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.     

Upregulation of steroidogenic enzymes and ovarian 17beta-estradiol in human granulosa-lutein cells by Cordyceps sinensis mycelium

There is increasing evidence that 17beta-estradiol (E2) directly influences the quality of maturing oocytes and thus the outcome of assisted reproduction treatment. Although Cordyceps sinensis (CS) mycelium, a Chinese herbal medicine, is believed to enhance libido and fertility in both sexes, the mechanism of its effect in women has not been determined. The aim of the present study was to evaluate the effects of CS on steroidogenic enzyme expression and E2 biosynthesis in human granulosa-lutein cells (GLC). We found that CS induced E2 production by GLC in a dose- and time-dependent manner and that a 3-h treatment with CS induced increased levels of mRNAs coding for the P450 side chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and aromatase. Western blot analysis demonstrated that, after treatment with CS for 3 h, protein levels of steroidogenic acute regulatory protein (StAR) and aromatase were upregulated while P450scc and 3beta-HSD levels showed no substantial change. New protein synthesis was required for CS-induced E2 production because it was abrogated by cycloheximide pretreatment. Addition of 22(R)-hydroxycholesterol, thus bypassing the need for StAR protein, did not induce as much E2 production as CS treatment, indicating that upregulation of StAR protein was not the only factor contributing to CS-induced steroidogenesis. Cotreatment of GLCs with CS and aminoglutethimide, an aromatase inhibitor, completely abolished CS-induced E2 production. In conclusion, treatment of GLCs with CS results in increased E2 production due, at least in part, to increased StAR and aromatase expression. These data may help in the development of treatment regimens to improve the success rate of in vitro fertilization.     

Modulation of porcine thecal cell aromatase activity by human chorionic gonadotropin, progesterone, estradiol-17 beta, and dihydrotestosterone

Porcine thecal cells synthesize estradiol, which may function as an intraovarian regulator of follicular growth. Production of estradiol by granulosa-cell aromatase is modulated by gonadotropins and local steroidal and nonsteroidal factors. Therefore, the effect of human chorionic gonadotropin (hCG) and physiological concentrations of steroids on aromatase activity of the thecal cells was determined. Theca was excised from large porcine follicles (greater than 10 mm diameter) and plated as monolayer cultures in 1 ml of serum-free medium. Twenty-four hours after culture, cells were treated as follows: 1) control; 2) hCG (5 IU); 3) progesterone (P, 3 micrograms), estradiol-17 beta (E, 4 micrograms), or dihydrotestosterone (DHT, 1 microgram); 4) hCG + P, E, or DHT. After 27, 30, 36, 48, and 72 h of culture, media were assessed for levels of P and E. Aromatase activity was determined by a radiometric assay. Levels of P in control media increased from 27 to 72 h. hCH significantly (p less than 0.01) increased P levels from 27 to 72 h of culture. Estrogen decreased (p less than 0.05) P levels at 36, 48, and 72 h compared to controls and also prevented the hCG-induced increase in P levels at these times. DHT significantly increased (p less than 0.05) P levels at 48 and 72 h. DHT + hCG reduced the hCG-associated increase in P concentration at 36 h and 72 h, but enhanced the hCG-induced increase in P levels at 48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol as an anti-aromatase agent in human breast cancer cells

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.     

Effects of arecoline on testosterone release in rats

Arecoline is one of the major components of betel nuts, which have been consumed as chewing gum in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 microg/kg) plus hCG via a jugular catheter. Blood samples were collected at several time intervals subsequent to the challenge. Rat anterior pituitary was treated with gonadotropin-releasing hormone in vitro with or without arecoline, and then the concentrations of luteinizing hormone (LH) in the medium were measured. Rat Leydig cells were purified by Percoll density gradient centrifugation and incubated with arecoline, hCG, forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or tetrandrine at 34 degrees C for 1 h. A single intravenous injection of arecoline resulted in an increase of the hCG-induced level of plasma T. Administration of arecoline (10(-8) to 10(-6) M) in vitro increased T production in Leydig cells. The stimulatory effect of arecoline on T release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10(-6) M), or 8-Br-cAMP (10(-5) M). By contrast, nifedipine, nimodipine, or tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute regulatory (StAR) protein expression compared with vehicle. These results suggested that arecoline stimulates testosterone production by acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17beta-hydroxysteroid dehydrogenase and enhancing the expression of StAR.     

Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate

Human breast cancer tissue contains enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol (E(2)) formation. In this tissue, E(2) can be synthesized by two main pathways: (1) sulfatase-transforms estrogen sulfates into bioactive E(2), and the (2) aromatase-converts androgens into estrogens. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization.In the present study, we demonstrated in T-47D and MCF-7 human breast cancer cells that norelgestromin (NGMN) (a metabolite of norgestimate) is a potent inhibitory agent of the estrone sulfatase activity. After 24h incubation of physiological concentrations of E(1)S (5 x 10(-9)mol/l) the inhibitory effect of NGMN at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 43+/-7, 74+/-4 and 97+/-2%, respectively, in T-47D cells; 25+/-4, 57+/-5 and 96+/-2% respectively, in MCF-7 cells. Comparative studies using medroxyprogesterone acetate (MPA) showed that this progestin also has an inhibitory effect on sulfatase activity, but significantly less intense than that of NGMN. The inhibition for MPA at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 31+/-5, 47+/-3 and 61+/-3%, respectively, for T-47D cells; 6+/-3, 20+/-3 and 63+/-4%, respectively, for MCF-7 cells.In conclusion, the present data show that NGMN is a very potent inhibitory agent for sulfatase activity in the hormone-dependent breast cancer cells, resulting in decreased tissue concentration of E(2). The clinical significance of this finding remains to be elucidated.     

The determination of the relationship between p97/VCP,small VCP-interacting protein,two ERAD proteins and steroidogenesis in Leydig cell lines

Background

In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse.

Methods and results

HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells.

Conclusion

We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.

     

Role of estrogen receptor-alpha and -beta in regulating leptin expression in 3T3-L1 adipocytes

We investigated the effects of the estrogen receptor-alpha (ERalpha) and -beta (ERbeta) in the regulation of leptin, resistin, and adiponectin expression in 3T3-L1 adipocytes. Mature adipocytes were exposed to estradiol (E2), ERalpha agonist (PPT (4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol)), ERbeta agonist (DPN (2,3-bis(4-Hydroxyphenyl)-propionitrile)), E2 with ERalpha antagonist (MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride)), and E2 with ERbeta antagonist (R,R-THC ((R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol)) at different concentrations. To clarify the expression and regulation of adipokines by ER subtypes, total RNA was extracted from cells and measured using quantitative PCR. Western blot analysis was performed to evaluate the protein expression of adipokines, ERalpha, and ERbeta. The leptin expression was significantly increased in the cells treated with high concentrations (10(-5) and 10(-6) mol/l) of the PPT (P < 0.01, P < 0.05). By contrast, the leptin expression decreased in a dose-dependent manner in the MPP-treated groups (P < 0.05). High concentrations (10(-5) mol/l) of R,R-THC with E2 (10(-7) mol/l) caused a significant increase of the leptin expression (P < 0.01). The leptin mRNA levels were positively correlated with the ERalpha mRNA levels (r = 0.584, P < 0.01) and negatively correlated with the ERbeta mRNA levels (r = -0.236, P = 0.03) in the adipocytes. The ratio of the ERalpha to ERbeta mRNA levels in the adipocytes was significantly associated with leptin mRNA levels (r = 0.454, P < 0.01). ERalpha induced leptin expression and ERbeta inhibited its expression in 3T3-L1 adipocytes. The ratio of the ERalpha-to-ERbeta expression in 3T3-L1 adipocytes may be an important potential regulatory factor in leptin expression.     

DEHP对中国林蛙精巢中StAR、CYP17和CYP19基因表达的影响

为探讨邻苯二甲酸二(2-乙基己基)酯(di-2-ethylhexyl phthalate,DEHP)对两栖动物精巢类固醇激素合成的影响,将中国林蛙(Rana chensinensis)雄性成体分别暴露于浓度为10-7、10-6、10-5、10-4mol·L-1DEHP的水体,分别在暴露20、30和40d取其精巢,提取精巢总RNA,逆转录合成cDNA,通过荧光实时定量PCR检测StAR、CYP17和CYP19mRNA表达相对值。结果表明:与对照组相比,DEHP处理组StAR和CYP19基因表达均上调,CYP17基因表达下调;比较不同DEHP浓度和不同暴露时间对StAR、CYP17和CYP19mRNA表达相对值的影响,显示DEHP浓度变化对3个基因表达影响的规律性不强,而DEHP暴露时间的累积效应较明显;提示DEHP可通过干扰中国林蛙精巢中StAR、CYP17和CYP19基因表达,影响其相应关键酶的表达,从而干扰类固醇激素的合成,产生雌激素效应。     

Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells

The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.     

Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles

The objective of this study was to investigate the levels of expression of steroid biosynthetic enzymes and steroidogenic acute regulatory protein (StAR) at different stages of ovarian follicular development in zebrafish (Danio rerio), and to investigate the sites within the steroid biosynthetic pathway that may be regulated by gonadotropins. Ovarian follicles of sexually mature fish were separated into primary, previtellogenic, vitellogenic, and mature stages and the expression of StAR, P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 hydroxylase/lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), and P450 aromatase (P450aromA) was determined by Real time RT-PCR. The expression of all genes changed significantly as follicles grew, with a decrease in the expression of StAR, P450scc, 3beta-HSD and P450c17 with maturation, and an increase in the expression of 17beta-HSD3 during vitellogenesis and 17beta-HSD1 and P450aromA during previtellogenesis. In vitro incubation of vitellogenic follicles demonstrated that the expression of StAR, 17beta-HSD3, and P450aromA increased in response to hCG, and decreased in the absence of hCG. In contrast, the expression of P450scc, 3beta-HSD, P450c17, and 17beta-HSD1 remained constant between treatments and over time. Testosterone and estradiol production in the culture medium was stimulated by human chorionic gonadotropin (hCG). These experiments aid in the characterization of the roles and regulation of steroids throughout ovarian development, and suggest that gonadotropins play a key role in the regulation of StAR, 17beta-HSD3, and P450aromA in zebrafish.     

Interferon-gamma inhibits steroidogenesis and accumulation of mRNA of the steroidogenic enzymes P450scc and P450c17 in cultured porcine Leydig cells

The inhibitory effects of recombinant porcine interferon-gamma (IFN gamma) on human CG (hCG)-stimulated testosterone production, and on mRNA concentrations of cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20lyase (P450c 17) were investigated using porcine primary Leydig cell culture as a model. After preincubation of Leydig cells for 24 h with 1000 pM IFN gamma, hCG-stimulated (10 ng/ml, 2 h) testosterone production was inhibited by 50%, whereas no significant changes were seen in hCG-stimulated cAMP production. Incubation with 10 microM 5-cholestene-3 beta,22(R)-diol or 10 microM 5-cholestene-3 beta,20 alpha-diol together with hCG (10 ng/ml, 2 h) reversed most of the inhibitory effect of IFN gamma, suggesting that IFN gamma inhibits P450scc activity, possibly by inhibiting the substrate (cholesterol) availability for P450scc. Incubation with IFN gamma also decreased basal concentrations of P450scc (45%) and P450c 17 (35%) mRNA, although these changes probably did not contribute to the decreased testosterone production. Long-term treatment with hCG (100 ng/ml, 24 h) increased P450scc mRNA (3- to 4-fold) and P450c 17 mRNA (4- to 5-fold) concentrations. Simultaneous treatment with IFN gamma attenuated these hCG-induced increases in P450scc mRNA (50%) and P450c 17 mRNA (40-100%) concentrations, as well as in testosterone production (77%). This inhibition of testosterone production could only be partly reversed by the hydroxylated cholesterol derivatives. This suggests that in addition to possible suppression of cholesterol availability, decreased P450scc and/or P450c 17 activities (through decreased mRNA concentrations) were also involved in the IFN gamma suppressed steroidogenic capacity of porcine Leydig cells during long-term hCG stimulation.     

Effects of amphetamine on steroidogenesis in MA-10 mouse Leydig tumor cells

Amphetamine influences plasma and testicular testosterone levels. However, there is no evidence that amphetamine can directly influence Leydig cell functions. In the present study, a MA-10 mouse Leydig tumor cell line was used to determine whether and how amphetamine affected Leydig cell steroidogenesis. MA-10 cells were treated with different concentrations of amphetamine without or with human chorionic gonadotropin (hCG) and/or enzyme precursors over different time durations. Steroid production, enzyme activities and StAR protein expression were determined. Amphetamine alone had no any effect on MA-10 cell steroidogenesis. However, amphetamine (10(-11)M and 10(-10)M) significantly enhanced hCG-treated progesterone production at 3 hr in MA-10 cells (p < 0.05). Furthermore, amphetamine significantly induced more progesterone production upon treatment with 22R-hydroxycholesterol (p < 0.05), a precursor of P450 side-chain cleavage enzyme (P450scc). However, amphetamine did not induce more progesterone production when treated with pregnenolone (p > 0.05), a precursor of 3beta-hydroxysteroid dehydrogenase. In addition, the expressions of StAR protein and P450scc enzyme were not significantly different between hCG alone and hCG plus amphetamine treatment in MA-10 cells (p > 0.05). These results suggested that amphetamine enhanced hCG-induced progesterone production in MA-10 cells by increasing P450scc activity without influencing StAR protein and P450scc enzyme expression or 3beta-HSD enzyme activity.     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries

Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection. At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts. In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells

Steroidal regulation of gene expression in follicular cells is not completely defined. Granulosa cells from 5 mm bovine follicles were cultured and treated and steady-state mRNA levels determined for FSHR (follicle-stimulating hormone receptor) and CYP19A1 (aromatase). Cells were treated for 5 days with (0.1-300 ng/ml) 17beta-estradiol (E2), testosterone (T), or 5alpha-dihydrotestosterone (DHT). FSHR mRNA was increased by T and DHT but not E2. In contrast, CYP19A1 mRNA was induced by all doses of E2 but only high doses of T and DHT. Similarly, varying treatment duration (1-5 days) showed that FSHR was increased by T and DHT and CYP19A1 mRNA increased by E2 and T at all times. Synergism between steroid hormones and FSH or forskolin was also evaluated. FSH or E2 did not alter FSHR mRNA and did not enhance DHT stimulation of FSHR mRNA. In contrast, DHT alone had no effect on CYP19A1 mRNA but synergized with FSH plus E2 to increase CYP19A1 mRNA, probably due to induction of FSHR by DHT. Effects of E2 and T on CYP19A1 were blocked by ICI 182,780, indicating mediation by estrogen receptors. However, the specific androgen receptor antagonist bicalutamide did not block E2 or T effects on CYP19A1 but did block T and DHT stimulation of FSHR. Thus, FSHR is specifically regulated through androgen receptor, whereas CYP19A1 is regulated by multiple pathways, including estrogen receptors and cAMP/protein kinase A induced by FSHR activation in granulosa cells. These inter- and intracellular regulatory mechanisms may be critical for normal follicle growth and dominant follicle selection.     

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.     

Involvement of nitric oxide in endothelium-dependent arterial relaxation by leptin

Leptin is a polypeptide, mainly produced in white adipose tissue, and increases sympathetic nerve activity. A few studies investigated leptin's effect on peripheral vessels. We examined the vasorelaxant effects of human leptin on rat arteries. Arterial rings were precontracted with 1 x 10(-6) mol/l of phenylephrine, and leptin was superfused. Leptin relaxed phenylephrine-precontracted arterial rings in a dose-dependent manner. ED50 was calculated to 8.4 microg/ml. Removal of endothelium abolished the effects of leptin. Indomethacin (1 x 10(-5) mol/l) did not affect the vasorelaxation by leptin, whereas 1 x 10(-4) mol/l of N(omega)-nitro-L-arginine methyl ester (L-NAME) completely suppressed it. The inhibition was antagonized by 1 x 10(-4) mol/l of L-arginine. Leptin normally relaxed arterial rings during superfusion of K channel blockers, including 3 x 10(-5) mol/l of glibenclamide, 1 x 10(-6) of mol/l apamin, and 5 x 10(-7) mol/l of charybdotoxin. Low Cl(-) solution (8. 3 mmol/l) inhibited leptin-induced relaxation, but endothelium-independent vasodilatation by nitroprusside was not impaired at low Cl(-) solution. These results suggest that arterial relaxation by leptin is mediated by nitric oxide released from endothelium, and Cl(-) plays an important role in leptin-induced nitric oxide release.