Steroid metabolism in corpora lutea of the western spotted skunk (Spilogale putorius latifrons).
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The present study reports steroid metabolism by corpora lutea (CL) obtained from skunks with diapausing embryos ('delay' CL) and with activated embryos (activated CL). CL from both reproductive periods were incubated with various radioactive precursors. Control incubations without any tissue or with 50 microliter of packed skunk blood cells were also conducted simultaneously. Incubation of skunk CL with [3H]-pregnenolone for 3 h resulted in 36% of the precursor accumulating as progesterone. Metabolism of [3H]dehydroepiandrosterone (DHEA) to androstenedione proceeded with approximately the same amount of product accumulating (34-46%) as was observed in the conversion of pregnenolone to progesterone. These results suggest that delta 5 isomerase, 3 beta-hydroxysteroid dehydrogenase, is the most prominent enzyme in skunk CL. Metabolism of [3H]pregnenolone to 17 alpha-hydroxypregnenolone and [3H]progesterone to 17 alpha-hydroxyprogesterone occurred at low rates (1-7%), suggesting the presence of C21 steroid 17 alpha-hydroxylase in skunk CL. Aromatase activity, as estimated by measuring accumulation of oestradiol-17 beta from [3H]testosterone, was demonstrated in activated CL. These results suggest that skunk CL appear to metabolize steroids in a manner similar to CL of other mustelids such as the ferret and American badger. (+info)
Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor.
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The MA-10 line is a clonal strain of Leydig tumor cells that has receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). These cells respond to hCG, cholera toxin, and 8-Br-adenosine 3':5'-monophosphate with increased steroid production. It is reported herein that exposure of the MA-10 cells to mEGF results in a substantial (80 to 90%) reduction in the number of hCG receptors per cell. The loss of hCG receptors is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. The steroidogenic responses to cholera toxin and 8-Br-adenosine 3':5'-monophosphate, however, are not affected. Other results presented show that mEGF is not a mitogen for these cells. (+info)
LH receptors in ovine corpora lutea in relation to various physiological states and effects PGF-2 alpha on LH-induced steroidogenesis in vitro.
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The LH binding properties (determined using tritiated methylated LH) and the in-vitro steroidogenic activity of CL from ewes in the oestrous cycle or early pregnancy (Day 18) were compared. No significant alteration in the Kd values was observed. However, the number of sites was maximal at Day 10 of the cycle and in early pregnant animals which had not been pregnant for at least 3 months (dry ewes). Non-lactating or suckling ewes had half the numbers of binding sites. The increase of the number of receptor sites was accompanied by a steroidogenic response at lower LH concentration. During incubation or superfusion for 5 h, a refractoriness to LH stimulation appeared after 1 h with high LH concentrations and after 3 h with low concentrations. The opposite effect of the addition of indomethacin or PGF-2 alpha suggests the intervention of PGs in this phenomenon. (+info)
A role for calmodulin in the regulation of steroidogenesis.
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TWO APPROACHES WERE USED TO STUDY THE POSSIBLE ROLE OF CALMODULIN IN THE REGULATION OF STEROID SYNTHESIS BY MOUSE ADRENAL TUMOR CELLS: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents. (+info)
Biphasic effect of gonadotropin releasing hormone on progestin secretion by rat granulosa cells.
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The effect of an agonistic gonadotropin releasing hormone (GnRH)-analog (D-Ala6, des-Gly10-NH2-GnRH-ethylamide, GnRHa) on granulosa cell steroidogenesis in the presence or absence of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) was studied. Granulosa cells, isolated from preovulatory follicles of pregnant mare's serum gonadotropin (PMSG)-treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20 alpha-dihydro-progesterone (20 alpha-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH-stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20 alpha-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Furthermore, the response is also to some extent determined by the maturational stage of the granulosa cells. (+info)
Dose-related effects of luteinizing hormone on the pattern of steroidogenesis and cyclic adenosine monophosphate release in superfused preovulatory rat follicles.
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The secretion of steroids and the release of cAMP in response to repeated luteinizing hormone (LH) stimulation were examined during superfusion of isolated preovulatory rat follicles. A high dose of ovine LH (1 microgram/ml for 20 min) caused a prolonged increase in the secretion of progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OHP) and a transient increase in the secretion of testosterone (T) and estradiol-17 beta (E2), and was accompanied by a peak of cAMP release. A single pulse of LH at a low dose level (10 mg/ml for 20 min) gave a limited increase in T secretion, but no clear change in P, 20 alpha-OHP and E2 secretion or cAMP release. When the follicles were challenged with a second pulse of LH (at 1 microgram/ml), the response varied according to the dose of LH delivered in the preceding pulse. Following exposure to the high dose of LH, the follicles were partially refractory to the second LH challenge in terms of cAMP and P and the secretion of T and E2 remained low. The low dose of LH, however, had a conditioning effect on the follicles since the response to the second LH challenge was amplified in terms of P, 20 alpha-OHP and cAMP. In this case a secondary increase in T and E2 secretion was found. The differential response to varying doses of LH are likely to reflect the physiological control of steroidogenesis during final follicular maturation. (+info)
Effect of infusion of PGI-2, 6-keto-PGF-1 alpha and PGF-2 alpha on luteal function in the pregnant rat.
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Prostacyclin (PGI-2), 6-keto-PGF-1 alpha and PGF-2 alpha were infused continuously for 6 h into the dorsal aorta of rats 8 days pregnant. PGF-2 alpha (10 micrograms/h) significantly reduced plasma progesterone concentrations by 66% and luteal tissue concentrations of pregnenolone and progesterone by 78% and 95% respectively. Plasma concentrations of 20 alpha-dihydroprogesterone remained unchanged whilst luteal tissue concentrations rose 2-fold. Plasma progesterone concentrations were significantly reduced to 50% by PGI-2 (10 micrograms/h) but were unaffected by 6-keto-PGF-1 alpha (10 or 100 micrograms/h). Neither PGI-2 (10 micrograms/h) nor 6-keto PGF-1 alpha (10 or 100 micrograms/h) had any significant effect on plasma concentrations of 20 alpha-dihydroprogesterone or on luteal tissue concentrations of pregnenolone, progesterone or 20 alpha-dihydroprogesterone. Arterial blood pressure was unaffected by PGF-2 alpha and 6-keto-PGF-1 alpha, but was significantly reduced by PGI-2 at infusion rates greater than or equal to 60 micrograms/h. (+info)
Selective release of follicle-stimulating hormone by 5 alpha-dihydroprogesterone in immature ovariectomized estrogen-primed rats.
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The effect of 5 alpha-dihydroprogesterone (5 alpha-DHP) on gonadotropin release was examined in the immature acutely ovariectomized (OVX) rat primed with a low dose of estradiol (E2). Treatment with various doses of 5 alpha-DHP given in combination with E2 increased levels of follicle-stimulating hormone (FSH) but had no effect on serum luteinizing hormone (LH). A single injection of a maximally stimulating dose of 5 alpha-DHP (0.4 mg/kg) stimulated increases in serum FSH at 1200 h and, 6 h later, at 1800 h. Pituitary LH and FSH content was dramatically enhanced by 1600 h and levels remained elevated at 1800 h. The administration of pentobarbital at 1200 h, versus 1400 h or 1600 h, prevented the increase in basal serum FSH levels at 1800 h, implying that the release of hypothalamic LH releasing hormone (LHRH) is modulated by 5 alpha-DHP. In addition, changes in pituitary sensitivity to LHRH as a result of 5 alpha-DHP were measured and a significant increase in the magnitude of FSH release was observed at 1200 h and 1800 h. Although the LH response to LHRH in 5 alpha-DHP-treated rats was not different from controls, the duration of LH release was lengthened. These results suggest that 5 alpha-DHP may stimulate FSH release by a direct action at the pituitary level. Together, these observations support the theory that 5 alpha-DHP mediates the facilitative effect of progesterone on FSH secretion and further suggests an action of 5 alpha-DHP in this phenomenon at both pituitary and hypothalamic sites. (+info)