believed to have a low level of ovarian steroidogenic activity during prepubertal development. In order to study the rate limiting factors associated with the low level of steroidogenesis, ovaries from prepubertal rhesus monkeys were quartered and incubated for 48 h at 37 C in minimum essential medium. These ovaries secreted 687 +/- 347 pg estradiol/mg ovary and 299 +/- 35 pg progesterone/mg ovary during 48 h of incubation. The addition of 100 ng luteinizing hormone (LH) or 1 mM dibutyryl (Bu)2 cAMP failed to increase significantly estradiol or progesterone secretion. Furthermore, the addition of either progesterone or androstenedione failed to augment estradiol secretion. The presence of either LH or (Bu)2 cAMP with the steroidal substrates also failed to augment estradiol secretion. In contrast, the addition of (Bu)2 cAMP with lipoprotein-derived cholesterol significantly stimulated a two-fold increase in progesterone secretion. The presence of LH in the lipoprotein-supplemented medium failed to augment progesterone secretion. These results suggest that prepubertal monkey ovaries lack the ability to respond to LH, probably due to a lack of gonadotropin receptors or failure of the receptor to stimulate cAMP synthesis. Furthermore, the failure of progesterone and androstenedione to augment estradiol secretion suggests that some cellular components needed to induce aromatase activity are not functional in the prepubertal primate ovary.     

Protein synthesis and steroidogenesis in amphibian (Rana pipiens) ovarian follicles: studies on the conversion of pregnenolone to progesterone

Previous experiments demonstrated that protein synthesis was involved in frog pituitary homogenate (FPH)-induced follicular progesterone production. In this study the metabolic conversion of pregnenolone to progesterone, and involvement of protein synthesis in this specific step of the progesterone synthetic pathway, was investigated in vitro cultured ovarian follicles of Rana pipiens. Fully grown follicles were incubated with frog pituitary homogenates or exogenous pregnenolone and progesterone content of follicle extracts and medium were measured by radioimmunoassay. In the absence of FPH, fully grown follicles converted exogenously added pregnenolone into progesterone in a dose-dependent fashion. Follicular progesterone concentrations were consistently higher than medium levels of steroid throughout the culture period. The conversion of pregnenolone to progesterone at different stages of follicle development was also investigated. The amount of follicular progesterone accumulated after culture with exogenous pregnenolone increased proportionally with follicle size. When fully grown follicles were cultured in the presence of cycloheximide, the inhibitor of protein synthesis blocked FPH-induced progesterone production, but conversion of exogenously added pregnenolone to progesterone was not affected. However, progesterone production was inhibited when cyanoketone (CK), an irreversible inhibitor of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), was added in combination with FPH or exogenous pregnenolone. FPH addition after CK pretreatment did not restore the capacity of follicles to convert pregnenolone to progesterone. These results suggest that conversion of pregnenolone to progesterone occurs efficiently even in the absence of FPH over the course of follicle and oocyte growth (vitellogenesis). Furthermore, in fully grown follicles the 3 beta-HSD activity is independent of protein synthesis. The dependence on protein synthesis in the acute action of FPH appears to be prior to conversion of pregnenolone to progesterone and does not involve de novo synthesis of 3 beta-HSD.     

Regulation by plasma lipoproteins of progesterone biosynthesis and 3-hydroxy-3-methyl glutaryl coenzyme a reductase activity in cultured human choriocarcinoma cells.

The regulation of both the activity of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG CoA) reductase [mevalonate-NADP+ oxidoreductase (CoA-acylating) EC 1.1.1.34] and the secretion of progesterone by human plasma lipoproteins has been investigated in human choriocarcinoma cells in culture. HMG CoA reductase activity was computed from the rate of formation of [14C]mevalonolactone from [14C]HMG CoA. The activity of HMG CoA reductase was expressed as nanomoles of mevalonolactone formed/min . mg solubilized cell protein. An inverse relationship was found between the presence of lipoprotein in the culture medium and the activity of HMG CoA reductase in these cells. In cells maintained in the presence of lipoprotein-enriched culture medium containing 840 micrograms cholesterol/ml, the average activity of HMG CoA reductase was 0.25 nmol/min . mg protein. After removal of lipoprotein, the activity of HMG CoA reductase increased to 1.3 nmol/min . mg protein. The average activity of HMG CoA reductase in cells maintained in lipoprotein-deficient culture medium was 1.5 nmol/min . mg protein but fell to 0.3 nmol/min . mg protein after addition of lipoprotein to the medium. When cells were maintained in the presence of lipoprotein, the rates of section of progesterone and pregnenolone into the culture medium were 2-8 times greater than the rates of secretion of these steroids by cells maintained in the absence of lipoprotein. On the basis of these results, it is concluded that lipoproteins control the rate of cholesterol biosynthesis in cultured choriocarcinoma cells by regulating the activity of HMG CoA reductase, and control the rate of synthesis of progesterone by providing the precursor, cholesterol. We suggest that progesterone synthesis by the trophoblast of the human placenta may also be regulated by the uptake of lipoprotein from maternal blood.     

Phosphatidyl-inositol-3 kinase-independent insulin action pathway(s) in the human ovary.

Hyperandrogenism observed in women with a variety of insulin-resistant states is thought to be due to a stimulatory effect of insulin on ovarian steroid hormone production. However, it is not known what mechanisms could allow the ovary to remain sensitive to insulin while classical target organs for insulin action (liver, fat, and muscle) exhibit insulin resistance. One hypothesis proposed to explain this paradox suggests that a postbinding divergence of insulin receptor signaling occurs in the ovary and that signaling pathways for steroid hormone synthesis and other ovarian effects of insulin may be distinct from classical glucose signaling pathways. We now report that activation of phosphatidyl-inositol-3 (PI-3) kinase, which is crucial for glucose transport, is not necessary for the insulin-induced stimulation of progesterone production or for the insulin-induced inhibition of insulin-like growth factor binding protein 1 (IGFBP-1) production in cultured human ovarian cells. Human granulosa cells obtained during in vitro fertilization procedures were cultured with 10, 10(2), 10(3), or 10(4) ng/mL insulin with or without preincubation with 100 nM wortmannin, a specific irreversible inhibitor of PI-3 kinase. IGFBP-1 concentration in the conditioned medium was measured using immunoradiometric assay or by Western blot analysis. Progesterone concentration was measured using RIA. Additional studies were carried out in cultures of human ovarian cells prepared from homogenized whole ovarian tissue of a woman with a family history of breast cancer and a mutation of BRCA-1 gene who underwent bilateral oophorectomy. These cells were cultured with 10(3) ng/mL insulin with or without preincubation with 100 nM wortmannin. Two-way ANOVA was used to compare mean values of IGFBP-1 and progesterone according to insulin dose and the use of wortmannin. In cultured granulosa cell medium, progesterone production was stimulated by insulin in a dose-related manner up to 175% of control (P < 0.0001). In tissue culture medium from ovarian cells obtained from a patient with BRCA-gene mutation, concentration of progesterone in the tissue culture medium increased from 2.5 +/- 0.2 ng/mL for control to 5.4 +/- 0.3 ng/mL for cells incubated with insulin (P < 0.001). IGFBP-1 production in tissue culture medium from human granulosa cells was inhibited by insulin to the nadir of 45% of control (P < 0.0001). Preincubation with wortmannin, despite complete inhibition of PI-3 kinase in both cell systems confirmed by Western blot analysis, failed to significantly alter these results. We conclude that inhibition of PI-3 kinase by wortmannin fails to abolish stimulatory effect of insulin on progesterone production or inhibitory effect of insulin on IGFBP-1 production in cultured human ovarian cells. These findings suggest that activation of PI-3 kinase, an enzyme crucial for insulin-stimulated glucose transport, is not necessary for the above effects of insulin in the ovary. These data provide evidence for the presence of PI-3 kinase-independent insulin signaling pathway(s) in human ovarian cells.     

The effects of experimental hyperinsulinemia on steroid secretion, ovarian [125I]insulin binding, and ovarian [125I]insulin-like growth-factor I binding in the rat

We randomized 32 cycling female Sprague-Dawley rats (82 days old) into experimental and control groups (16 animals/group). Hyperinsulinemia was induced and maintained for 22 days in the experimental group with NPH human insulin (Novolin, Squibb-Novo, Princeton, NJ) as previously described. Controls received an identical volume of vehicle. Fifteen minutes before death, each rat received a sc injection of 100 ng synthetic GnRH (Factrel, Ayerst Laboratories, New York, NY). The mean serum insulin level was significantly higher in the insulin-treated group than in the control group (165 +/- 57 vs. 49 +/- 9 microU/ml; P less than 0.05). The mean final weight also was significantly higher in the insulin-treated group (283 +/- 4 vs. 242 +/- 7 g; P less than 0.001). There were no significant differences in mean final serum levels of testosterone, estradiol, estrone, or androstenedione or in GnRH-stimulated serum levels of LH or FSH. The androstenedione to estrone ratio, however, was significantly lower in the insulin-treated group (2.5 +/- 0.3 vs. 3.4 +/- 0.2; P less than 0.01), suggesting that aromatase activity increased with hyperinsulinemia. Specific [125I]insulin binding to ovarian tissue homogenates was lower in the insulin-treated group (1.7 +/- 0.1% vs. 2.6 +/- 0.6%/0.2 mg protein; P greater than 0.05), suggesting that ovarian insulin receptors tended to down-regulate with hyperinsulinemia. Specific [125I]insulin-like growth factor I [( 125I]IGF-I) binding to ovarian tissue homogenates, in contrast, was significantly higher in the insulin-treated group (13.3 +/- 1.4% vs. 7.2 +/- 0.6%/0.2 mg protein; P less than 0.05), suggesting that ovarian IGF receptors up-regulated with hyperinsulinemia. The affinity of neither [125I]insulin binding nor that of [125I]IGF-I binding changed significantly, with the 50% inhibition point remaining between 2.0 and 5.0 ng/ml for each peptide in both groups. We conclude that hyperinsulinemia increases ovarian [125I]IGF-I binding and stimulates aromatase activity in the rat. These phenomena, if also true in women, could be important factors contributing to the ovarian hyperstimulation observed in various hyperinsulinemic states.     

The role of thyroid hormone as a biological amplifier of the actions of follicle-stimulating hormone in the functional differentiation of cultured porcine granulosa cells

To characterize thyroid hormone action on the ovary, the direct effects of T4 or T3 were investigated in vitro using a monolayer culture system of porcine granulosa cells. Monolayer cultures were maintained for 6 days in 4% serum-supplemented medium in the absence or presence of porcine FSH (20 ng/ml), with or without graded doses of T4 or T3. Combined treatment with FSH and T4 (10(-7) M) induced morphological alternation resembling epithelioid cells, while FSH alone or T4 alone failed to bring about the epithelioid morphology. Concomitant treatment with FSH and T4 (10(-7) M) markedly increased FSH-stimulated induction of [125I]iodo-human CG binding to cultured granulosa cells obtained from small follicles. The combined treatment with FSH and T4 (10(-7) M) also resulted in a significant increase in progesterone and estrogen secretion by the cultured cells relative to treatment with FSH alone. Increases in progesterone, 17 beta-estradiol, and estrone secretion caused by the combined treatment with FSH and T4 (10(-7) M) were further augmented in response to the addition of exogenously provided substrate pregnenolone, testosterone, and androstenedione, respectively. Furthermore, aromatase activity assessed by the release of [3H]water from [1 beta-3H, 4-14C]androstenedione was significantly higher in cells treated concomitantly with FSH and T4 (10(-7) M) than that in cells treated with FSH alone. All the stimulatory effects of T4 (10(-7) M) on the morphological and functional differentiation of cultured granulosa cells were also found in combined treatment with FSH and T3 (10(-9) M). Either treatment with higher or lower concentrations of T4 or T3 gave attenuated effects, and T4 or T3 alone without FSH was incapable of exhibiting these stimulatory effects. These findings suggest that thyroid hormones synergize with FSH to exert direct stimulatory effects on granulosa cell functions, including morphological differentiation, LH/human CG receptor formation and steroidogenic enzyme (3 beta-hydroxysteroid dehydrogenase and aromatase) induction. Hence, decreases in ovarian functions during the states of hypo- or hyperthyroidism may account for diminished responsiveness of the granulosa cells to FSH.     

Steroidogenesis by cultured granulosa cells aspirated from human follicles using pregnenolone and androgens as precursors

Human granulosa cells from Graafian follicles aspirated 3-4 h before the expected time of ovulation were incubated with various steroid substrates, including pregnenolone, androstenedione, testosterone and dehydroepiandrosterone (DHA). Steroid production after 3 and 10 h of incubation was determined by radioimmunoassay. Progesterone and 17alpha-hydroxyprogesterone were the major products of granulosa cells in control short-term cultures with endogenous substrates. The addition of pregnenolone increased the synthesis of progesterone and 17alpha-hydroxyprogesterone compared with the controls, although the response varied considerably between paired short-term cultures. Little or no oestradiol-17beta was produced from endogenous precursors or short-term cultures to which pregnenolone had been added; one follicle, however, produced similar amounts of oestradiol-17beta in the control cultures and after incubation with pregnenolone. When granulosa cells were cultured with various amounts of androstenedione, DHA or testosterone, large amounts of oestradiol-17beta were produced, especially in short-term cultures in which larger amounts of substrate were added. Progesterone production continued and progesterone was synthesized more rapidly or in greater amounts in some short-term test cultures than in the controls. The results indicate that human granulosa cells are one source of oestradiol-17beta during the preovulatory phase. The data support the two-cell theory for oestradiol synthesis, for granulosa cells do not appear to undertake steroid conversion via the 5-unsaturated pathway, but aromatize androgens known to be produced by thecal cells. It is also suggested that either androgens or oestradiol-17beta stimulate progesterone production by granulosa cells, at least in vitro.     

Steroid metabolism in the yolk sac placenta and endometrium of the tammar wallaby, Macropus eugenii

Yolk sac and endometrial tissue were obtained from tammar wallabies between 11 and 25 days after the removal of pouch young. Tissues were examined histologically and steroid-metabolizing enzymes were identified by incubation for 3 h at 37 degrees C in Medium 199 containing labelled steroid precursors. Yolk sac membrane (YSM) incubated with labelled pregnenolone produced a small amount of progesterone and pregnanediols; 80.5 +/- 8.4 (S.E.M.) % of the original substrate remained unmetabolized. Labelled androstenedione was metabolized to 5 alpha-androstane-3,17-dione and androsterone, and only 5.8 +/- 3.8% of the original substrate remained at the end of incubation. Incorporation of androstenedione or dehydroepiandrosterone (DHA) into phenolic compounds was low (0.5 +/- 0.1%). There was no evidence for the enzymes, arylsulphatase or sulphotransferase, in YSM. Endometrial tissue from the same animals metabolized pregnenolone, DHA and androstenedione, converted progesterone to androstenedione, and produced aqueous-soluble steroid conjugates. The results demonstrated that YSM contains enzymes associated predominantly with steroid catabolism and with incipient progesterone synthesis. The findings are discussed in relation to the histological appearance of the tissues and compared with placental steroid synthesis in eutherian mammals.     

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)     

Correlation of human follicular fluid inhibin activity with spontaneous and induced follicle maturation

We sought to correlate the inhibin activity of individual ovarian follicles (greater than 16 mm in diameter) from untreated (7 patients; 7 follicles), clomiphene-stimulated (150 mg/day; menstrual cycle days 5-9; 9 patients, 14 follicles), and human menopausal gonadotropin (hMG)-stimulated (150 IU/day; menstrual cycle days 3-11; 8 patients; 23 follicles) ovarian cycles and to correlate these results with the follicular fluid (FF) steroid concentration. Follicular aspirates were obtained via laparoscopy from 24 regularly menstruating patients when the diameter of the largest follicle reached 20 mm, as determined by serial ultrasonography. FF concentrations of estradiol, progesterone, testosterone, 17-hydroxyprogesterone, and androstenedione were determined by RIA. Inhibin activity was determined using the inhibition of basal 24-h FSH secretion by dispersed rat anterior pituitary cells. Inhibin values were highest among the follicles aspirated from those patients who received hMG [277 +/- 31 (+/- SE) U/ml] compared to untreated subjects (51 +/- 13 U/ml) or those who received clomiphene (96 +/- 14 U/ml). Estradiol was highest in FF from untreated patients (2295 +/- 1155 ng/ml) compared to levels in patients who received hMG (368 +/- 1.76 micrograms/ml) or clomiphene (1049 +/- 174 ng/ml). FF progesterone values were highest in untreated patients (9.4 +/- 2.59 micrograms/ml) compared to those in hMG-treated (5.04 +/- 1.76 micrograms/ml) and clomiphene-treated patients (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values were similarly higher in the untreated (1.55 +/- 0.21 micrograms/ml) and clomiphene-treated (2.54 +/- 0.27 micrograms/ml) patients than in the hMG-treated group (0.73 +/- 0.09 micrograms/ml). FF androstenedione (untreated, 50.7 +/- 30 ng/ml; clomiphene-treated, 73.4 +/- 23.4 ng/ml; hMG-treated, 60.2 +/- 19.8 ng/ml) and testosterone (6.66 +/- 2.45, 5.98 +/- 1.46, and 6.39 +/- 2.16 ng/ml, respectively) concentrations in all three patient groups were similar. In untreated patients, there was a highly significant positive correlation between intrafollicular inhibin activity and FF estradiol, testosterone, and androstenedione concentrations and a statistically significant negative correlation between intrafollicular inhibin activity and FF progesterone concentrations. Patients receiving clomiphene therapy demonstrated at least two different response patterns, one with a positive and one a negative correlation between intrafollicular inhibin activity and FF steroid concentrations. The patients receiving hMG therapy had no statistically significant correlation between intrafollicular inhibin     

Increased steroid production by the ovarian stromal tissue of postmenopausal women with endometrial cancer.

An increase in ovarian steroid secretion could play a role in the pathogenesis of endometrial cancer in postmenopausal women. The present study was undertaken to investigate steroid production by isolated ovarian stromal tissues of postmenopausal women with endometrial cancer and to study the effect of LH and insulin on ovarian steroidogenesis in postmenopausal women. Ovarian stromal tissue was obtained from 10 postmenopausal women with endometrial cancer and 8 women without cancer. The stroma was incubated in either the medium alone or the medium to which was added LH (50 ng/mL) or insulin (500 ng/mL). The ovarian stroma of postmenopausal women with cancer released significantly more androstenedione (A), testosterone, and dehydroepiandrosterone than that of women without cancer. Addition of LH resulted in a significant increase in A, testosterone, dehydroepiandrosterone, and progesterone release compared to that with vehicle alone. Addition of insulin stimulated the release of A from the ovarian stroma of women with cancer, but had no effect on the normal postmenopausal ovarian stroma. These results indicate that the ovarian stroma of postmenopausal women with endometrial cancer secrete significantly greater amounts of androgens than those of women without cancer and that both LH and insulin may be important factors contributing to this increase in ovarian steroidogenesis.     

Pathways of testosterone synthesis in decapsulated testes of mice.

In order to study the temporal relations in the biogenesis of testosterone, decapsulated testes of adult mice were incubated with carbon-14-labelled sodium acetate and attempts were made to isolate the most likely intermediates. Considerable quantities of radiochemically homogeneous squalene, lanosterol, cholesterol, testosterone and androstenedione, but no pregnenolone, progesterone, 17-hydroxypregnenoline, 17-hydroxyprogesterone, dehydroepiandrosterone, pregnenolone sulphate or dehydroepiandrosterone sulphate were isolated. The same pattern of incorporation was found when gradually increasing amounts of non-labelled pregnenolone, progesterone, 17-hydroxypregenenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate or testosterone were added to the system as "trapping agents" or when Leydig cell preparations rather than decapsulated testes were used. The presence of 10 mIU of HCG greatly enhanced the de novo formation of testosterone, androstenedione and 5alpha-dihydrotestosterone but did not change the pattern of acetate incorporation. Radioimmunoassays of the incubation medium with or without added HCG, and carried out at different periods of time indicated the presence of gradually increasing amounts of testosterone and androstenedione together with some 5alpha-dihydrotestosterone, whereas only trace amounts of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and dehydroepiandrosterone were present. An analysis of the incubated testes revealed that the addition of HCG significantly enhanced the content of testosterone, androstenedione and 5alpha-dihydrotestosterone. Little or no increase was observed as far as pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone or dehydroepiandrosterone were concerned. It is concluded that decapsulated testes of mice synthesize de novo testosterone from sodium acetate under conditions in which the formation of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnenolone sulphate and 17-hydroxypregnenolone sulphate cannot be demonstrated.     

EFFECT OF OESTROGEN TREATMENT ON TESTICULAR LH/hCG RECEPTORS AND ENDOGENOUS STEROIDS IN PROSTATIC CANCER PATIENTS

LH/hCG binding and concentrations of pregnenolone, progesterone, 17-hydroxy-progesterone, androstenedione, testosterone and 5 alpha-dihydrotestosterone were measured in testicular tissue obtained from patients undergoing orchiectomy for prostatic cancer. One group of the orchiectomized patients had received an injection of 80 mg polyestradiol phosphate 1-9 days before the operation, another group were treated with monthly injections of the oestrogen for several months, and the remainder were not treated with oestrogen. In non-oestrogen treated patients (n = 8) the hCG-binding capacity was 28.0 +/- 15.0 (SD) ng/g and the equilibrium association constant of binding was 0.69 +/- 0.38 x 10(10) M-1. The binding capacity was significantly (P less than 0.025) lower (10.3 +/- 8.7 ng/g) in patients (n = 6) receiving the first oestrogen injection 3-9 days prior to the operation. A further decline to the level of 1.77 +/- 1.03 ng/g occurred in patients (n = 3) treated over 3 months with the oestrogen injections. At the same time, no significant changes were observed in peripheral serum LH and FSH levels. In the testicular endogenous steroid levels, statistically significant decreases were seen 9 days after the oestrogen injection in pregnenolone (from 505 +/- 315 ng/g to 138 +/- 86 ng/g) and in testosterone (from 669 +/- 243 ng/g to 254 +/- 49 ng/g) whereas no significant changes could be seen in the levels of the other steroids analysed. This study gives further evidence for the direct inhibitory action of oestrogens on human testicular steroidogenesis and suggests that the loss of testicular luteinizing hormone receptors may be one facet of this inhibitory action.     

Inhibin bioactivity and pituitary cell mitogenic activity from cultured chicken ovarian granulosa and thecal/stromal cells

A sensitive bioassay for inhibin based on the suppression of FSH release from cultured sheep anterior pituitary cells was used to determine whether inhibin is present in the preovulatory follicle in the domestic hen. Granulosa and thecal/stromal layers were separated from the five largest (F1-F5) yellow yolky follicles in the ovary and incubated in culture medium for 18 h. Inhibin was found predominantly in the media in which granulosa layers had been incubated. There was a progressive increase in the amount of inhibin produced per mg granulosa layer protein during the 5-6 days before ovulation. The ovary was observed to contain a growth factor which stimulated the proliferation of ovine pituitary cells. Thecal/stromal layer-conditioned medium (ThCM) but not granulosa layer-conditioned medium had a dose- and time-dependent mitogenic effect on cultured sheep pituitary cells. The maximal mitogenic effect achieved for ThCM was four to fivefold greater than control media and was significantly higher than the maximal mitogenic effects of epidermal growth factor (250 ng/ml; 1.5 x control) and transforming growth factor-beta (500 ng/ml; 1.2 x control). It is concluded that inhibin is produced by the granulosa layers in the large yellow yolky preovulatory ovarian follicles of the domestic hen. The thecal/stromal layers in these follicles produce a potent mitogenic factor, not produced by the granulosa layers, which stimulates the division of ovine anterior pituitary cells in vitro.     

Regulation of steroid production in cultured porcine thecal cells by transforming growth factor-beta.

Evidence that transforming growth factor-beta (TGF beta) is produced by porcine thecal cells and acts upon porcine granulosa cells suggests that this peptide may be a local regulator of follicular function in this species. The objective of the present study was to investigate the effects of TGF beta on steroidogenesis in thecal cells from 4-6 mm follicles of prepubertal gilts. In this culture system, cells undergo functional luteinization such that production of androstenedione, the major steroid product in 24 h incubations, declines, and in the presence of luteinizing hormone (LH) (250 ng/ml) and insulin (1 micrograms/ml), progesterone production increases over a 3-day culture period. TGF beta (0.1-10 ng/ml) had no effect on production of androstenedione from endogenous precursors in the presence or absence of LH, although there was a slight inhibition of androstenedione production in the presence of exogenous progesterone (up to 23%). As the cells luteinized in culture, the increase in progesterone production in response to LH increased (day 1, 4.4-fold; day 3, 13-fold). TGF beta at concentrations as low as 0.1 ng/ml caused marked (up to 90%) inhibition of LH-stimulated progesterone production in day 3 cultures. In the presence of TGF beta (10 ng/ml), the response to LH was completely abolished, and the response to dibutyryl cAMP was considerably attenuated (25% of controls). Since the primary site of action of TGF beta appeared to be distal to cAMP formation, the effect of TGF beta on conversion of exogenous 22-hydroxy-cholesterol and pregnenolone to progesterone was determined in day 3 cultures. 22-Hydroxycholesterol and pregnenolone restored progesterone production to at least 80% and 89% of controls, respectively. While the primary inhibitory action of TGF beta appears to be exerted distal to cAMP formation, neither cholesterol sidechain cleavage nor the 3 beta-hydroxysteroid dehydrogenase: delta 5-delta 4 isomerase reactions are primary targets of this factor. Together with evidence of thecal production of TGF beta, the results of this study indicate that this peptide may be an autocrine regulator of thecal steroidogenesis.