Interleukin-2 affects steroidogenesis and numbers of bovine ovarian granulosa cells but not thecal cellsin vitro

The effect of recombinant bovine interleukin-2 (IL-2) on steroidogenesis and numbers of bovine ovarian granulosa and thecal cells has been studied. Granulosa cells have been examined from both small (surface diameter ≤5 mm) and large (≥8 mm) follicles, whereas thecal cells from only large follicles were utilized. Estradiol and progesterone production per cell by granulosa cells from large follicles was 2- to 3-times greater than those from small follicles. Increasing doses of IL-2 significantly attenuated FSH-induced estradiol production by cells from small follicles but not large follicles. In general, progesterone production per cell by granulosa cells was almost double that of thecal cells. Moreover, IL-2 significantly attenuated FSH-induced progesterone production by granulosa cells from small and large follicles but had no effect on LH-induced progesterone or and-rostenedione production by thecal cells. Co-treatment of TNFα with IL-2 enhanced the responsiveness of granulosa cells to IL-2. The effect of IL-2 on the numbers of granulosa and thecal cells were studied independently under serum-free conditions and media enriched with 10% fetal calf serum. In serum-free medium containing insulin, IL-2 dosage significantly increased numbers of granulosa cells from large follicles, whereas IL-2 had no effect on numbers of granulosa cells from small follicles or thecal cells from large follicles. When cells were grown in medium enriched with serum, increasing doses of IL-2 significantly inhibited numbers without affecting viability of granulosa cells from small follicles, but had no effect on numbers of thecal cells. Thus, it appears that granulosa cells are more sensitive to IL-2 than are thecal cells. Approved for publication by the Director, Oklahoma Agriculture Experiment Station. This research was supported in part under project H-2088.     

Site at which FSH regulates estradiol-17beta biosynthesis in Sertoli cell preparations in culture.

Sertoli cells were isolated from testes of 20-day-old rats and were maintained in primary culture. The ability of these cells to synthesise estradiol-17beta from a variety of exogenous substrates, progesterone, testosterone,androstenedione, 19-hydroxyandrostenedione and 19-hydroxytestosterone in the presence and absence of follicle-stimulating hormone (FSH) was examined. In the presence of each of the substrates alone for 24 h the rate of estradiol-17beta synthesis was very low. FSH (NIH-FSH-S11, 5 mug/ml) stimulated estradiol-17beta synthesis 75-fold when added to medium containing testosterone (5 X 10(-7)M) but caused only marginal stimulation when added to medium containing progesterone (5 X 10(-7) M). Both FSH and dibutyryl cyclic AMP (bu2cAMP) stimulated the conversion of each of the substrates, androstenedione, 19-hydroxyandrostenedione and 19-hydroxytestosterone to estradiol-17beta, and the effects were similar to those observed in the presence of testosterone. These data indicate that, under the culture conditions employed, progesterone is not an effective substrate for conversion to estradiol-17beta by Sertoli cells. Estradiol-17beta synthesis was stimulated by FSH in the presence of the C19 steluences the conversion of androgens to estrogens, either directly or indirectly, at the aromatisation step (i.e. the conversion of 19-hydroxylated androgens to estrogens).     

Tumor necrosis factor-α (TNF-α) inhibits steroidogenesis of bovine ovarian granulosa and thecal cells in vitro

The effect of recombinant bovine tumor necrosis factor-α (TNF-α) on steroidogenesis and numbers of bovine ovarian granulosa and thecal cells has been studied, and specific binding sites for ¹²⁵I-TNF-α on ovarian cells have been determined. Granulosa cells have been examined from small (surface diameter 1–5 mm) follicles, whereas thecal cells from large (≥ 8 mm) follicles were utilized. Increasing doses of TNF-α significantly attenuated insulin- and IGF-I-induced estradiol production by granulosa cells from small follicles, but had no effect on basal estradiol production. Moreover, TNF-α significantly attenuated insulin- and LH-induced androstenedione production by thecal cells from large follicles. TNF-α had little or no effect on the numbers of granulosa and thecal cells in these same studies. Specific high-affinity, low-capacity binding of ¹²⁵I-TNF-α was also demonstrable in granulosa and thecal cells. Thus, it appears that TNF-α inhibits insulin-and IGF-I-induced estradiol production by granulosa cells and androstenedione production by thecal cells via TNF-α binding to its own receptor.     

Effect of androgens on the biosynthesis of estradiol-17beta by isolated periovulatory rat follicles.

To analyse the mechanism for the earlier reported decline in estrogen synthesis by the periovulatory rat follicle, prepubertal rats injected with 8 IU PMS on day 28 were killed following the endogenous gonadotrophin surge (15.00–18.00) on day 30. Isolated preovulatory follicles were incubated for 2 h in a chemically defined medium. Steroids were measured by specific RIA methods. Follicles exposed in vivo to the gonadotrophin surge and extirpated 19.00–22.00 h on day 30 secreted significantly lower amounts of androstenedione, testosterone and estradiol-17β but significantly higher amounts of progesterone than did follicles extirpated from rats in which the gonadotrophin surge had been prevented by a Nembutal injection. Secretion of estradiol-17β by follicles isolated following the endogenous gonadotrophin surge remained low when LH, FSH or dibutyryl cyclic 3′,5′-AMP was added to the medium. However, the addition of testosterone (0.1–1 μg/ml) or androstenedione (1 μg/ml) to the incubation medium restored estradiol biosynthesis to values similar to those seen prior to gonadotrophin exposure. There was no effect of 5α-dihydrotestosterone or 17α-hydroxyprogesterone on the estradiol-17β synthesis. The results indicate that cleavage of the 17:20 sidechain rather than the aromatase enzyme limits estradiol synthesis in the periovulatory follicle following the gonadotrophin surge. It is suggested that the combined action of LH and FSH of the gonadotrophin surge might explain the lack of inhibitory effect on the aromatase enzyme recently reported by Katz and Armstrong (1976) 6–8 h after the injection of LH.     

In vitro estradiol-17 beta and testosterone production by ovarian follicles of the goldfish, Carassius auratus

In vitro production of estradiol-17 beta and testosterone by ovarian follicles of goldfish (Carassius auratus) at different developmental stages in response to human chorionic gonadotropin (HCG) was examined using 18-hr incubations. The vitellogenic follicles (primary and secondary yolk stage) produced estradiol-17 beta in response to HCG; follicles at the secondary yolk stage produced about five times more estradiol-17 beta than the primary yolk stage follicles. However, tertiary yolk stage follicles, which could be induced to mature in vitro by HCG treatment, did not produce estradiol-17 beta. HCG also stimulated testosterone production by follicles at all stages of development; maximal production was observed in tertiary yolk stage follicles. An indirect assessment of aromatase activity at each stage was made. Vitellogenic follicles (primary and secondary yolk stage) produced estradiol-17 beta when incubated with testosterone, the larger follicles producing about three times more estradiol-17 beta. Only low levels of estradiol-17 beta were produced by tertiary yolk stage follicles. Addition of HCG to primary yolk stage follicles slightly but significantly enhanced estradiol-17 beta production; however, no enhancement was seen with secondary and tertiary stage follicles. These results suggest that the decrease in estradiol-17 beta production in tertiary yolk stage follicles may be partly due to a decrease of aromatase activity at this stage.     

Regulation of citrate metabolism by androgen in the LNCaP human prostate carcinoma cell line

Citrate production and accumulation are characteristic physiological functions of the prostate gland that are regulated by testosterone and prolactin. Results reported here show that treatment of LNCaP cells with dihydrotestosterone (DHT) resulted in increased intracellular citrate and increased citrate accumulation in the medium. Moreover, DHT also caused an increase in both mitochondrial aspartate aminotransferase (mAAT) activity and the steady state level of pmAAT (precursor) mRNA. Androgen treatment increased the rate of citrate oxidation by LNCaP cells as it does in rat ventral prostate which suggests that DHT increased aconitase activity in LNCaP cells. The results reported here are consistent with the operation of the glutamate-aspartate-citrate pathway that we described for rat ventral prostate. In addition, these results provide the first evidence that androgen responsive functions associated with citrate metabolism are retained in LNCaP cells. In addition, and more important, these results suggest that the more aggressive PC-3 carcinoma cell line has a higher rate of citrate oxidation than the less aggressive LNCaP cell line. This could have significant implications for our understanding of the relationship between alterations in prostate citrate metabolism and expression of the malignant phenotype.     

Influence of cortisol on insulin- and insulin-like growth factor 1 (IGF-1)-induced steroid production and on IGF-1 receptors in cultured bovine granulosa cells and thecal cells

During stress, hyperactivity of the adrenal gland can directly and indirectly inhibit ovarian function. However, little evidence existed to support the notion that glucocorticoids could influence insulin-like growth factor 1 (IGF-1) action within the ovary. Therefore, the effect of cortisol on IGF-1-induced granulosa and thecal cell function was evaluated. Granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in the presence of 10% fetal calf serum and then cultured for an additional 2 d in serum-free medium with added hormones. Cortisol had little or no effect (p>0.05) on IGF-1-induced progesterone production by granulosa cells from both small (1–5 mm) or large (≥8 mm) follicles. Also, cortisol had little or no effect (p>0.05) on basal, insulin-, or IGF-1-induced estradiol production by granulosa cells from small or large follicles, or on the number of IGF-1 receptors in granulosa cells from small follicles. Cortisol had no effect (p>0.10) on insulin-induced granulosa cell numbers, but increased IGF-1-induced granulosa cell numbers. In thecal cells, doses of 1–100 ng/mL of cortisol increased (p<0.05) insulin- and IGF-1-induced thecal cell numbers by 10–20%, progesterone production by 18–36%, and androstenedione production by two- to fourfold. The estimated dose of cortisol necessary to stimulate 50% of the maximum androstenedione production in the presence of IGF-1 was 7 ng/mL. In contrast, cortisol decreased (p<0.05) the number of IGF-1 receptors in thecal cells by 45%. In conclusion, cortisol at physiological levels can directly influence ovarian follicular function in cattle, especially thecal androstenedione production.     

Inhibition of ovarian estradiol-17beta secretion by luteinizing hormone in prepubertal, pregnant mare serum-treated rats.

Serum estradiol-17beta levels, elevated prior to the luteinizing hormone (LH) surge, decline abruptly following the release of endogenous LH or the injection of exogenous LH. To investigate the mechanism of this decline, bovine LH (NIH-LH-B8) was administered to immature rats, in which follicular maturation and estrogen biosynthesis were induced by a non-ovulating dose of pregnant mare serum gonadotropin (PMS). Serum and ovarian estradiol-17beta concentrations fell detectably by 4h, and reached levels around 20% of the controls by 8h after iv injection of 10 mug LH. Concomitant decreases occurred in ovarian androgen concentrations, following an initial rise, and in the in vitro ovarian testosterone aromatizing enzyme activity. The LH-induced inhibition of the aromatase activity was found to be of a non-competitive type. It is proposed that two enzyme systems are inhibited as a result of the LH treatment: the C17,20-lyase and the C19 androgen aromatase, thereby leading to decreased concentrations of estrogens in the ovaries and blood.     

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.     

Comparison of the in vitro conversion of estradiol-17 beta to estrone of normal and neoplastic human breast tissue.

Specific activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) was measured in 48 tissue specimens of human female breast cancer and, in addition, 48 nonmalignant tissue specimens obtained in each case from the same cancer-bearing breast. In all cases the nonmalignant tissue showed greater conversion of estradiol-17 beta into estrone than the neoplastic tissues. In normal human breast tissue of premenopausal women specific enzyme activity depended on the phase of the MENSTRUAL CYCLE: the highest values of 17 beta-HSD activity were found in the early secretory phase. To determine the intracellular distribution of the 17 beta-HSD, purified microsomes, mitochondria, peroxysomes, lysosomes, nuclei and cytosol fractions were prepared. The purity of each fraction was monitored by marker enzymes. It was found that the 17 beta-HSD was mainly located in mitochondria and microsomes. Furthermore it could be demonstrated that the microsomal enzyme was bound tightly to the membranes of the endoplasmic reticulum, while the mitochondrial 17 beta-HSD was mainly associated with the outer membranes of the organelle. Kinetic parameters (Km-values, coenzyme requirements and maximal velocities) of a cytoplasmic, nuclear, mitochondrial and microsomal 17 beta-HSD of normal and neoplastic human mammary tissue were compared. Maximal velocity was highest in enzyme preparations of normal mammary tissue obtained from premenopausal women in the early secretory phase. Km-values wrere nearly identical in normal and neoplastic mammary tissue preparations (approx. 1 X 10(-6) M). NAD was more efficient than NADP as a cofactor. For the conversion of estradiol to estrone the optimum temperature was approximately 40 degrees C and the optimum pH 9.5. For the reduction of estrone the optimum pH was 6.5. Sulphydryl groups were shown to be essential for catalysis.     

Inhibition of aldosterone production by pinacidil in vitro.

Pinacidil, an antihypertensive agent that opens potassium channels, lowers plasma aldosterone levels in hypertensive patients by an unknown mechanism. In the present study, pinacidil's direct effects on production of aldosterone were assessed using isolated cells from bovine adrenal glomerulosa. Pinacidil was found to inhibit aldosterone production, both basally and during stimulation with either potassium, angiotensin II (Ang II), or adrenocorticotropic hormone (p less than 0.001), with half maximal inhibition occurring at 10(-5) M. As assessed by the exclusion of trypan blue from cells, pinacidil did not inhibit secretion through injurious effects on glomerulosa cells. Also, washing of cells previously exposed to pinacidil restored secretory responsiveness. Pinacidil did not alter cytosolic calcium (Ca2+) concentrations when aequorin was used as a photoluminescent indicator of Ca2+ levels, suggesting that pinacidil acted by a non-Ca(2+)-mediated mechanism. Consistent with direct inhibition of the late pathway in steroidogenesis was that pinacidil decreased conversion of pregnenolone and corticosterone to aldosterone. Pinacidil did not block binding of Ang II to its receptor, nor did it appear to affect adrenocorticotropic hormone-receptor binding, since stimulation by cyclic AMP, the post-receptor second messenger of adrenocorticotropic hormone, was also inhibited. In summary, pinacidil inhibited directly the adrenal's production of aldosterone. The mechanism whereby the inhibition occurred was unclear.     

Modulation of HLA-DR expression in epithelial cells by interleukin 1 and estradiol-17 beta

Both ultrapure human interleukin-1 (IL-1) and Escherichia coli derived recombinant IL-1 alpha and beta consistently induced the expression of major histocompatibility class II (HLA-DR) molecules in a human endometrial and a breast carcinoma cell line. [35S]Methionine incorporation into IL-1 induced, immunoprecipitable HLA-DR molecules demonstrated de novo synthesis of both light and heavy chains of the HLA-DR molecules. Lipopolysaccharide, recombinant interleukin-2 and recombinant interleukin-6 failed to induce HLA-DR expression in these epithelial cells. In contrast to the dramatic effect on HLA-DR expression, IL-1 had no effect on the epithelial cell proliferation. Pretreatment of T47D cells with estradiol-17 beta significantly decreased the IL-1 induced HLA-DR expression, and pretreatment of IL-1 with an IL-1 specific antibody, neutralized IL-1 action. These studies demonstrate that a cytokine (IL-1) and a sex steroid hormone estradiol-17 beta can interact to regulate the expression of HLA-DR molecules in epithelial cells.     

Stimulatory action of follice-stimulating hormone on estradiol-17 beta secretion by hypophysectomized rat ovaries in organ culture.

Ovaries of rats explanted in organ culture 2-3 days after hypophysectomy at 26 days of age synthesize ans secrete estradiol-17beta into the culture medium at approximately linear rates for 48 h. Addition of testosterone (5 times 10- minus 7M) to the culture medium occasionally caused small increases of up to 50 percent in rate of estradiol production. Significant increases (60-200 percent) in estradiol secretion resulted from addition of two different FSH preparations, in concentrations as low as 0.25 mug/ml in the absence of testosterone. In the presence of testosterone, the same FSH preparation caused much more marked increases in estradiol secretion of up to 900 percent. In the absence of testosterone, two different luteinizing hormone preparations failed to increase estradiol secretion significantly, although in the presence of testosterone, small increases were sometimes observed. It is concluded that FSH regulates estradiol biosynthesis in the hypophysectomized rat ovary by a specific stimulatory action on the aromatizing enzyme system.     

Melatonin directly suppresses steroid production by preovulatory follicles in the cyclic hamster

Abstract: The purpose of these studies was to investigate the effects of melatonin on the production of steroids (progesterone, testosterone, and estradiol) and cAMP by preovulatory follicles and to examine changes in melatonin concentrations in the ovary during the estrous cycle. Adult cyclic hamsters were used in this study. Melatonin concentrations in the ovary, pineal gland, and serum were measured at mid-light and mid-dark during the estrous cycle. Effects of melatonin on steroidogenesis by preovulatory follicles, thecae, and granulosa cells were examined, and its effect on cAMP production by preovulatory follicles was also investigated. Melatonin (0.1–10 ng/ml) had no effect on steroid production in the absence of hCG, but melatonin decreased progesterone and estradiol production by preovulatory follicles in a dose-dependent and time-dependent manner in the presence of hCG (100 mlU/ml). The target of melatonin was thecae but not granulosa cells, and melatonin significantly reduced cAMP production by preovulatory follicles. Melatonin concentrations in the ovary showed a similar phasic variation with high levels during mid-dark and low during mid-light, as in the pineal gland and serum. These results show that the ovarian melatonin levels also exhibit a circadian rhythm and suggest that the high melatonin milieu in the ovary may induce gonadal regression in the cyclic hamster.     

In vitro effects of thyroid hormones on gonadotropin-induced estradiol-17 beta secretion by ovarian follicles of rainbow trout, Salmo gairdneri

Ovarian follicles isolated from rainbow trout during early exogenous vitellogenesis were used to study the in vitro effects of thyroid hormones on salmon gonadotropin (GtH)-induced estradiol-17 beta (E2) secretion. Triiodothyronine (T3) alone did not alter E2 secretion but T3 in the presence of GtH (0.5 micrograms/ml) modified E2 secretion according to a biphasic dose-response curve. Maximum E2 secretion occurred at 1.9 x 10(-8) M T3; a concentration of 3.0 x 10(-7) M was inhibitory. T3 was more potent in stimulating GtH-induced E2 secretion than thyroxine. The stimulatory and inhibitory effects of T3 were consistent over a range of GtH concentrations (0.1-1.0 micrograms/ml). Cycloheximide (0.1-10 microM) decreased E2 secretion by GtH-treated follicles in a dose-dependent manner, but failed to overcome all the stimulatory effects of T3. Time course studies with follicles incubated with GtH, GtH + T3, GtH + cycloheximide, or GtH + T3 + cycloheximide indicated that T3 stimulation of GtH-induced E2 secretion occurs within 6 hr. It is concluded that thyroid hormones amplify the effects of GtH on E2 secretion by isolated ovarian follicles; at least a part of this effect does not require de novo protein synthesis.     

The in vitro effects of cyclic nucleotides, cyanoketone, and cycloheximide on the production of estradiol-17 beta by vitellogenic ovarian follicles of goldfish (Carassius auratus)

The effects of human chorionic gonadotropin (HCG), forskolin, cyclic nucleotides, the phosphodiesterase inhibitors IBMX and theophylline, cyanoketone, and cycloheximide on the production of estradiol-17 beta by isolated ovarian follicles of vitellogenic goldfish (Carassius auratus) were examined using 18-hr incubations. HCG and all test agents which are known to increase intracellular concentrations of cAMP significantly stimulated the production of estradiol-17 beta. However, dibutyryl cGMP was unable to stimulate estradiol-17 beta production at any concentration used (1-10 mM). Cyanoketone at a concentration of 1 micrograms/ml completely blocked forskolin-induced estradiol-17 beta production. Even in the presence of cyanoketone, however, forskolin stimulated conversion of exogenous testosterone to estradiol-17 beta in a dose-dependent manner, suggesting the involvement of an adenylate-cyclase system in the induction of aromatase activation by vitellogenic follicles of goldfish. Cycloheximide also completely abolished HCG-induced estradiol-17 beta production when this inhibitor was added within the first 1 hr after the addition of HCG. These results provide evidence that the stimulation of estradiol-17 beta by goldfish vitellogenic follicles in response to HCG is dependent upon the synthesis of new protein.     

Melatonin increases cleavage rate of porcine preimplantation embryos in vitro

Melatonin has been used to promote in vitro embryo development in different species. This study determined the effects of melatonin on in vitro porcine embryo development; in particular, cleavage rate, blastocyst rate, and blastocyst cell number. Starting 5 hr after insemination, porcine zygotes were cultured in porcine zygote medium 3 (PZM-3) culture medium supplemented with melatonin at increasing concentrations (10(-12) M, 10(-9) M, 10(-6) M, 10(-3) M). Melatonin at a concentration of 10(-9) M had a positive effect on cleavage rates, while the highest concentration of melatonin (10(-3) M) significantly decreased cleavage rates. Although blastocyst rates were not increased by 10(-9) M melatonin, blastocyst cell numbers were significantly higher for embryos subjected to 10(-9) M melatonin. The expression levels of the pro-apoptotic gene BAX and anti-apoptotic gene BCL2L1 in blastocysts were not affected by the presence of melatonin in the culture medium. To further study the protective properties of 10(-9) M melatonin against stressful conditions, hydrogen peroxide (0.01 mm) and heat (40 degrees C) were used during embryo culture. The addition of melatonin to embryos subjected to 40 degrees C for 3 hr increased cleavage rates, but had no protective effect for embryos subjected to 0.01 mm H(2)O(2), probably because the physiological levels of melatonin could not counteract the pharmacological levels of H(2)O(2). Our data indicate that 10(-9) M melatonin has a positive effect on porcine embryo cleavage rates and blastocyst total cell numbers and it might have a protective effect against heat stress.