Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

Deglycosylated human chorionic gonadotropin. An antagonist to desensitization and down-regulation of the gonadotropin receptor-adenylate cyclase system

Human chorionic gonadotropin (hCG) was deglycosylated with anhydrous HF and compared with native hCG for binding and biological activity. The deglycosylated hormone (DG-hCG) had the same affinity as hCG for gonadotropin receptors in murine Leydig tumor cells (MLTC-1) but was less than 1% as potent as hCG in stimulating cyclic AMP production in these cells. Exposure of MLTC-1 cells for 30 min to hCG caused a desensitization of hCG-stimulated adenylate cyclase activity, whereas DG-hCG did not induce desensitization even after 4 h. hCG induced down-regulation of hCG receptors; by 4 h, 40% of the receptors had disappeared, whereas there was no receptor loss in cells exposed to DG-hCG for the same time. By 6 h, receptor down-regulation began to occur in the DG-hCG-treated cells and could be mimicked by exposing the cells to dibutyryl cyclic AMP or cholera toxin. Thus, the small increase in cyclic AMP generated by DG-hCG appears to result in some loss of receptors. Cells were incubated with iodinated hCG or DG-hCG for 30 min, washed, and incubated in fresh medium. Both bound ligands were degraded as measured by disappearance of cell-associated radioactivity and appearance of trichloroacetic acid-soluble label in the medium. The half-lives were 3 and 6 h for hCG and DG-hCG, respectively. Our results indicate that DG-hCG in contrast to hCG does not cause either rapid desensitization of hCG-stimulated adenylated cyclase or rapid down-regulation of hCG receptors. Therefore, receptor occupancy alone is insufficient to induce these phenomena.     

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened alpha-subunits. II. Stimulation of adenylate cyclase activity and depletion of cytochrome P-450 in the rat testis

Human choriogonadotropin (hCG) analogues, containing the native beta-subunit and alpha-subunits enzymatically shortened by 2-3 amino acid residues, were used for studying the influence of hCG on the content of microsomal progesterone-binding cytochrome P-450 in rat testis. When 2-3 residues have been removed from the alpha-subunit, the ability of the hormone analogue to stimulate adenylate cyclase of isolated rat Leydig cells was diminished by 55%. When the hCG analogue containing a des-(88-92)-alpha chain was applied, the residual activity of the adenylate cyclase was negligible. 18 h after administration to rats in vivo, the hormone species containing des-(Lys-91-Ser-92)-alpha or des-(90-92)-alpha, respectively, were found to have induced a decrease in microsomal cytochrome P-450 content with an effectiveness corresponding to their ability of stimulating the adenylate cyclase in vitro. However, when assayed 48 h after application, the desensitization of the microsomal cytochrome P-450 system had persisted in case of the hCG species containing a des-(90-92)-alpha chain but not in case of hCG consisting of des-(Lys-91-Ser-92)-alpha and a native beta-subunit. From these results, it is concluded that short-term effects of hCG on the microsomal content of progesterone-binding cytochrome P-450 are mediated by the stimulation of adenylate cyclase. In contrast, the long-lasting action of hCG on this system seems not to be exclusively mediated by the increase in intracellular cAMP.     

Cell-free lutropin-dependent desensitization of the lutropin-sensitive adenylate cyclase of pig ovarian follicles is dependent on ATP.

Experiments were conducted to clarify the nucleotide requirements for lutropin (LH)-dependent adenylate cyclase desensitization in a cell-free membrane preparation derived from a thecal-cell-enriched component of preovulatory pig ovarian follicles. The follicular membranes were extensively washed in 2M-urea to remove endogenously bound GTP, and ATP devoid of GTP was utilized. Results conducted in the presence of 60 microM-GTP and various concentrations of ATP confirm the dependence of LH-stimulated adenylate cyclase activation and desensitization on millimolar concentrations of ATP. In experiments in which adenylate cyclase activation was supported by Mg2+, LH and adenosine 5'-[beta, gamma-imido]triphosphate, GTP did not support the desensitization response. Moreover, although GTP increased both basal and LH-stimulable adenylate cyclase activities in a concentration-dependent manner, the percentage desensitization was not significantly modified by the presence of 10nM-10mM-GTP. These results demonstrate that, even in the presence of exogenous GTP and Mg2+, activation of adenylate cyclase by saturating concentrations of LH in the presence of adenosine 5'-[beta, gamma-imido]triphosphate is not sufficient to initiate desensitization; millimolar concentrations of ATP are also required for the adenylate cyclase desensitization response.     

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened α-subunits. II. Stimulation of adenylate cyclase activity and depletion of cytochromeP-450 in the rat testis

Human choriogonadotropin (hCG) analogues, containing the native β-subunit and α-subunits enzymatically shortened by 2–3 amino acid residues, were used for studying influence of hCG on the content of microsomal progesterone-binding cytochromeP-450 in rat tests. When 2–3 residues have been renuwed from the α-subunit, the ability of the hormone analogue to stimulate adenylate cyclase of isolated rat Leydig cells was diminished by 55%. When the hCG analogue containing a des-(88–92)-α chain was applied, the residual activity of the adenylate cyclase was negligible. 18 h after administration to rats in vivo, the hormone species containing des-(Lys-91-Ser-92)-α or des-(90–92)-α, respectively, were found to have induced a decrease in microsomal cytochromeP-450 content with an effectiveness corresponding to their ability of stimulating the adenylate cyclase in vitro. However, when assayed 48 h after application, the desensitization of the microsomal cytochromeP-450 system had persisted in case of the hCG species containing a des-(90–92)-α chain but not in case of hCG consisting of des-(Lys-91-Ser-92)-α and a native β-subunit. From these results, it is concluded that short-term effects of hCG on the microsomal content of progesterone-binding cytochromeP-450 are mediated by the stimulation of adenylate cyclase. In contrast, the long-lasting action of hCG on this system seems not to be exclusively mediated by the increase in intracellular cAMP.     

Ovulation and Embryonic Developmental Rate Following hCG-stimulation in Sows

The hCG induced ovulation in sows was studied by use of ultrasonography, and an investigation of the development and diversity of the zygotes/embryos was performed at 24 h after ovulation. Crossbred sows (N=48) were weaned (day 0) and checked for heat twice daily from day 3 onwards. From day 4, the ovaries were transrectally scanned twice daily On day 4, the sows were given an injection of 750 iu hCG im and inseminated 27 ± 2 h (X ± SD) and 38 ± 1 h later. From 38 to 48 h after the hCG injection, the ovaries were scanned at 60 to 90 min intervals. At 24 h after ovulation the oviducts were surgically flushed in 18 sows. Out of the 48 sows, 34 showed heat at 12–36 h after the hCG-treatment and 14 showed heat before the hCG treatment. In the former group of sows, 20 (59%) ovulated within the interval of 38 to 48 h after the hCG treatment, and the follicular size immediately before ovulation was 7.8 ± 0.6 mm. Among the sows which showed heat before hCG treatment only 7 (50%) ovulated within the above interval and the preovulatory follicle size was larger (8.3 ± 0.5, p<0.05) than in the former group of sows, which showed heat after the hCG treatment. The flushing of 18 sows yielded a total of 243 ova, 70 (29 %) 1-cell stages, 160 (66 %) 2-cell stages and 13 (5%) 4-cell stages. A pronounced difference in the degree of variation in embryonic development was seen between sows: 4 animals yielded 1- to 4-cell stages, one exclusively 2-cell stage. In conclusion, the control of ovulation in sows by hCG treatment will affect the follicular growth and the exact timing of ovulation can not always be relied on. It is strongly recommended to use ultrasonography to monitor the time of ovulation if this parameter is important. Ova recovered at 24±1 h after the median time of ovulation revealed a pronounced diversity (1- to 4- cell stage) within sows. No obvious relation with this embryonic diversity and the follicular size at ovulation was seen in these data.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation.

Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

The effect of age on beta-adrenergic receptors and adenylate cyclase activity in rat erythrocytes.

Beta-adrenergic receptors and catecholamine-sensitive adenylate cyclase activity were studied in erythrocytes obtained from rats 6 weeks, 6 months, and 15 months of age. Intact erythrocytes from 6 week old rats contained significantly more beta receptors (411 ± 31 sites/cell) than 6 month (328 ± 21) or 15 month old rats (335 ± 16), as determined by binding of [¹²⁵I] iodohydroxybenzylpindolol. Erythrocytes from 6 week old rats also contained significantly greater isoproterenol-sensitive adenylate cyclase activity (95.0 ± 9.4pmoles/10⁹ cells) than erythrocytes from 6 month (27.9 ± 3.3) or 15 month old rats (23.7 ± 3.6). The erythrocyte population of 6 week old rats was bigger (mean corpuscular volume = 62 ± 2μ^3/cell) than the older rat erythrocytes (47 ± 1μ³ and 48 ± 1μ³). When the data were expressed relative to a unit of cell volume, there was no difference in the density of beta receptors among all three populations but a progressive and significant fall in hormone-sensitive adenylate cyclase activity. In the rat erythrocyte, the age-related loss of adenylate cyclase activity is not accompanied by changes in β-receptor density.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation

Culture of preovulatory rat follicles with luteinizing hormone, folliclestimulating hormone or prostaglandin E₂ for 24 h reduced the subsequent response of adenylate cyclase to the homologous hormone by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone or prostaglandin E₂, those refractory to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E₂, and those refractory to prostaglandin E₂ could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8−2.0 μg/ml in the mediem; a lower dose of luteinizing hormone (0.4 μg/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E₂ caused partial refractoriness at dose levels of 0.1–0.25 μg/ml; higher dose levels were more effective. These findings suggest that continued exposure of the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

Pulsatile secretion of luteinizing hormone and follicle stimulating hormone and their relationship to secretion of estradiol and onset of estrus in weaned sows

The objective of this experiment was to characterise temporal changes in estradiol and pulsatile secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) during the interval between weaning and estrus in the sow. Five multiparous sows were sampled at 10-min intervals for 3 h beginning 8 h after weaning and continuing every 12 h until estrus. Interval to estrus was 102 ± 2 h (range 96–108) after litters were weaned, and interval to preovulatory LH and FSH surges was 109 ± 5 h (range 92–116). With the exception of the period of the preovulatory surge, neither average nor basal concentrations of LH or FSH changed over time. Number of LH peaks per 3 h reached a maximum of 2.8 at 48 h before the preovulatory surge, then declined to 0.8 at 12 h before the surge. Peak amplitude for LH and peak frequency and amplitude for FSH also declined with time before preovulatory surges. Relative ranks were computed for individual sows based on the mean concentration of LH or FSH for each bleeding period. Rankings were consistent over the periods, but were not correlated with interval to estrus. Estradiol concentrations peaked (88 ± 7 pg/ml) at 36 h before preovulatory surges, coincident with the decline in peak frequency of LH. We conclude that pulsatile secretion of LH and FSH changes during the interval between weaning and estrus but secretion of these two hormones may be controlled by different mechanisms.     

Changes in follicular steroidogenic enzymes following the preovulatory surge of gonadotropins and experimentally-induced atresia

Atresia that is induced experimentally and the preovulatory surge of gonadotropins stimulate similar changes in follicular steroidogenesis in the rat, i.e., both enhance production of progesterone and reduce production of androgen and 17 beta-estradiol. In this study, mature cycling rats were either stimulated with human chorionic gonadotropin (hCG) or atresia was induced by blocking the proestrous surge of gonadotropins through the use of pentobarbitone or hypophysectomy. Changes in activity of C17,20-lyase (lyase) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) were estimated from homogenates of 10-15 Graafian follicles by evaluating conversion of precursors to products that were separated and quantified by high performance liquid chromatography (HPLC). Within 3 h of administration to proestrous rats, hCG reduced follicular lyase activity (pmole androstenedione produced per mg protein during 30 min incubation) from (mean +/- SEM) 221.3 +/- 24.2 to 120.2 +/- 30.4, and to 8.5 +/- 0.1 after 9 h. By contrast, 20 alpha SDH activity increased somewhat after hCG stimulation. Similar changes were observed after follicular atresia was induced, with hypophysectomy causing the most striking changes. Lyase was reduced to 60% within 6 h after the operation, and to 2% within 24 h. Activity of 20 alpha SDH was doubled within 6 h of hypophysectomy and remained high even 24 h later. Thus, in preovulatory rat follicles, luteinizing hormone (LH)/hCG reduces lyase activity and similar changes occur in such follicles undergoing atresia. There was no clear correlation between 20 alpha SDH and lyase activities; our results did not support the argument that 20 alpha SDH products regulate lyase following the ovulatory stimulus and atresia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

Role of carbohydrate in human chorionic gonadotropin: Deglycosylation uncouples hormone-receptor complex and adenylate cyclase system

Previous work has shown that deglycosylation of human chorionic gonadotropin (hCG) does not affect its receptor binding characteristics, but its ability to stimulate intracellular cyclic AMP accumulation and steroidogenesis in ovarian cells is abolished. To identify the site at which carbohydrate of hCG is involved in the mechanism of action of the hormone, we have studied adenylate cyclase activity in ovarian membrane preparations in response to deglycosylated and native hCG. The deglycosylated hCG does not stimulate adenylate cyclase of ovarian membrane preparation and also it acts as an inhibitor of hCG action. Data are presented to show that both hCG- and catecholamine receptors are coupled to the same adenylate cyclase complex. Since adenylate cyclase activity in the presence of deglycosylated hCG remains still responsive maximally to catecholamines, it indicates that the adenylate cyclase complex is functional and is unaffected by the interaction of deglycosylated hCG to its receptor. This is further supported by the fact that the deglycosylated hCG does not impair the maximal stimulation of adenylate cyclase by guanine nucleotides. Thus, the site of action of the carbohydrate of hCG is prior to the coupling of the hormone-receptor complex and the adenylate cyclase system.     

Neuroblastoma cell adenylate cyclase: direct activation by adenosine and prostaglandins.

—Adenylate cyclase activity of permeabilized neuroblastoma cells was measured by the conversion of [α³²P]ATP into labelled cyclic AMP. Adenosine (10^?6 - 10^?4m ) induced a dose-dependent increase in cyclic AMP formation. This effect could not be accounted for either by an adenosine-induced inhibition of the phosphodiesterase activity present in the enzyme preparation, or by a direct conversion of adenosine into cyclic AMP. This indicates that the observed increase in cyclic AMP accumulation reflected an activation of adenylate cyclase. Adenosine is partially metabolized during the course of incubation with the enzyme preparation. However, none of the identified non-phosphorylated adenosine metabolites were able to induce an adenylate cyclase activation. This suggests that adenosine itself is the stimulatory agent. The apparent K_m of the adenylate cyclase for adenosine was 5 ± 10^?6-10^?5m . Maximal activation represented 3-4 times the basal value (10-100 pmol cyclic AMP formed/10 min/mg protein). The adenosine effect was stereospecific, since structural analogues of adenosine were inactive. Adenosine increased the maximal velocity of the adenylate cyclase reaction. The stimulatory effect of adenosine was inhibited by theophylline. Prostaglandin PGE₁ had a stimulatory effect much more pronounced than that of adenosine (6-10-fold the basal value at 10^?6m ). Dopamine and norepinephrine induced a slight adenylate cyclase activation which was not potentiated by adenosine. It is concluded that adenosine is able to activate directly neuroblastoma cell adenylate cyclase. It seems very likely that such a direct activation is also present in intact nervous tissue and account, at least partly, for the observed cyclic AMP accumulation in response to adenosine.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E₁ or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue.In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

Protease inhibitors block hormonal activation of adenylate cyclase

To investigate the mechanism of serine protease stimulation of rat ovarian adenylate cyclase, a variety of synthetic protease inhibitors were used. These inhibitors blocked trypsin, chymotrypsin and hCG stimulation of adenylate cyclase in nearly the same manner. The inhibition of hormone stimulated adnylate cyclase could not be explained by a loss of [¹²⁵I]hCG binding. Cholera toxin and epinephrine stimulation of adenylate cyclase were similarly inhibited, whereas basal and fluoride-stimulated activities were only affected by higher doses of the inhibitors. The results suggest that adenylate cyclase in the ovary may be regulated by membrane protease activity.     

Synchronous generation of ovarian hCG binding sites and LH-sensitive adenylate cyclase in immature rats following treatment with pregnant mare serum gonadotropin.

A concomitant increase in the activity of LH-senstive adenylate cyclase and in the number of LH/hCG binding sites was induced in ovaries of immature rats upon administration of pregnant mare serum gonadotropin (PMSG), a hormone preparation known to have predominantly follicle stimulation (FSH-like) activity. When an optimal dose of PMSG (15 i.u./rat) was administered to 25-day-old rats, specific activity of LH-dependent adenylate cyclase and the number of binding sites for LH/hCG per mg protein remained unchanged during the first 24h, but 48h after injection a 2-to 4-fold increase in both parameters was observed. By contrast, there was no change in basal adenylate cyclase activity or in the response of the enzyme to the stimulatory action of guanosine-5'-(beta gamma-imino) triphosphate (Gpp (NH)p), GTP, or NaF. Specific activity of succinate cytochrome c reductase, glucose-6-phosphatase and 5'-nucleotidase were found to be unaffected by the hormonal pretreatment, although total protein determined in these homogenates increased 3-fold in the course of this treatment. It is inferred that during follicular maturation, FSH enhances the responsiveness of ovarian adenylate cyclase to LH by stimulating the insertion of LH/hCG-receptors into the cell membrane.     

Treatment with human chorionic gonadotropin (hCG) for ovulation induction is associated with an immediate 17beta-estradiol decrease and a more rapid LH increase in mares

The effect of treatment with an ovulation-inducing dose of human chorionic gonadotropin (hCG) on 17beta-estradiol (estradiol) and LH concentrations was studied in mares. In Experiment 1, treatment with hCG resulted in ovulation in approximately 48 h. The LH surge centralized to ovulation and the preovulatory decline in estradiol were not different between hCG-treated (n=15) and control (n=13) groups. In Experiment 2, concentrations of hCG decreased 92% between 1 and 48 h after hCG treatment, estradiol decreased (P<0.003) within 6h, and LH increased at a greater (P<0.02) rate after 12h (n=14). Results indicated: (1) hCG induced a decrease in estradiol and a subsequent greater rate of increase in LH and (2) when centralized to ovulation, the endogenous LH surge and the estradiol decrease were similar between hCG-treated and control groups.