Androgen metabolites in bovine follicular fluid

The values of C21-steroids, Delta4-androgens, estrogens as well as 5alpha-reduced steroids have been determined in follicular fluid obtained from superovulated and untreated cows. In the three cows treated with a hormone regimen to induce superovulation, the levels of progesterone and estradiol determined in 3 to 6 follicles per cow ranged from 65 to 448 ng/ml and 1.9 to 8.6 ng/ml, respectively while the concentrations of androstenedione and testosterone varied between 1.5 to 2.5 ng/ml. Low levels of dihydrotestosterone and androstane-3alpha, 17beta-diol (approximately 30 to 50% of Delta4-androgens) were found in the bovine follicular fluid. In untreated cows, the follicular steroid concentrations were divided into two groups on the basis of the ratio between estrogen and Delta4-androgen concentrations. In estrogen-rich follicles, the ratio of estrogens Delta4- androgens was higher than 1 and in estrogen-poor follicle, the ratio of estrogens Delta4- androgens was lower than 1. Pregnenolone, dehydroepiandrosterone, androst-5-ene-3beta, 17beta-diol, progesterone, androstenedione and testosterone levels were not significantly different in the two groups while the levels of estradiol and estrone were approximately 100-fold higher in the estrogen-rich group. The concentrations of 5alpha-reduced steroids particularly, dihydrotestosterone, androstane-3alpha, 17beta-diol and androsterone as well as their glucuronides which were found at values extremely low (under 1 ng/ml) were not significantly different in both groups. The results indicate that low levels of 5alpha-reduced steroids and their glucuronides are present in bovine follicular fluid and their concentrations remained fairly stable either in estrogen-rich or estrogen-poor groups.     

Influence of diet on plasma steroids and sex hormone-binding globulin levels in adult men

Several experimental studies have suggested that diet can alter the production and metabolism of steroids in men. The purpose of this study was to determine the levels of unconjugated steroids and steroid glucuronides as well as sex hormone-binding globulin (SHBG) among normal adult men who were either omnivorous or vegetarians. The participants were white volunteers ranging from 25-35 years of age and the blood samples were taken between 0900 h and 1000 h and between 1600 h and 1700 h for two consecutive days. No significant statistical change was found in plasma dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone and estradiol levels. Vegetarian group showed a higher levels of sex hormone-binding globulin (SHBG) while the free androgen index (FAI; calculated by the ratio testosterone/SHBG) was lower in this group. Although the concentrations of androsterone glucuronide were higher in vegetarian group, the vegetarians had a 25-50% lower level of androstane-3 alpha, 17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide. Our data further indicate that both, androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide concentrations are significantly correlated with SHBG levels and with the FAI values. The increases in androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide levels in the omnivorous group are probably a consequence of the elevation of the FAI. Our data suggest that in a vegetarian group, less testosterone is available for androgenic action.     

Steroid glucuronides: Human circulatory levels and formation by LNCaP cells

We studied the relationship between circulating androsterone glucuronide, androstane-3,17β-diol glucuronide and androstane-3β,17β-diol glucuronide concentrations and adrenal as well as testicular C-19 steroids in men. Among the three 5-reduced steroid glucuronides, androsterone glucuronide is the predominant C-19 steroid measured in plasma and its levels are markedly elevated compared to those of the non-conjugated steroid. The marked rise in testosterone during puberty was strongly correlated with the increase in both androsterone glucuronide and androstane-3,17β-diol glucuronide, thus suggesting that testicular C-19 steroids are the main precursors of the steroid glucuronides. We also found that the presence of testicular androgen in plasma contributes to approx. 70% of plasma androsterone glucuronide and androstane-3,17β-diol glucuronide. Our data suggest that the adrenal C-19 steroids remaining in circulation after castration in men are converted into potent androgen which are then glucuronidated by UDP-glucuronyltransferase. We also demonstrated that the human prostate cell line LNCaP is capable of converting to a large extent androstenedione into androsterone glucuronide. Our data further confirm that glucuronidation is a major pathway of steroid metabolism in steroid target tissues.     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Effects of flutamide and aminoglutethimide on plasma 5 alpha-reduced steroid glucuronide concentrations in castrated patients with cancer of the prostate

Plasma levels of androstane-3 alpha, 17 beta-diol glucuronide (3 alpha-diol-G) and androsterone glucuronide (ADT-G) have been found to be effective markers of C-19 steroid metabolism in periphery in man. The present study has been performed in order to study in castrated patients the effect of antiandrogen administered alone or in combination with aminoglutethimide (AG) on the metabolism of adrenal C-19 steroid. Ten castrated patients with prostatic cancer received flutamide (FLU) alone for 2 months and, afterwards, the combined therapy of FLU and AG for 2 months. Antiandrogen treatment alone reduces the levels of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone glucuronide (DHEA-G) and androstenedione (4-ene-dione) by 43, 34 and 38% (P less than or equal to 0.01) respectively while dehydroepiandrosterone (DHEA), androst-5-ene-3 beta,17 beta-diol (5-ene-diol) and androst-5-ene-3 beta,17 beta-diol-glucuronide (5-ene-diol-G) levels show a nonsignificant inhibition. In these patients, plasma 3 alpha-diol-G and ADT-G concentrations are nonsignificantly stimulated to 122 and 143%. Moreover, when patients were receiving the combined administration of FLU and AG, adrenal C-19 steroids were further inhibited while both 3 alpha-diol-G and ADT-G show a small but nonsignificant decrease. Our data indicate that the antiandrogen increases the formation and/or the metabolism of adrenal C-19 steroids into steroid glucuronides.     

Relationships between unconjugated and sulphated steroids in porcine primary Leydig cell culture

Steroidogenesis in immature porcine Leydig cells was investigated in primary culture at 48-84 h under basal conditions and in the presence of hCG. The basal accumulation of unconjugated steroids was close to linear only during the first 4 h of study, whereas the sulphate-conjugated steroids accumulated essentially linearly over the 36 h experimental period. At the last time point, 95% of the steroids measured were sulphated. Stimulation with hCG (1 ng/ml) led to a still more pronounced sulphate conjugation, and approx 99% of the steroids measured were sulphated at 36 h. Under maximal stimulation with hCG (100 ng/ml) the sulphates accounted for 74% of the total steroids measured at 36 h. Testosterone, androstenedione, dehydroepiandrosterone, 5-androstene-3 beta, 17 beta-diol and estrone were usually quantitatively the most important unconjugated steroids, and sulphated dehydroepiandrosterone, estrone, testosterone and 5-androstene-3 beta, 17 beta-diol were the most important steroid sulphates, especially following maximal stimulation of the cultures. These data emphasize the importance of steroid sulphates in porcine testicular steroid metabolism. Under stimulation with hCG, there was a rapid response in testicular steroidogenesis, initially seen as a rapid increase in the secretion of unconjugated and sulphated steroids. At approx 4-12 h, the rate of sulphate conjugation appeared to reach or even to exceed that of steroid biosynthesis, which lead to stabilisation or a decrease in the concentrations of unconjugated steroids. Only high doses of hCG, 10-100 ng/ml, were then able to lead to a net accumulation of unconjugated steroids, at 24-36 h of incubation with hCG.     

A physiological dose of estradiol with progesterone affects striatum biogenic amines

The acute effect of estradiol and progesterone on dopamine and serotonin metabolism in rat striatum was studied. One subcutaneous injection of 17 beta-estradiol (300 ng) and progesterone (150 micrograms) into intact male rats increased plasma levels of these steroids, while testosterone, corticosterone, and estrone remained unchanged. Dehydroepiandrosterone, androstane-3 beta, 17 beta-diol and dihydrotestosterone remained undetectably low. Prolactin decreased and androstane-3 alpha, 17 beta-diol, and 17-OH progesterone increased, but less than estradiol and progesterone. Peak levels of striatal dopamine, dihydroxyphenylacetic acid, and homovanillic acid were observed 15-45 min after steroid injection with a return to control values after 45-60 min, while serotonin and 5-hydroxyindoleacetic acid levels were slightly decreased. An injection of estradiol (70 ng) with progesterone (70 micrograms) to ovariectomized female rats left plasma prolactin levels unchanged, while striatum dopamine and serotonin as well as their metabolite concentrations peaked 15-60 min after steroid injection and returned to control values after 45-75 min. To allow for a better comparison of the action of these steroids, the effect of estradiol or progesterone alone and in combination on the brain of ovariectomized rats was compared in the same experiment. A similar increase in metabolites of dopamine levels was observed after these steroids alone or in combination, while dopamine levels were increased only after progesterone alone or in combination with estradiol. An injection of estradiol or progesterone to ovariectomized rats led to peak steroid concentrations at approximately the same time in the brain and plasma. In addition, plasma and brain steroid levels were significantly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ceramide increases steroid hormone production in MA-10 Leydig cells.

Ceramide is known to have major roles in the control of cell proliferation, differentiation, and apoptosis. Recent studies also have shown that ceramide affects steroid production by JEG-3 choriocarcinoma cells, acutely dispersed rat Leydig cells, and ovarian granulosa cells, but the mechanism by which this occurs is unknown. Because ceramide induces apoptosis in many different cell types, we hypothesized that ceramide might affect steroidogenesis and/or induce apoptosis in MA-10 murine Leydig cells. To test this, MA-10 cells were incubated with either the water soluble C2-ceramide, (N-acetyl-sphingosine, 0.01-10 cm); bacterial sphingomyelinase (1-100 mU/ml); or C2-dihydroceramide (N-acetyl-sphinganine, 0.1-10 microM). The data show that N-acetyl-sphingosine significantly increased basal (0.87 +/- 0.2 vs. 0.42 +/- 0.09 ng/mg cell protein, P < 0.01) and human chorionic gonadotropin (hCG) stimulated progesterone (P) synthesis (204 +/- 12 vs. 120 +/- 5 ng/mg cell protein, P < 0.001); as did sphingomyelinase (basal P = 0.83 +/- 0.1 ng/mg cell protein, P < 0.01; hCG stimulated P = 173 +/- 7 ng/mg cell protein, P < 0.001). C2-dihydroceramide also increased basal P synthesis but was less effective than ceramide on a molar basis. Neither sphingomyelinase (100 mU/ml) nor ceramide (10 microM) had any effect on cAMP production or human chorionic gonadotropin binding; and neither induced any signs of apoptosis (FragEL DNA fragmentation assay and electron microscopy). Cells incubated with anti-Fas (300 ng/ml) demonstrated DNA fragmentation, nuclear condensation, and frequent apoptotic bodies, but had no change in P synthesis. These data show that ceramide significantly increases MA-10 Leydig cell P synthesis but does not induce apoptosis. The mechanism by which ceramide increases steroid hormone synthesis remains unknown but does not appear to be linked to the induction of apoptosis in MA-10 cells.     

Synthesis of deuterium-labeled 5 alpha-androstane-3 alpha, 17 beta-diol and its 17 beta-glucuronide.

Using unlabeled androsterone as starting material, 5 alpha-[16,16-2H2]androstan-3 alpha-ol-17-one was synthesized by exchange using deuterated potassium methoxide. This labeled androsterone product was reduced by sodium borodeuteride, which gave predominantly trideuterated 5 alpha-androstane-3 alpha, 17 beta-diol. The labeled androstanediol was conjugated with glucuronide by using the Koenig-Knorr reaction with methyl-1-bromo-1-deoxy-2,3,4-tri-O-acetyl-alpha-D-glucopyranosuronate . The dominant product was identified by thermospray high-performance liquid chromatography/mass spectrometry (MS) and electrospray MS as 5 alpha-[16,16,17-2H3]androstane-3 alpha, 17 beta-diol, 17 beta-glucuronide.     

Induction of ovulation and spermatogenesis by hMG/hCG in hypogonadotropic GH-deficient patients.

Nine female and 20 male hypogonadotropic GH-deficient patients were studied for sexual development by hCG/hMG. In the female patients, gonadotropin therapy was started at the mean age of 22.7 +/- 2.1 years. The administration of progesterone induced withdrawal bleeding at an average of 2.77 +/- 1.94 years after the initiation of hMG/hCG therapy in 8 of the 9 patients studied. Of 6 patients who had been confirmed as positive in a gestagen test, induction of ovulation by hMG/hCG was observed in 5 patients at an average of 5.58 +/- 1.23 years after the onset of therapy, but not in the remaining patient who had been given estrogen and progesterone 4 years 9 months prior to the initiation of the gonadotropin therapy. In male patients, gonadotropin therapy was started at the mean age of 23.6 +/- 5.7 years. Seminal fluid was obtained by masturbation and brought to our clinic in the morning. Of the 20 patients, 19 patients could be observed once a month regularly. Of the 19 patients, spermatozoa could be detected at a mean period of 2.19 +/- 0.87 years after initiation of hCG/hMG therapy in 18, but not in the remaining patient, after 5 years of therapy, who did not receive hCG/hMG regularly. The sperm count exceeded 20 x 10(6)/ml and more in 12 and was lower than that in 8 patients after 3 years of the therapy. No side effects were observed in female patients, but gynecomastia developed in 2 of the 20 male patients. These data suggest that gonadotropin therapy for hypogonadotropic GH-deficient patients is effective in promoting ovulation and spermatogenesis despite the initial replacement therapy with sex hormones.     

Effects of hyper- and hypoprolactinemia on gonadotropin secretion, rat testicular luteinizing hormone/human chorionic gonadotropin receptors and testosterone production by isolated Leydig cells

The effect of prolactin (Prl) on gonadotropin secretion, testicular luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors, and testosterone (T) production by isolated Leydig cells has been studied in 60-day-old rats treated for 4 days, 4 and 8 weeks with sulpiride (SLP), a dopaminergic antagonist, or for 4 days and 4 weeks with bromocriptine (CB), a dopaminergic agonist. Plasma Prl concentrations were significantly greater in the SLP groups (204 +/- 6 ng/ml) and lower in the CB groups (3.0 +/- 0.2 ng/ml) than those measured in the control groups (54 +/- 6 ng/ml). The plasma concentrations of gonadotropin were not affected by a 4-day treatment with SLP or CB, nor were they after a 4-week treatment with CB. However, the hyperprolactinemia induced by an 8-week treatment with SLP was associated with a reduced secretion of gonadotropin (LH, 16 +/- 4 vs. 35 +/- 6 ng/ml; FSH, 166 +/- 12 vs. 307 +/- 14 ng/ml). In SLP-induced hyperprolactinemia, a 30% increase in the density of the LH/hCG testicular binding sites was observed (178 +/- 12 fmol/mg protein), whereas a 60% decrease was measured in hypoprolactinemia (55 +/- 5 vs. control 133 +/- 5 fmol/mg protein). Plasma T levels were increased in 4-day and 4-week hyperprolactinemic animals (4.3 +/- 0.4 and 3.9 +/- 0.4 ng/ml, respectively), but returned to normal levels in the 8-week group (3.0 +/- 0.5 vs. C: 2.3 +/- 0.2 ng/ml). No T modifications were observed in hypoprolactinemic animals. Two distinct populations of Leydig cells (I and II) were obtained by centrifugation of dispersed testicular cells on a 0-45% continuous Metrizamide gradient. Both possess LH/hCG binding sites. However, the T production from Leydig cells of population II increased in the presence of hCG, whereas that of cell population I which also contain immature germinal cells did not respond. The basal and stimulated T secretions from cell populations I and II obtained from CB-treated animals were similar to controls, whereas from 4 days to 8 weeks of hyperprolactinemia, basal and hCG induced T productions from cell population II decreased progressively. These data show that hyperprolactinemia causes, in a time-dependent manner, a trophic effect on the density of LH/hCG testicular receptors; reduces basal and hCG-stimulated T production from isolated Leydig cells type II; and results in an elevated plasma T concentration which decreases with time. The latter suggests a slower T catabolism and/or an impaired peripheral conversion of T into 5 alpha-dihydrotestosterone (DHT). Although hypoprolactinemia is associated with a marked reduction in testicular LH receptors, it does not affect T production.     

Estrogen-stimulated glucuronidation of dihydrotestosterone in MCF-7 human breast cancer cells

The non-aromatizable androgen dihydrotestosterone (DHT) has been shown to exert a potent inhibitory effect on the proliferation of some human breast cancer cell lines. DHT, however, has little or no significant inhibition on MCF-7 cell proliferation in either the presence or absence of estradiol (E₂). Since the metabolism of DHT into non-active compounds may be responsible for the observed lack of androgenic effect in this cell line, we have investigated the metabolic fate of labeled DHT in MCF-7 cells. A time course incubation was performed with 1 nM [³H]DHT and analysis of the various metabolites formed revealed a time-dependent increase in glucuronidated steroids which was stimulated more than 4-fold by 0.1 nM E₂. The major glucuronidated steroid was androstane-3, 17β-diol in both control and E₂-stimulated cells, comprising 22 ± 1.2% and 30 ± 0.6% of the total radioactivity in the medium, respectively. Other steroid glucuronides observed included DHT, androstane-3β, 17β-diol, and androsterone, all of which were elevated in the E₂-treated cells relative to control values. The present data show that E₂ exerts a stimulatory effect on the glucuronidation of androgens and their metabolites in the estrogen-dependent breast cancer celll line MCF-7. Since glucuronidation is an effective means of cellular elimination of active steroids, such a pathway may be considered as a possible site of regulation of breast cancer cell growth by hormones.     

A simple technique for collecting blood of testicular origin: application to in vivo studies on testicular steroidogenesis in rats and Macaca fascicularis

A technique for rapidly collecting blood of testicular origin is described, one which can provide sufficient plasma amounts to investigate some steps of testicular steroid biogenesis in vivo in 2 species. In adult male rats, testosterone (T), androstenedione (4A) and 5-androstenediol (5AD) were determined in pampiniform plexus testicular venous blood (PPTV) and peripheral (PV) blood samples before and 2 h after human Chorionic Gonadotropin (hCG). PPTV concentration of 5AD was 0.83 +/- 0.1 ng/ml (mean +/- SEM) with a PPTV/PV ratio of 7.0 +/- 1.0, comparable to a PPTV/PV ratio for 4A of 5.8 +/- 1.8. After hCG, PPTV concentration of 5AD significantly increased to 1.28 +/- 0.15 ng/ml (P less than 0.05). Those data are in favor of a participation of 5-ene pathway to testicular biogenesis of T associated to a 4-ene pathway which is predominant. In adult male Macaca fascicularis, spermatic vein (SV) concentrations of 5AD and 4A were comparable (3.0 +/- 1.2 vs 4.3 +/- 1.0 ng/ml) as well as SV/PV ratios under basal conditions (3.5 +/- 0.9 vs 5.1 +/- 0.1), as well as 48 h after hCG, confirming in vivo that both 5-ene and 4-ene pathways are involved in testicular T biogenesis. Testicular production of estradiol (E2), estrone (E1) and their sulfates E2S and E1S showed a SV/PV ratio significantly higher than 1 (3.4 +/- 0.6; 2.4 +/- 0.1; 1.7 +/- 0.2 and 1.6 +/- 0.2, respectively).     

Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles

It was hypothesized that growth divergence of dominant and subordinate follicles during Wave 1 and growth termination of the dominant follicle would be associated with changes in the number of gonadotropin receptors on granulosa cells and estradiol in follicular fluid. To test this hypothesis, follicular development of 16 Holstein heifers was monitored by ultrasound, and follicles were collected on Days 2,4,6 and 10 (Day 0 = ovulation). Dominant follicles were compared across days, whereas dominant and largest subordinate follicles were compared on Days 2 and 4 only. The numbers of LH and FSH receptors on the granulosa cells of dominant follicles did not differ significantly over Days 2, 4, 6 and 10. In contrast, concentrations of estradiol in follicular fluid decreased (P < 0.05) from Days 2 to 10 (373 +/- 150 to 42 +/- 12 ng/ml) and concentrations of progesterone in follicular fluid increased (P < 0.05) from Days 2 to 10 (12.2 +/- 2.3 to 24.4 +/- 4.8 ng/ml). Correspondingly, the ratio of estradiol:progesterone in the dominant follicles decreased (P < 0.05) from Days 2 to 10. Comparisons between dominant and subordinate follicles indicated greater (P < 0.05) estradiol concentrations in the dominant follicle on Day 2, but the number of gonadotropin receptors was not different until Day 4. Thus, differences in concentrations of follicular fluid estradiol, but not numbers of granulosa cell gonadotropin receptors, were associated with the early growth divergence of dominant and subordinate follicles (Day 2) and the eventual growth termination of the dominant follicle (Day 10). Late divergence (Day 4) was associated with higher gonadotropin receptor numbers and follicular estradiol concentrations in the dominant than in the subordinate follicles. These results indicate that an increase in estradiol productivity of the selected dominant follicle occurred before an increase in the number of gonadotropin receptors.     

Stimulation of folliculogenesis in domestic cats with human FSH and LH

Folliculogenesis in response to exogenous stimulation by human urinary follicle stimulating hormone (huFSH) and human menopausal gonadotropin (hMG) was evaluated in the domestic queen (Felis catus). The role of LH and/or FSH in folliculogenesis was examined by measuring concentrations of estradiol 17beta (E(2)) and progesterone (P) in the serum. Additionally, changes in the number and size of follicles from before the administration of exogenous hormones to surgical oocyte collection were monitored. Findings indicated that in queens receiving huFSH or hMG followed by human chorionic gonadotropin (hCG) to induce ovulation, the numbers of follicles from 1 to 3 mm increase with statistical significance (P<0.005) from before the initiation of treatment to surgical collection of oocytes. Although E(2) concentrations in cats receiving hMG increased above baseline by the third exogenous hormone injection, mean E(2) concentrations did not increase in the groups that received both huFSH and hCG, or hCG only, until after the administration of hCG. This suggests that the exogenous administration of LH contained in both hMG and hCG was necessary for E(2) to rise to levels associated with estrus.     

Androstenedione metabolism in epithelial cells derived from early-lactation human milk

Epithelial cells derived from duct epithelium were cultured from early lactation human milk in medium supplemented with 15% fetal calf serum, insulin (0.3 u/ml), cortisol 21-sodium succinate (6 micrograms/ml) and amikacin (50 micrograms/ml). The capacity of these cells to metabolize androstenedione to estrone, estradiol and C19 metabolites was studied during continuous culture. After extraction of the medium, the products were subjected to phenolic partition and separated by thin-layer and paper chromatography, followed by recrystallization to constant specific activity. The study demonstrated a progressive increase in the formation of estrone and testosterone over the first 24 h in culture, while estradiol formation showed an initial 2-4 h lag, then increased slowly. The C19 compounds identified were androsterone, 5 alpha-androstanedione, epiandrosterone, dihydrotestosterone and etiocholanolone. 5 alpha-Androstanedione and androsterone were the major 5 alpha-reduced metabolites. Since these cells are derived from normal duct epithelium, their metabolic characteristics may be more representative of normal breast tissue than those of tissue removed from patients with pathological breast disorders.     

Secretion of follicle-regulatory protein by porcine granulosa cells

Follicle-regulatory protein (FRP) affects ovarian steroidogenesis and thus follicular maturation. However, secretion of FRP by cells from different-sized follicles as well as the modulation of FRP production by gonadotropins and locally produced steroids are unknown. To evaluate which cell type secretes FRP, theca and granulosa cells were obtained from porcine follicles. In addition, the effects of follicle-stimulating hormone (FSH) and steroids on FRP secretion from granulosa cells of small (less than 3 mm), medium (3-6 mm), and large (greater than 8 mm) porcine follicles and theca cells of large follicles were determined. Granulosa cells were obtained from follicular aspirates, whereas theca cells were recovered after digestion of the stereomicroscopically removed thecal layer. Both were cultured in monolayer in serum-free medium. Granulosa cells were treated as follows: 1) control; 2) FSH (250 ng/ml); 3) progesterone (500 ng/ml, 3 micrograms/ml), or estradiol-17 beta (500 ng/ml, 4 micrograms/ml), or dihydrotestosterone (500 ng/ml, 1 microgram/ml); 4) FSH + progesterone, or estradiol-17 beta, or dihydrotestosterone. Theca cells received the same treatment except that human chorionic gonadotropin (hCG) (5m IU/ml) was used in place of FSH. At 48 or 96 h, media were removed and FRP was quantitated by an Enzyme-Linked Immunosorbent Assay (ELISA). FRP was identified in granulosal medium from follicles of all sizes, but was not present in thecal cultures. At 48 h, granulosa cells from small and medium-sized follicles produced more FRP (20.04 +/- 4.4, 35.42 +/- 4.1 immunoreactive units [IRU]) than cells from large (3.53 +/- 0.97 IRU) follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.     

Effects of prolactin on progestin secretion by human granulosa cells in culture

Concentrations of human prolactin (hPrl) greater than or equal to 600 ng/ml produced inhibition of progestin production in cultures of granulosa cells pooled from follicles of women stimulated with clomiphene citrate-human chorionic gonadotropin (hCG). However, cells collected from follicles of human menopausal gonadotropin (HMG)-hCG-treated patients did not demonstrate a significant reduction in progestin secretion in response to hPrl. We conclude that high concentrations of hPrl can result in inhibition of steroidogenesis, but the expression of the inhibitory effects of Prl depends upon the hormonal treatments used to stimulate follicular growth.