Receptor-mediated gonadotropin action in the ovary. Demonstration of acute dependence of rat luteal cells on exogenously supplied steroid precursor (sterols) for gonadotropin-induced steroidogenesis

Incubation of luteal cells with human, horse and rat sera, but not bovine sera resulted in enhanced basal and hCG-stimulated progesterone accumulation. The stimulatory effect of human or rat sera on basal, hCG- or 8 Br-cyclic AMP-induced progesterone synthesis in luteal cells was evident within 15-30 min after incubation, reaching a maximum after 3-4 h. The stimulatory effects of hCG and/or sera were blocked by inhibitors of RNA and protein synthesis. Similarly, lysosomotropic agents, chloroquine (100 microM) and ammonium chloride (10 mM), partly blocked the steroidogenic response of luteal cells to hCG and/or human or rat sera. Incubation of cells in the presence of 2-deoxyglucose, sodium azide and phenylmethylsulfonyl fluoride resulted in partial inhibition of progesterone secretion in response to hCG or sera. Fractionation of human or rat sera into various lipoprotein fractions demonstrated that LDL and HDL most effectively supported and potentiated the steroidogenic response to hCG. Lipoprotein-deficient serum, however, did not alter gonadotropin-induced steroid production. Incubation of luteal cells with increasing concentrations of h-LDL and h-HDL enhanced both basal and hCG-mediated steroidogenesis in a dose-related manner, although very high concentrations of these lipoproteins were inhibitory. Further, [3H]cholesterol from [3H]cholesteryl linoleate-LDL was incorporated into luteal cell progesterone and the extent of this incorporation was enhanced by hCG. Addition of excess unlabeled h-LDL, h-HDL, as well as r-HDL, drastically reduced the incorporation of radioactive label into progesterone. These studies suggest that (a) serum potentiation of steroidogenesis was due to presence of lipoproteins, mainly LDL and HDL, and (b) the lipoprotein-bound cholesterol is delivered into the luteal cells and utilized for steroidogenesis.     

Receptor-mediated gonadotropin action in ovary. Differential effects of various gangliosides and cholera enterotoxin on 125I-choriogonadotropin binding, production of adenosine 3':5'-monophosphate and steroidogenesis in rat ovarian cells.

Poly(A)-containing RNA isolated from liver nuclei of untreated rats and 3 h or 12 h after partial hepatectomy or sham operation was hybridized to the complementary DNAs (cDNAs). In the homologous reactions two major components could be seen. When compared to normal liver, the complexity of the least abundant class was lower in nuclei from livers 3 h after partial hepatectomy and was higher in those isolated 12 h after operation. The heterologous reactions revealed an increase of some abundant poly(A)-containing sequences and a loss or dilution of rare sequences 3 h after operation. The latter effect was not specific to the regeneration process but occurred after laparotomy as well. 12 h after partial hepatectomy, however, about 10% new poly(A)-containing sequences were detected, corresponding to about 5000 molecules of 4500 nucleotides length, which are unique to regenerating nuclei.     

GnRH action on luteal steroidogenesis during pregnancy.

The results of our study presented here establishes that gonadotropin-releasing hormone (GnRH) acts directly on the corpus luteum, leading to suppressed production and release of progesterone and thus disrupting pregnancy. A GnRH-agonist (GnRH-Ag) treatment suppressed the luteal and serum progesterone levels. This suppression is neither mediated by a fall in ovarian testosterone production nor its conversion to estradiol. Although the treatment suppressed the nuclear estradiol-receptor content and binding sites for LH in the corpus luteum, it had no effect on the luteal binding sites for GnRH and prolactin within 24 h. GnRH-Ag augmented the plasma levels of luteinizing hormone, decreased the magnitude of nocturnal surges of prolactin, and had no effect on luteal cyclic adenosine 5'-monotriphosphate levels. Yet, the treatment had no effect on the luteal content of free cholesterol. We have also demonstrated, for the first time, the presence of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the rat corpus luteum, and the suppression of these proteins by GnRH-Ag leads to reduced steroidogenesis by the corpus luteum. Concomitantly, P450 side-chain cleavage enzyme, its activity, and its mRNA content and 3beta-hydroxy-steroid dehydrogenase content in the corpus luteum decreased. The treatment suppressed the plasma levels of pregnenolone and 20alpha-dihydroprogesterone. These data suggest that the suppression of luteal steroidogenesis by GnRH-Ag may be due to its inhibitory effect on the cholesterol transport and/or on the enzymes involved in the steroidogenic pathway. Furthermore, based on other observations made in our laboratory, we propose a hypothesis that an endogenous GnRH is present in the corpus luteum/ovary during pregnancy in the rat and that this GnRH may play a physiological role in the regulation, maintenance, and/or termination of pregnancy.     

Action of ACTH in the luteal ovary.

Oestrous rats and golden hamsters were anesthetized with pentobarbital, one of the femoral arteries and veins and one of the ovarian veins were cannulated. Blood fractions were collected from the ovary. After the first two fractions synthetic adrenocorticotropic hormone (ACTH) or human chorionic gonadotropin (hCG) was injected i.v. Blood pressures and ovarian blood flow were continuously recorded. Progesterone (P) and oestradiol-17 beta (E2) were determined from the ovarian venous blood by radioimmunoassay (RIA). ACTH induced a temporary elevation in the ovarian blood flow, P and E2 secretion both in rats and hamsters. In rats and hamsters hCG induced a continuous elevation in P secretion but the ovarian blood flow and E2 secretion remained unchanged. Luteal cells from pseudopregnant rats or oestrous hamsters were dispersed with collagenase and incubated with ACTH or hCG. A sample of the cells was preincubated with polymixin-B, indomethacin or ibuprofen. P and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) contents of the medium and cyclic 3,5 adenosine monophosphate (cAMP) content of the cells were determined by RIA. ACTH stimulated the release of 6-keto-PGF1 alpha and the secretion of P from the luteal cells of both species, which was inhibited by indomethacin or ibuprofen, but ACTH did not alter the cAMP content of luteal cells. The polymixin-B prevented ACTH to stimulate P secretion, but it did not elevate the 6-keto-PGF1 alpha release, while the cAMP content of the cells remained unchanged. It is supposed that the polyphosphoinositol-Ca(2+)-protein kinase-C second messenger system is involved in the ACTH induced stimulation of P secretion.     

Receptor-mediated gonadotropin action in the ovary. Inhibitory actions of concanavalin A and wheat-germ agglutinin on gonadotropin-stimulated cyclic AMP and progesterone responses in ovarian cells.

Pretreatment of ovarian cells with concanavalin A and wheat-germ agglutinin blocked the gonadotropin-induced cyclic AMP and progesterone responses and this effect was time- and concentration-dependent. Basal production of either cyclic AMP or progesterone, however, was not affected by treatment of cells with lectin. The effect of concanavalin A on gonadotropin-mediated cyclic AMP and progesterone responses was blocked by alpha-methyl D-mannoside and alpha-methyl d-glucoside. Similarly the inhibitory effect of wheat-germ agglutinin was reversed by N-acetyl-D-glucosamine. Pretreatment of ovarian cells with concanavalin A or wheat-germ agglutinin had no effect on protein synthesis in the ovary as monitored by [3H]proline incorporation studies. Concanavalin A and wheat-germ agglutinin did not affect steroid production in response to dibutyryl cyclic AMP and 8-bromo cyclic AMP, indicating that the inhibitory action of lectin was occurring at a step before cyclic AMP formation. Lectins specific for L-fucose, D-galactose and N-acetyl-D-galactosamine, gorse seed agglutinin, peanut agglutinin and Dolichos biflorus agglutinin respectively, did not interfere with gonadotropin-induced cyclic AMP and progesterone responses. The present studies suggest that gonadotropin receptors may be glycoprotein in nature or closely associated with glycoprotein structures with the carbohydrate chain containing N-acetyl-D-glucosamine, mannose and possibly N-acetylneuraminic acid.     

Effects of methylxanthines on gonadotropin-induced steroidogenesis and protein synthesis in isolated testis interstitial cells.

The effects of 1-methyl 3-isobutyl xanthine (MIX) and theophylline on basal and gonadotropin-stimulated production of cyclic AMP and testosterone were evaluated in enzyme-dispersed testicular interstitial cells. The actions of these compounds upon precursor incorporation into RNA and protein were also examined in the same cell preparation. The considerable higher potency of MIX as a phosphodiesterase inhibitor was accompanied by a steeper dose-response curve for cyclic AMP recovery in incubation media of hormone-treated cells. Both inhibitors caused increases in basal and hormone-stimulated cyclic AMP levels. Although low concentrations of theophylline and MIX had no effect on the maximum levels of hCG-stimulated testosterone production, 10 mM theophylline and 1 mM MIX significantly inhibited steroidogenesis. Conversely, basal testosterone levels were increased by MIX and theophylline. The higher concentrations of MIX and theophylline also significantly inhibited precursor incorporation into RNA and protein. These actions of phosphodiesterase inhibitors upon RNA and protein synthesis could contribute to their inhibitory effects at high concentrations upon gonadotropin-induced steroidogenesis.     

The role of plasma lipoproteins in steroidogenic response of rat luteal cells during gonadotropin-induced refractory states

Administration of human chorionic gonadotropin (hCG) to pregnant mare's serum gonadotropin--hCG primed rats results in the loss of in vitro responsiveness of the ovaries to exogenous gonadotropins for progesterone production. This state is associated with a loss of membrane receptors for hCG and a concomitant increase in lipoprotein receptors. Although lipoproteins potentiated gonadotropin response in ovaries from saline-injected rats, no stimulation was observed in hCG-desensitized ovarian cells. Examination of the time course for the loss of lipoprotein response after hCG injection revealed that injection with 50 IU of hCG results in a loss of gonadotropin response as early as 1 h after injection, but exogenous cholesterol-carrying lipoprotein fractions, LDL and HDL, were capable of stimulating progesterone production up to 4 h after hormone injection. Measurement of endogenous cholesteryl ester content showed that there was a 72% decline during this period with a concomitant increase in the basal progesterone production. One hour after hCG injection there was no stimulation of steroidogenesis by hCG in the presence or absence of exogenous lipoproteins. The refractoriness to exogenous hCG appeared only 4 h later when the hCG dose was reduced to 10 IU, whereas with 25 IU of hCG, the effect was similar to that observed using 50 IU of hCG. Such diverse steroidogenic stimuli as hCG, LH, LDL, cAMP, and cholera enterotoxin failed to stimulate progesterone synthesis in vitro in luteal cells of rats injected with 50 IU of hCG 48 h prior to sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA影响大鼠卵巢黄体细胞孕酮的生成

实验用离体培养方法观察ＧＡＢＡ对大鼠黄体细胞孕酮及羟自由基（．ＯＨ）生成的影响。结果表明：ＧＡＢＡ抑制黄体细胞孕酮的生成,同时也促进黄体细胞．ＯＨ的生成。ＧＡＢＡ对孕酮的抑制作用可能与腺苷酸环化酶系统及ＧＡＢＡＡ型受体有关,而与蛋白质合成无关。     

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.     

Control of steroidogenesis in small and large bovine luteal cells

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the oestrous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that: (1) the recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.     

细胞渗透性神经酰胺可抑制大鼠离体黄体细胞甾体激素生成并诱导细胞凋亡

本文旨在观察神经酰胺对离体孵育的大鼠黄体细胞孕酮分泌及细胞凋亡的影响,以PMSG－hCG处理的雌性Wistar大鼠为模型,分离制备黄体细胞,将外源性细胞渗透性神经酰胺与黄体细胞共同孵育,分别用放免法和流式细胞仪分析神经酰胺对黄体细胞孕酮生成和凋亡的影响,同时还检测了一氧化氮合酶（NOS）活性和一氧化氮（NO）水平的变化,结果显示,神经酰胺可以剂量相关方式抑制hCG－诱导的孕酮分泌,而对基础孕酮没有显著影响,离体孵育12h的大鼠黄体细胞存在自发性凋亡,5umol/L神经酰胺能显著增加亡率（P＜0．05）,流式细胞仪分析可见增强的凋亡蜂,实验还发现,50umol/L神经酰胺能明显促进NOS活性（P＜0．01）和NO生成（P＜0．01）,结果提示,神经酰胺可能通过调节甾体激素生成和细胞凋亡而作为一种重要的信息分子参与黄体退化等卵巢的生理过程。