共查询到20条相似文献,搜索用时 46 毫秒
1.
Recent studies have demonstrated the ability of somatomedin-C (Sm-C) to synergize with follicle-stimulating hormone (FSH) in the activation of cultured rat granulosa cell progesterone biosynthesis as well as the induction of luteinizing hormone (LH) receptors. Neither effect could be attributed to Sm-C-enhanced granulosa cell survival or replication, but could be accounted for, in part, by increased adenosine 3',5'-cyclic monophosphate (cAMP) generation. The present study was undertaken to determine if the synergistic property of Sm-C is FSH-selective and hence limited in relevance to follicular maturation, as well as to clarify further the role of cAMP in Sm-C-amplified agonist action. To this end, the ability of Sm-C to modulate the hormonal action of a series of physiologic as well as pharmacologic granulosa cell agonists was examined in vitro using cultured granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats. Concurrent treatment with highly purified Sm-C (50 ng/ml) resulted in marked increases over controls in the LH-stimulated [1 ng human chorionic gonadotropin (hCG)]-and beta 2-adrenergic-stimulated (10(-6) M terbutaline) accumulation of cAMP (3.8- and 2.6-fold, respectively and progesterone (3.2- and 7.4-fold, respectively). Similarly, concurrent treatment with Sm-C also augmented the vasoactive intestinal peptidergic stimulation of granulosa cell cAMP generation (4.1-fold) and progesterone biosynthesis (2.1-fold). In contrast, Sm-C was incapable of enhancing progesterone accumulation in response to stimulation with rat prolactin, a cAMP-independent granulosa cell agonist.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
Transforming growth factor beta regulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation 总被引:1,自引:0,他引:1
The effects of transforming growth factor beta (TGF-beta) on epidermal growth factor (EGF) receptor content and EGF action were studied in cultured granulosa cells from immature diethylstilbestrol-implanted rats. During follicle-stimulating hormone (FSH)-induced differentiation in vitro, EGF receptors increased by 20-fold as measured by the binding of 125I-EGF to the intact cells. Addition of TGF-beta during the 48-h culture period amplified the stimulatory effects of FSH on EGF receptors up to 2-fold, with ED50 and maximal concentrations of 2.5 and 8 pM, respectively. Also TGF-beta alone in amounts from 1.6 to 16 pM increased EGF receptor content 4-fold. The stimulatory effects of TGF-beta were due to increased numbers of EGF receptors/cell, since the growth factor had no effect on the Kd (3-5 X 10(-11) M) of the high-affinity EGF binding site. TGF-beta action was observed within 20 h of granulosa cell culture and was maximal by 48 h of a 96-h culture. The stimulatory actions of TGF-beta in gonadotropin-induced cells were exerted through the cAMP effector system of the granulosa cell, since the growth factor also amplified the induction of EGF receptors by cholera toxin, forskolin, and 8-bromo-cAMP. The augmentation of EGF receptors by TGF-beta resulted in a parallel 2-fold increase in the inhibitory effects of EGF on FSH-induced cAMP production and luteinizing hormone receptor expression during granulosa cell development. TGF-beta did not increase granulosa cell numbers during culture although it elevated [3H]thymidine incorporation into DNA by 2-fold over that of FSH-treated cells. These results indicate that TGF-beta regulates the effects of both FSH and EGF during granulosa cell differentiation and provides evidence that ovarian function may be controlled by the combined actions of gonadotropins and multiple growth factors. 相似文献
3.
The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system. 相似文献
4.
We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation. 相似文献
5.
The role of adenosine 3',5'-cyclic monophosphate (cAMP) as an intracellular second messenger of luteinizing hormone (LH) was reinvestigated in vitro with diterpene forskolin, a highly specific activator of adenylate cyclase. Treatment of cultured testicular cells from adult hypophysectomized rats with increasing concentrations (10(7)-10(-4) M) of forskolin produced dose-dependent increments in cAMP and testosterone accumulation. Concomitant blockade of cAMP-phosphodiesterase activity with 3-isobutyl-1-methyl-xanthine (10(-4) M) resulted in significant (P less than 0.05) enhancement of the forskolin effect for all but the 10(-4) M forskolin dose. Potency evaluation as judged by half-maximal stimulation of testosterone accumulation revealed median effective doses (mean +/- SE) of 1.25 +/- 0.2 x 10(-5), 1.7 +/- 0.5 x 10(-5), and 2.5 +/- 0.4 x 10(-10) M for forskolin, N6, O2'-dibutyryl cAMP (Bt2cAMP), and human chorionic gonadotropin (hCG), respectively. Examination of the time requirements of forskolin disclosed time-dependent increments in the accumulation of extracellular cAMP and testosterone, the earliest significant (P less than 0.05) increases being noted by 6 hr of treatment. In comparison, a minimal time requirement of less than or equal to 12 hr was noted for hCG- and choleragen-stimulated androgen biosynthesis, whereas the apparent onset of action of Bt2cAMP was delayed to the 24-hr time point. Although 10(-7) M of forskolin by itself did not alter the accumulation of testosterone, its addition resulted in substantial amplification of the hCG effect, producing a 4.6-fold reduction in the median effective dose (ED50) of hCG. Moreover, concurrent treatment with this functionally inert dose of forskolin rendered steroidogenically inert doses of hCG (eg, 10(-11) or 3 x 10(-11) M) steroidogenically potent. However, combined treatment with maximally stimulatory doses of Bt2cAMP (10(-4) M) and one of several testicular cell agonists [forskolin (10(-4) M), choleragen (10(-9) M) or hCG (10(-9) M)] did not prove additive. Taken together, our findings indicate that forskolin, like LH, is capable of stimulating testicular cAMP generation as well as androgen biosynthesis and that a functionally inert low dose of forskolin can significantly amplify LH hormonal action. Inasmuch as forskolin-stimulated and forskolin-amplified hormonal action are acceptable as novel criteria of cAMP dependence, our observations provide new evidence in keeping with the notion that cAMP may be in intracellular second messenger of LH. 相似文献
6.
R Procházka E Nagyová Z Rimkevicová T Nagai K Kikuchi J Motlík 《Journal of reproduction and fertility》1991,93(2):569-576
Oocyte-cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0.01-1.0 micrograms/ml) or forskolin (50-100 mumol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0.1 microgram/ml) or forskolin (100 mumol/l) increased the contents of intracellular adenosine 3',5'-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0.1 microgram/ml) or forskolin (100 mumol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P less than 0.01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte. 相似文献
7.
Cultures of granulosa cells from small (less than 3 mm), medium (3-6 mm), or large (8-10 mm) pig follicles were treated as follows: (1) basal controls, (2) cyclic adenosine 3',5'-monophosphate (cAMP) pathway agonists (pig FSH: 100 ng/ml; forskolin: 10 microM; dibutyryl cAMP; 1 mM), (3) calcium ionophore A23187 (0.005-1 micrograms), or (4) phorbol 12-myristate 13-acetate (TPA; 0.05-4 ng/ml). The combination of A23187 or TPA together with cAMP agonists was also examined in cultures of granulosa cells from follicles of different sizes. All substances were added at the time of culture, and oestradiol and progesterone were measured in the culture media after 48 h. All cAMP agonists were most potent in their stimulation of steroidogenesis (as a % of control) in cells from small follicles (P less than 0.05) with the exception of forskolin, which increased oestradiol in cells from large follicles to a greater extent than in cells of small follicles (P less than 0.05) (cells from medium follicles demonstrated less stimulation than those from small follicles except in progesterone production, for which FSH was equipotent). With the exception of forskolin, however, granulosa from large follicles showed little (oestradiol) or no stimulation (progesterone) with cAMP agonists. Under basal conditions, A23187 inhibited progesterone in all groups (P less than 0.05), and oestradiol production was reduced in granulosa cells from small follicles (P less than 0.05), unchanged in cells from medium follicles, and significantly stimulated in cells from large follicles. A23187 inhibited the enhanced production of both hormones after administration of cAMP agonists from cells of small and medium follicles (P less than 0.05), with inhibition significantly greater in cells of small follicles compared with medium. In cells from large follicles challenged with cAMP agonists, A23187 inhibited progesterone but stimulated oestradiol production; substitution of TPA (a protein kinase C stimulator) for A23187 gave identical results under basal or FSH-treated cultures of granulosa cells from small-, medium- or large-sized follicles. Our results suggest that TPA, A23187 and cAMP agonists modulate steroidogenesis differently in pig granulosa cells, depending on the stage of maturation of the follicle. Oestradiol production in granulosa cells from large preovulatory follicles may come under the stimulatory control of regulators of protein kinase C as in follicles near ovulation. 相似文献
8.
9.
I J Gonzalez-Robayna T N Alliston P Buse G L Firestone J S Richards 《Molecular endocrinology (Baltimore, Md.)》1999,13(8):1318-1337
The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells. 相似文献
10.
P E Gottschall G Katsuura R R Dahl S T Hoffmann A Arimura 《Biology of reproduction》1988,39(5):1074-1085
Recombinant human interleukin-1 (IL-1) inhibits the follicle-stimulating hormone (FSH)-induced development of luteinizing hormone (LH) receptors and suppresses progesterone secretion in cultured rat granulosa cells. Since activation of adenylate cyclase by FSH is considered to be the primary second messenger system responsible for differentiation of granulosa cells, we examined whether IL-1 could alter the FSH, cholera toxin, or forskolin-induced accumulation of cyclic adenosine 3', 5'-monophosphate (cAMP) from these cells. In addition, we sought to determine if IL-1 could influence differentiation induced by the cAMP analog, 8-bromo cAMP. Cells collected from ovaries of immature, diethylstilbestrol-treated rats were stimulated to differentiate by addition of FSH, cholera toxin, forskolin, or 8-bromo cAMP to the cultures. IL-1 or interleukin-2 (IL-2) was added to some of the tubes, and the primary cultures were incubated for various periods of time. At the end of the culture, the tubes were centrifuged, the medium was saved for progesterone and cAMP radioimmunoassay, and the cells were assayed for specific 125I-human chorionic gonadotropin (hCG) binding to determine the number of LH receptors. In the presence of FSH, IL-1, at a dose as small as 5 ng/ml, but not IL-2, significantly inhibited LH receptor formation and suppressed progesterone secretion in a dose-related manner. IL-1 also significantly suppressed FSH-induced cAMP accumulation after 72 h of incubation but did not appear to do so in a dose-related fashion. In the presence of FSH, IL-1 did not significantly alter the protein content of granulosa cells at the end of culture. During stimulation of granulosa cells with cholera toxin, forskolin, or 8-bromo cAMP, IL-1 significantly reduced LH receptor formation compared to that observed in the absence of IL-1. However, in contrast to IL-1 in the presence of FSH, IL-1 significantly augmented the forskolin-induced secretion of progesterone and accumulation of cAMP after 72 h at subsaturating doses of forskolin. Thus, IL-1 appeared to inhibit forskolin-induced and cholera toxin-induced formation of LH receptors even when cAMP levels were elevated. Similar to forskolin, 8-bromo cAMP-stimulated progesterone secretion was significantly enhanced by IL-1, but LH receptor formation was inhibited. Over a 72-h time course at single doses of FSH or forskolin, IL-1 did not affect cAMP accumulation until 48 h of culture, at which time IL-1 significantly suppressed FSH-induced, but augmented forskolin-induced, accumulation of cAMP.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
11.
Insulin, somatomedin-C, human chorionic gonadotropin, and forskolin enhance glucose oxidation by granulosa cells 总被引:1,自引:0,他引:1
The long exposure times required to observe stimulatory effects of insulin on steroidogenesis and protein synthesis in granulosa cells suggested that these effects might be secondary to stimulation of another metabolic process. The present studies examined the effects of insulin, the insulin-like growth factor somatomedin-C (Sm-C), human chorionic gonadotropin (hCG), and forskolin, a compound that activates adenylyl cyclase independently of a receptor, on glucose metabolism. Granulosa cells from preovulatory porcine ovarian follicles were incubated at 37 degrees C in Dulbecco's phosphate-buffered saline supplemented with bovine serum albumin, vitamins, amino acids, and glucose (0.01-20 mM). Cells were incubated with [14C]glucose for up to 23 h with or without a prior 20-h preincubation. Oxidation of glucose, assessed by quantitation of 14CO2 produced, was dependent on time and concentration of glucose. Optimal glucose concentrations for glucose oxidation were 3 mM in the absence or presence of insulin and correlated well with the measured glucose concentrations in follicular fluid (3 mM). After a 20-h preincubation in the absence or presence of insulin (1 microM), the rates of CO2 production were 10.6 and 21.6 pmol/micrograms DNA/h for control and insulin-treated cells, respectively. Insulin had an EC50 of 164 nM. Sm-C and hCG were more potent stimulators than insulin with EC50s of 768 pM and 161 pM, respectively. The greater sensitivity of granulosa cells to Sm-C than to insulin supports the concept that insulin exerts its effect via reactivity with the Sm-C receptor. The effect of hCG may have been mediated by cyclic adenosine 3',5'-monophosphate (cAMP), since forskolin also enhanced 14CO2 production.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Cholinergic inhibition of follicle-stimulating hormone-induced progestin production by cultured rat granulosa cells 总被引:1,自引:0,他引:1
The influence of cholinomimetics on follicle-stimulating hormone (FSH)-induced progestin production was studied in a primary culture of rat granulosa cells. Cells were cultured for 2 days with FSH and delta 4-androstenedione in the presence or absence of increasing concentrations of cholinergic agonists. Although ineffective as stimulators of steroidogenesis by themselves, the three nicotinic receptor-selective agonists lobeline, dimethylphenylpiperazinium iodide (DMPP), and phenyltrimethylammonium iodide (PTMA) inhibited FSH-induced progesterone and 20 alpha-hydroxypregn-4-en-3-one production in dose-dependent fashions. The rank order of inhibitory potencies was lobeline greater than DMPP greater than PTMA with IC50 values of 2 X 10(-6) M, 3 X 10(-5) M, and 3 X 10(-4) M, respectively. In contrast, the muscarinic receptor-selective agonists muscarine and bethanechol failed to inhibit steroid production. The inhibitory effect of lobeline on the time course of FSH-induced induced steroid production indicated an immediate inhibitory action; however, this inhibition was readily reversed upon removal of the drug. Further studies demonstrated that the FSH-stimulated increase in intracellular cAMP levels, as well as progesterone production induced by cholera toxin and forskolin (agents that stimulate cAMP production) and by dibutyryl cAMP (a cAMP analog), were also suppressed by lobeline. The present observations indicate that nicotinic, but not muscarinic, cholinergic agonists inhibit progesterone biosynthesis in cultured granulosa cells and suggest that endogenous acetylcholine may play a modulatory role in ovarian steroidogenesis. 相似文献
13.
Interleukin-1 beta (IL-1 beta), one of the polypeptide lymphokines released in response to antigen, toxins, injury or inflammation by nearly all cell types, has multiple systemic effects. In the present study the effect of IL-1 beta on follicle stimulating hormone (FSH)-induced estrogen production in primary culture was investigated. Granulosa cells obtained from immature estrogen-treated female rats were cultured for 3 days with increasing doses of FSH (1-30 ng/ml) with or without increasing doses of IL-1 beta (2-20 U/ml). The FSH stimulated estrogen production is dose-dependent, whereas IL-1 beta alone did not affect estrogen biosynthesis. In contrast, simultaneous treatment with IL-1 beta caused a dose-dependent inhibition of FSH action. This inhibitory effect of IL-1 beta was evident 48 h after the treatment. Furthermore, IL-1 beta inhibited forskolin (10(-5) mmol/L) and (Bu)2 cAMP (10(-2) mmol/L)-stimulated estrogen production, indicating a post-cyclic AMP site of action. The present study suggests that IL-1 beta is a potent modulator of granulosa cell steroidogenesis. Decreased estrogen formation may contribute to the follicle atresia and the impaired reproductive functions during injury and inflammation. 相似文献
14.
15.
Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells 总被引:4,自引:0,他引:4
The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation. 相似文献
16.
J M Darbon F Oury J Laredo F Bayard 《Biochemical and biophysical research communications》1989,163(2):1038-1046
We have investigated the effects of TNF-alpha on FSH-induced LH receptor expression, cAMP and progesterone production in cultured rat granulosa cells. TNF-alpha (0.5-100 ng/ml) inhibits the stimulating action of FSH on LH receptor formation in a dose-dependent manner with an IC50 of 1 ng/ml and an almost complete suppression of LH receptor induction for 50-100 ng/ml TNF-alpha. The inhibitory effect of TNF-alpha is not due to variations in cell number or viability but rather to a reduction of the LH receptor content per cell with no change in binding affinity (KD = 0.8 x 10(-10)M). TNF-alpha also inhibits the FSH-induced cAMP production but at a lower extent, with a maximum reduction of 60% for 100 ng/ml TNF-alpha. Moreover, TNF-alpha impairs the LH receptor formation induced by forskolin, cholera toxin or 8-Bromo-cAMP, indicating that the cytokine also acts at a step distal to FSH receptor and to cAMP formation. Finally, TNF-alpha decreases dramatically the progesterone synthesis that is stimulated by FSH, with a reduction to undetectable levels on and after 10 ng/ml TNF-alpha. These results suggest that TNF-alpha may drastically reduce the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the physiological ovarian follicular maturation. Such anti-gonadotropic action of TNF-alpha on granulosa cell differentiation may be also relevant to the alteration of ovarian function during physiopathological processes like inflammatory or infection diseases. 相似文献
17.
Differential actions of phorbol ester and diacylglycerol on inhibition of granulosa cell maturation 总被引:1,自引:0,他引:1
O Shinohara M Knecht K J Catt 《Biochemical and biophysical research communications》1985,133(2):468-474
Hormonal induction of granulosa cell maturation is inhibited by phorbol esters and permeant synthetic diacylglycerols, but these activators of protein kinase C differ in their effects on cAMP production and actions. Both agents prevented the induction of luteinizing hormone receptors and progesterone biosynthesis by follicle-stimulating hormone, choleragen, and forskolin, but only diacylglycerol abolished the cAMP responses to these stimuli. Granulosa cell aggregation and aromatase activity were inhibited by phorbol ester but not completely by diacylglycerol. In intact granulosa cells, cytosolic C kinase activity was rapidly decreased by phorbol ester but unaffected by diacylglycerol. Although diacylglycerol has a marked inhibitory action on cAMP production, the more prominent suppression of granulosa cell differentiation by phorbol ester may be related to its rapid and prolonged action on kinase C. 相似文献
18.
Effect of genistein on steroidogenic response of granulosa cell populations from porcine preovulatory follicles 总被引:2,自引:0,他引:2
Genistein affects reproductive processes in animals. However, the mechanism of its action is not fully elucidated and differs among species. The objectives of the current study were: 1/ to establish an in vitro model of granulosa cell culture for studying the intracellular mechanism of phytoestrogen action in porcine ovary; 2/ to determine an in vitro effect of genistein on basal and FSH-stimulated P(4) and E(2) production by porcine granulosa cell populations (antral, mural, total) isolated from large, preovulatory follicles. Granulosa cells were isolated from large (> or =8 mm), preovulatory follicles and separated into antral and mural cell subpopulations. Cells were allowed to attach for 72 h (37 degrees Celsius, 10% serum, 95% air/5% CO2) and than cultured for next 48 hours with or without serum (0, 5 and 10%), FSH (0, 10 or 100 ng/ml) and genistein (0, 0.5, 5 or 50 microM). Basal P(4) and E(2) production did not differ among antral, mural and unseparated granulosa cells isolated form porcine preovulatory follicles. Only mural cells tended to secrete less P(4) and E(2) than other cell populations. FSH stimulated P(4) production in a dose dependent manner in all cell populations and culture systems. Genistein inhibited in a dose dependent manner basal and FSH-stimulated P(4) production by antral, mural and unseparated granulosa cells. However, genistein did not affect E(2) production by granulosa cells. In addition, viability of porcine granulosa cells was not affected by the pyhytoestrogen except the highest dose of genistein. It appears that genistein may be involved in the regulation of follicular function in pigs. Moreover, unseparated porcine granulosa cells may provide a suitable in vitro model for studying the intracellular mechanism of phytoestrogen action in porcine ovary. 相似文献
19.
Little is known regarding the role of insulin-like growth factor 2 (IGF2) and the regulation of the IGF2 receptor (IGF2R) during follicular development. Granulosa cells were collected from small (1-5 mm) and large (8-22 mm) bovine follicles and were treated with IGF2 for 1-2 days in serum-free medium, and steroid production, cell proliferation, specific (125)I-IGF2 binding, and gene expression were quantified. IGF2 increased both estradiol and progesterone production by granulosa cells, and cells from large follicles were more responsive to the effects of IGF2 than those from small follicles. Abundance of aromatase (CYP19A1) mRNA was stimulated by IGF2 and IGF1. The effective dose (ED(50)) of IGF2 stimulating 50% of the maximal estradiol production was 63 ng/ml for small follicles and 12 ng/ml for large follicles, and these values were not affected by FSH. The ED(50) of IGF2 for progesterone production was 20 ng/ml for both small and large follicles. IGF2 also increased proliferation of granulosa cells by 2- to 3-fold, as determined by increased cell numbers and (3)H-thymidine incorporation into DNA. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell proliferation. Specific receptors for (125)I-IGF2 existed in granulosa cells, and 2-day treatment with estradiol, FSH, or cortisol had no significant effect on specific (125)I-IGF2 binding. Also, FSH treatment of small- and large-follicle granulosa cells had no effect on IGF2R mRNA levels, whereas IGF1 decreased IGF2R mRNA and specific (125)I-IGF2 binding. Granulosa cell IGF2R mRNA abundance was 3-fold greater in small than in large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R, thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R. 相似文献
20.
The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system. 相似文献