Multiple inhibitory effects of a luteinizing hormone-releasing hormone agonist on hCG-dependent steroidogenesis and on FSH-dependent responses in ovarian cells in vivo and in vitro

[125I] labelled [D-Leu6, des-Gly-NH10(2)] LH-RH ethylamide (LH-RHa), when injected into immature female rats, bound specifically not only to the pituitary but also to the ovaries. LH-RHa inhibited hCG-stimulated progesterone production and ovarian weight augmentation in hypophysectomized immature female rats in vivo. FSH-induced ovarian hCG receptors and ovarian weight gain in diethylstilbestrol (DES)-treated hypophysectomized immature female rats were also suppressed by LH-RHa. Progesterone production by rat luteal cells in vitro was inhibited by LH-RHa. LH-RHa did not change the affinity or population of LH/hCG receptor in porcine granulosa cells in short term incubation. However, LH-RHa inhibited induction of LH/hCG receptor stimulated by FSH and insulin in long term culture of porcine granulosa cells. LH-RHa delayed hCG-stimulated cyclic AMP accumulation in porcine granulosa cells. These findings suggest that LH-RHa inhibits hCG-stimulated cyclic AMP accumulation and subsequent progesterone production as well as FSH-stimulated LH/hCG receptor induction by acting directly on ovarian cells.     

The effect of epidermal growth factor (EGF) on progestin secretion and cyclic AMP synthesis in cultured luteal cells from pregnant rats

It is reported that steroid synthesis in ovarian cells is affected by epidermal growth factor (EGF). We cultured luteal cells from pregnant rats for 2 days with or without EGF, followed by incubation of the cells with or without stimulants (hCG, forskolin and dibutyryl cyclic AMP) for 5 hours. The levels of progesterone, 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) and cyclic AMP (cAMP) in the media were assayed. EGF had no effect on the basal levels of progesterone, 20 alpha-dihydroprogesterone and cAMP, but it suppressed these levels which were increased by the stimulants. We investigated binding capacity of [125I]-EGF to ovarian tissue of pregnant rats. Ovarian tissue had specific binding sites for EGF. The maximum number of binding sites was 2.38 fmol/mg tissue and the Kd value was 0.547 nM. It was indicated that EGF modified the reactivity of luteal cells to stimulants; counteracting the tropic effect of gonadotropins. It was shown that this effect of EGF might be exerted through its receptor in luteal cells.     

大鼠黄体细胞酪氨酸含量及hCG,cAMP和孕酮对含量的影响

经PMSG-hCG处理的未成年雌性大鼠卵巢,用胶原酶-DNA酶溶液消化,制成黄体细胞悬浮液。预培育1h后,加入不同浓度的hCG、cAMP及孕酮;并在加入hCG(10mIU/ml)、cAMP(2.5mmol/L)或孕酮(1nmol/L)的同时分别加入苯丙氨酸或放线菌酮,再培育1.5h,取细胞悬浮液400μl,用薄层层析扫描技术测其酪氨酸含量。结果:黄体细胞内有一定量内源酪氨酸,hCG,cAMP和孕酮均可明显促进酪氨酸的释放(P<0.05).苯丙氨酸对酪氨酸的含量无影响。酪氨酸释放也不依赖蛋白质合成过程。     

Studies of hCG binding and endocytosis in rat ovary by ultrastructural immunocytochemistry

Summary Localization of hCG binding sites and the process of endocytosis in pseudopregnant rat ovaries were investigated by indirect electron-microscopic immunocytochemistry. Immature female rats were treated with pregnant-mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovarian luteinization. Eight days after priming with PMSG-hCG and 1–6 h before sacrifice the animals were given another injection of hCG to bind the receptors. Receptor sites to hCG localized by reaction product were present in most luteal cells, but not in primary follicular cells. The receptor sites were distributed on luteal cell surfaces facing interstitial spaces. Endocytotic pits containing hCG binding sites were rarely seen 1 h after hCG injection. At 2 h, hCG and presumably its receptor were taken up within endocytotic vesicles with the evidence of reaction product coated on the vesicle wall. With time, fusion of endocytotic vesicles with lysosome occurred and the reaction product appeared in phagolysosomes. The reaction product was localized on phagolysosomal inner surface or in free granular form. These findings suggest that hCG and its receptors were internalized through endocytotic pits and endocytotic vesicles and delivered to lysosomes probably for degradation. An additional experiment for localization of acid phosphatase was also performed to delineate the lysosomes and phagolysosomes.     

The activity of receptor guanylyl cyclases in tissues of rats with neonatal streptozotocin diabetus mellitus and the influence of intranasal administration of insulin and serotonin

Functional activity of hormanal signaling systems and their sensitivity to regulatory actions of hormones in diabetes mellitus (DM) and its complications are altered. The activity of receptor forms of guanylyl cyclases (rGC) sensitive to natriuretic peptides, ANP and CNP, in tissues of female rats with 240-days neonatal streptozotocin DM and the influence of intranasal administration of insulin and serotonin (6 weeks, daily dose is 0.48 IU of insulin or 20 microg of serotonin to rat) on this activity were studied. In diabetic animals, the increase of the basal activity of rGC in the myocardium and its decrease in the uterus and ovaries were found, whereas the brain showed no differences from control animals. The treatment of diabetic rats with insulin led to a decrease in the basal activity of rGC in the myocardium and to its restoration to normal level in the ovaries. Serotonin treatment induced a less pronounced compared with insulin decrease in the basal activity of the enzyme in the myocardium and also a slight increase the activity in the brain. In the myocardium of diabetic rats, the weakening of GC stimulating effect of ANP and, on the contrary, the strengthening of CNP effect were observed. In the ovaries, GC stimulating effect of CNP and, to a lesser degree, the corresponding effect of ANP were decreased. In the uterus and the brain, the sensitivity of rGC to hormones was practically not changed. The administration of insulin to diabetic rats induced an increase in GC effect of ANP in the myocardium to its values in control animals and a decrease in CNP effect as well as in partially restored GC effect of CNP in the ovaries. Serotonin treatment led to some increase in the effects of natriuretic peptides in the brain of both control and diabetic animals. Summing up, in neonatal model of type 2 DM in the myocardium and the tissues of reproductive system of rats the functioning of natriuretic peptide-sensitive rGC is altered in the myocardium and the tissues of reproductive system of model rats with neonatal type 2 DM. Treatment of animals with insulin substantially restores rGC activity while intranasal administration of serotonin has little effect.     

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)     

Laparoscopic evaluation of the reproductive organs and abdominal cavity content of the lowland gorilla

Laparoscopy was used to examine the abdominal cavity and evaluate reproductive competence in six adult female gorillas which had not demonstrated copulatory activity within 12 months of examination. Blood samples were obtained on the day of laparoscopy and analyzed for serum estradiol-17β and progesterone. The uterus and oviducts of each gorilla appeared morphologically normal and free of lesions. The ovaries of three animals contained anatomical evidence of activity as indicated by the presence of luteal scars and a prominent corpus hemorrhagicum, corpus luteum, or developing follicles. No visible luteal or follicular tissue was observed on the ovaries of three gorillas. In the eldest female (age, 22 years) the ovaries were flaccid in texture and demonstrated irregularities in ovarian surface integrity, similar to that observed in women during reproductive senescence. Serum concentrations of estradiol-17β and progesterone on the day of laparoscopy corresponded with observed ovarian activity. All six females had extensive adhesions in the abdominal cavity exclusive of the reproductive organs. Numerous gut, omentum, and liver to peritoneum adhesions were observed. The results indicated that laparoscopy was a safe and effective method for evaluating anatomical competence in the gorilla. Uterine and ovarian integrity appeared morphologically normal, but only three of the six gorillas showed evidence of ongoing ovarian activity.     

Demonstration of luteinizing hormone/human chorionic gonadotrophin receptor-binding inhibitor in aqueous extracts of frozen human corpus luteum

Aqueous extracts of frozen human corpora lutea were tested for the presence of an inhibitor of luteinizing hormone-receptor site binding (LHRBI) and for the subsequent effect on the stimulatory response of luteinizing hormone (LH) on progesterone synthesis by sheep ovarian cells. In the presence of human corpus luteum extract of normal menstrual cycle (30,000-g supernatant), the binding of 125I human chorionic gonadotrophin (hCG) to granulosa and luteal cells of sheep ovaries was markedly reduced, but the ability of rat testicular LH receptors to bind labelled hCG was less affected. However, extracts of corpora lutea of the first trimester of pregnancy appeared to be less inhibitory on the binding of LH/hCG to ovarian cells and had no effect on the binding of rat testicular cells compared to those of normal menstrual cycle. Addition of both extracts separately inhibited the LH-stimulated in vitro progesterone synthesis by granulosa cell cultures and by incubated sheep corpus luteum slices. These findings provide evidence for the presence of LHRBI in human corpus luteum.     

Inhibitory effect of gossypol on human chorionic gonadotropin (hCG)-induced progesterone secretion in cultured bovine luteal cells

Inhibitory effects of gossypol on the female reproductive system have been recently reported. This study investigated a possible site of gossypol action on progesterone synthesis. Bovine luteal cells were cultured with hCG and forskolin in the presence or absence of gossypol. At 10 micrograms/ml, gossypol significantly inhibited hCG- and forskolin-stimulated progesterone secretion and intracellular cAMP formation; at 20 micrograms/ml, gossypol completely abolished the stimulative effect of hCG and forskolin. The results suggest that adenylate cyclase may be a site of gossypol action on steroidogenesis of bovine luteal cells.     

Follicle-stimulating hormone plays a role in the induction of ovarian follicular cysts in hypophysectomized rats.

Unabated stimulation by low doses of LH-like activity produces ovarian follicular cysts in both progesterone-synchronized immature rats and pregnant rats. Serum FSH is maintained in both of these models at values similar to those observed on diestrus. To determine whether unabated stimulation by basal serum FSH affects the ability of LH-like activity to induce cystic ovaries, immature hypophysectomized (HYPOXD) rats were given either no hormone (control); 2 micrograms ovine FSH (oFSH) once daily for 14 days beginning on Day 27; 0.5 IU hCG twice daily for 13 days beginning on Day 28 of age; or both oFSH and hCG (FSH + hCG) beginning on Day 27 and Day 28, respectively. By the end of the in vivo treatments (Day 40 of age), the largest follicles in the ovaries of control and hCG-treated HYPOXD rats were at the preantral stage of development, whereas the largest follicles present in ovaries from FSH-treated animals were atretic and at the small antral stage of development. In contrast, ovaries from rats treated with FSH + hCG displayed large follicular cysts by Day 37 of age. Of the serum steroids analyzed, only estradiol and androstenedione concentrations for animals treated with FSH + hCG were consistently elevated above values observed for control HYPOXD rats. Serum testosterone and dihydrotestosterone values were similar for hCG-treated and control HYPOXD rats throughout the in vivo treatments. In contrast, these steroids were elevated between Days 3 and 5 of FSH treatment (+/- hCG treatment). Serum progesterone and estrone values for all in vivo gonadotropin treatment groups were similar to those of controls. Serum insulin concentrations were not affected by any in vivo treatment. Incubates of follicles/cysts from FSH + hCG-treated HYPOXD rats contained more progesterone, androstenedione, and estradiol than incubates of follicles from any other in vivo treatment group. Follicles from all in vivo treatment groups responded to 8-bromo cAMP (cAMP) with increased in vitro progesterone accumulation. However, only follicles from FSH-treated and FSH + hCG-treated rats responded to cAMP with increased androstenedione and estradiol accumulation in vitro. Inclusion of 400 ng of either androstenedione or testosterone in the incubation medium enhanced progesterone accumulation in follicular incubates from control, hCG-treated, and FSH-treated HYPOXD rats, but did not enhance progesterone accumulation in follicular incubates from FSH + hCG-treated animals. Both androstenedione and estradiol production increased markedly under these conditions for follicles from all in vivo treatment groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

PACAP mRNA在大鼠妊娠黄体及离体细胞的表达与调节

目的:观察垂体腺苷酸环化酶激活肽(PACAP)mRNA在大鼠妊娠黄体中的表达及调节。方法:①于妊娠不同时期收集大鼠卵巢。用RT-PCR和原位杂交方法,观察妊娠过程卵巢PACAP mRNA表达的动态变化;②未成年雌性大鼠颈部皮下注射50IU孕马血清促性腺激素,48h后注射25IU人绒毛膜促性腺激素,第6天收集培养黄体细胞。用放免法测定给予不同处理后,培养液中孕酮的含量;用RT-PCR方法检测各组PACAP mRNA表达水平。结果:从妊娠11d起,PACAP mRNA表达逐渐增强,在妊娠19d达高峰;与对照组相比,血小板活化因子(PAF)、福司考林(forskolin)、佛波酯(PMA)均使培养黄体细胞孕酮分泌量及PACAP mRNA表达显著增高(P0.05)。结论:PACAP与中、晚期妊娠的维持密切相关;PAF可促进培养黄体细胞PACAP mRNA的表达,蛋白激酶C(PKC)和蛋白激酶A(PKA)途径都有可能参与了此过程。     

Activity of receptor guanylyl cyclases in rats with neonatal streptozotocin diabetes and effect of intranasal administration of insulin and serotonin

In diabetes mellitus (DM) and its complications the functional activity of hormonal signaling systems and their sensitivity to the regulatory action of hormones are changed. We studied the activity of receptor forms of guanylyl cyclase (rGC) sensitive to natriuretic peptides ANP and CNP in the tissues of female rats with 240 day neonatal streptozotocin DM and the effects of intranasal administration of insulin and serotonin (6 weeks, daily dose for rat is 0.48 IU insulin or 20 μg serotonin). In diabetic rats, the increase of the basal rGC activity in the myocardium and its decrease in the uterus and ovaries were found, while in the brain, there were no differences from the control. The treatment of diabetic rats with insulin led to a decrease of the basal rGC activity in the myocardium and its restoration to a normal level in the ovaries. The administration of serotonin produced a less pronounced decrease in the basal enzyme activity in the myocardium compared to insulin and an insignificant increase in the brain. In the myocardium of diabetic rats, the guanylyl cyclase (GC)-stimulating effect of ANP was attenuated, whereas the CNP effect was enhanced; in the ovaries, the GC-stimulating effect of CNP and, to a lesser degree, the effect of ANP were decreased. In the uterus and brain of a diabetic rats, the rGC sensitivity to hormones was practically did not change. The administration of insulin to diabetic rats induces an increase of GC effect of ANP in the myocardium to its values in control and a decrease of CNP effect, as well as partially restored GC effect of CNP in the ovaries under the influence of CNP. The administration of serotonin somewhat enhanced effect of natriuretic peptides in the brain of both control and diabetic animals. Thus, in the neonatal model of type-2 DM in the myocardium and the tissues of the reproductive systems of rats, the functioning of natriuretic peptide-sensitive rGC is changed. The treatment of animals by insulin substantially restores rGC activity, while the intranasal serotonin administration has a little effect.     

Binding of high-density lipoproteins to luteal membranes: the role of prolactin, luteinizing hormone, and circulating lipoproteins

Ovarian and adrenal membranes from immature gonadotropin-primed rats, treated with 4-amino-pyrazolopyrimidine (4APP) to reduce endogenous lipoprotein levels, displayed higher binding of porcine high-density lipoprotein (HDL) when compared to control rats. Immature, hypophysectomized (HYPOX) rats bearing corpora lutea (CL) on Day 5 after ovulation had lower levels of serum progesterone and reduced capacity for HDL and human chorionic gonadotropin (hCG) binding to ovarian membranes when compared with intact animals. Hypophysectomy also reduced the number of HDL binding sites in adrenal membranes. Treatment of HYPOX animals with luteinizing hormone (LH) and prolactin (Prl) alone or in combination increased the HDL binding sites in the ovary relative to HYPOX-untreated rats. Neither hormone affected binding to adrenals, where only adrenocorticotropic hormone (ACTH) enhanced HDL binding. LH treatment reduced the serum progesterone levels and hCG binding to the ovaries, whereas Prl administration increased progesterone levels with no effect on hCG binding. We conclude from this study that HDL binding in the luteinized ovary is regulated by Prl and LH and circulating lipoproteins, whereas in adrenals it is regulated by ACTH and circulating levels of lipoproteins.     

Effect of 80 kDa protein of porcine follicular fluid on gonadotropin stimulated progesterone production in rat granulosa cells in vitro.

We reported the presence of a 80 kDa polypeptide in porcine follicular fluid that inhibited the binding of 125I-radiolabelled hFSH as well as hCG to the rat ovarian gonadotropin receptors. In the present study, the biological activity of the receptor binding inhibitor is determined using an in vitro bioassay procedure. Granulosa cells isolated from PMSG primed immature rat ovaries respond to exogenously added gonadotropins in terms of progesterone production. Addition of fractions containing the gonadotropin receptor binding inhibitory activity inhibited progesterone production stimulated by the gonadotropins in a dose-dependent fashion. The receptor binding inhibitory activity was also capable of inhibiting progesterone production stimulated by PMSG, which has both FSH- and LH-like activities in rats. In contrast, progesterone production stimulated by dbcAMP was not inhibited by the receptor binding inhibitor. This result indicates that the site of action of the inhibitor is proximal to the formation of the cAMP. The above observations point out to a possible role for this factor in modulating gonadotropin activity at the ovarian level.     

Antigonadotropic effects of the bovine ovarian gonadotropin-releasing hormone-binding inhibitor/histone H2A in rat luteal and granulosal cells

A gonadotropin-releasing hormone (GnRH)-binding inhibitor (GnRH-BI) was purified from bovine ovaries and identified as histone H2A. In the present studies, the biological effects of partially purified and purified ovarian GnRH-BI, as well as calf thymus histone H2A, were examined in rat ovarian cells. Since GnRH has direct antigonadotropic actions on these cells, the effects on luteinizing hormone-stimulated cAMP accumulation in luteal cells and follicle stimulating hormone-induced cAMP and progesterone production in granulosal cells were evaluated. Antigonadotropic activity in both luteal and granulosal cells coeluted directly with GnRH-BI activity during purification from bovine ovaries, and the antigonadotropic effects were dose dependent and reversible. In contrast to GnRH, GnRH-BI maximally inhibited gonadotropin responses and the effects of GnRH-BI were not blocked by a GnRH antagonist. The purified ovarian GnRH-BI and calf thymus histone H2A had identical antigonadotropic properties, and the half-maximal concentrations for inhibiting the gonadotropin responses of granulosal and luteal cells was 2 and 5 microM, respectively. In conclusion, the ovarian GnRH-binding inhibitor, identified as histone H2A, not only inhibits the high affinity binding of GnRH to rat ovarian membranes but also evokes GnRH-like antigonadotropic responses in rat ovarian cells that do not appear to be mediated by binding to GnRH receptors.     

Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

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。     

Mechanisms for the antigonadotropic action of the ovarian gonadotropin-releasing hormone-binding inhibitor protein/histone H2A on ovarian cells.

A GnRH-binding inhibitor (GnRH-BI) was recently purified from bovine ovaries. On the basis of amino acid composition and partial sequence analysis this antigonadotropic GnRH-BI was identified as histone H2A. In the present study the mechanism for the antigonadotropic action of histone H2A was examined and compared to that of GnRH and poly-L-lysine. The potential sites examined were the receptor-coupled pathway of second message synthesis including receptor binding of hormone, G protein activation, and adenylyl cyclase activation. Histone H2A inhibited (ID50 = 2 microM) the binding of hCG by membrane receptors from luteinized rat ovaries in a noncompetitive and dose-dependent manner. The binding of FSH by membrane receptors from immature rat ovaries was not inhibited by histone H2A. Binding of GnRH by pituitary membrane receptors was inhibited by histone H2A, and the ID50 of 8 microM was similar to that previously observed for GnRH binding sites in rat ovarian membranes. No high-affinity binding of histone H2A by rat ovarian membranes was detected. Near-maximal doses of histone H2A (7 microM), poly-L-lysine (10 microM), and GnRH (1 microM) inhibited LH-stimulated cAMP production in isolated rat luteal cells. Inhibition by H2A and poly-L-lysine was larger than by GnRH. Furthermore, histone H2A and poly-L-lysine inhibited cholera toxin (CT)-stimulated cAMP production, but GnRH did not. Like GnRH, neither histone H2A nor poly-L-lysine inhibited forskolin (FK)-stimulated cAMP production. In isolated rat granulosa cells, histone H2A and poly-L-lysine inhibited FSH-, CT-, and FK-stimulated cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.