Progesterone Inhibition Research Articles

Corticotropin-releasing hormone in chimpanzee and gorilla pregnancies.

In humans, the length of gestation and the onset of parturition have been linked to the exponential production of placental CRH and a late gestational decline in maternal plasma CRH-binding protein (CRH-BP). CRH has been shown to have direct effects on the myometrium and on the fetal adrenal, where it stimulates production of the estrogen precursor dihydroepiandrosterone sulfate. In vitro placental CRH production is stimulated by cortisol and inhibited by progesterone. To determine whether this mechanism might operate in other apes, we sampled eight chimpanzees and two gorillas through their pregnancies for CRH, CRH-BP, cortisol, estradiol, progesterone, and alpha-fetoprotein. We show that both chimpanzee and gorilla maternal plasma CRH concentrations rise exponentially as observed in the human. The gorillas exhibited a human-like antepartum fall in CRH-BP, whereas CRH-BP in the chimpanzee remained stable. Pregnancy-associated changes in cortisol, estradiol, progesterone, and alpha-fetoprotein were qualitatively similar to those observed in humans. Maternal plasma cortisol correlated with plasma CRH in both gorillas (r = 0.60; P < 0.05) and chimpanzees (r = 0.36; P < 0.02). Further, there was a strong correlation between plasma estradiol and the log of plasma CRH in the gorilla (r = 0.93; P < 0.0001) and in the chimpanzee (r = 0.72; P < 0.001), which is consistent with the hypothesis that placental CRH determines the placental production of estradiol by stimulating the production of fetal adrenal dehydroepiandrosterone sulfate. Plasma CRH and progesterone were positively correlated providing no in vivo support for progesterone inhibition of CRH release.

Interferon-tau regulation of interleukin-2 and -10 gene expression in cultured bovine lymphocytes and endometrial cells

Three anti progestogens of the RU 38.486 type, RU 38.486, ZK 98.734, and ZK 98.299, were studied at different stages of pregnancy in the guinea pig. Treatment starting on postconception day 4 completely prevented nidation; all three compounds had comparable inhibitory potency. Treatment after nidation, starting on postconception day 8, induced decidual collapse and bleeding, but embryonic tissue was retained in nidation sites. In contrast to results in animals in nonfertile cycles, luteolysis was not induced, indicating that anti progestogens lack the ability to induce uterine prostaglandin synthesis/liberation, On postconception day 43, RU 38.486 showed marginal abortifacient activity. The other compounds induced expulsion more rapidly and at a higher rate. The comparatively pronounced antiglucocorticoid activity of RU 38.486 may account for this difference. With RU 38.486, a high level of uterine prostaglandin sensitivity and a cervical ripening were induced consistently and fast; spontaneous labor, on the other hand, occurred after several days, if at all. Complete uterine evacuation was induced within hours by otherwise inactive doses of sulprostone in various combinations with ZK 98.299 and RU 38.486 but surprisingly not with ZK 98.734. A single dose of ZK 98.299 induced an approximately thirtyfold increase in uterine prostaglandin sensitivity within 24 hours, exceeding that present before term, but did not induce spontaneous labor. This is evidence that endogenous prostaglandins were not activated, analogous to perinidation stages. Observation of antiluteolytic activity of anti progestogens in nonpregnant animals is considered of major theoretical importance in this context. It seems that inhibition of progesterone leads to suppressed uterine prostaglandin liberation. The same effect in pregnancy could explain the inability of the uterus to expel a seriously compromised conceptus. In conclusion, we suggest that progesterone is a stimulator rather than a depressor of uterine prostaglandins in the late luteal phase and pregnancy. The ability of the conceptus to neutralize this stimulatory action of progesterone is considered to be essential for the rescue of the corpus luteum and uterine motor quiescence in the guinea pig. The clinical significance of these findings is that the high frequency of incomplete abortions and protracted, sometimes heavy bleeding in pregnant women treated with RU 38.486 may reflect decidual compromise and simultaneous uterine prostaglandin deficiency, as found in our animal model after progesterone blockage.

Progesterone blockade of a luteinizing hormone surge blocks luteinizing hormone-releasing hormone Fos activation and activation of its preoptic area afferents

Progesterone is capable of facilitating or blocking the luteinizing hormone (LH) surge, depending on the timing of its administration. However, the precise targets of progesterone's actions are unknown. Since recent studies described the presence of a periventricular preoptic area (pePOA) neuron population afferent to LH-releasing hormone (LHRH) neurons that is co-activated to express c-Fos with LHRH neurons at the time of the LH surge, the present study was designed to determine if the pePOA neurons contain progesterone receptors (PRs) and whether progesterone inhibition is manifested by a failure of LHRH and pePOA neurons to become activated at the time of an LH surge. For progesterone facilitation, a group of immature rats each received a silastic capsule (1.57 mm i.d., 3.18 mm o.d., 1.5 cm long) containing estradiol-17 β (E 2) in peanut oil (150 μg/ml) at 09.00 h on postnatal day 28 followed 24 h later by a progesterone implant (crystalline, 1.57 mm i.d., 3.18 mm o.d., 1.5 cm long). For progesterone inhibition, a second group of rats received the estrogen capsule and a progesterone capsule (3.35 mm i.d., 4.65 mm o.d., 3.0 cm long) together at 09.00 h on day 28, and 24 h later received only a blank capsule. On the afternoon of postnatal day 29, all animals were anesthetized and perfused for localization of c-Fos and LHRH, PRs alone, or c-Fos and PRs. The present studies determined that following a progesterone-inhibition paradigm, along with blockade of the LH surge, both activation of LHRH and pePOA neurons was low or absent. Staining of PRs in progesterone-facilitated and progesterone-inhibited rats indicated that the pePOA neurons contained PRs in similar patterns. Double labeling of c-Fos and PRs in progesterone-facilitated rats indicated that nearly all the c-Fos-positive neurons of the pePOA (80±4.2%) co-expressed PRs; in progesterone-inhibited rats, only 32±12% of few c-Fos-positive neurons also contained PRs. In no instance were LHRH neurons found to contain PRs. Taken together, these data suggest that both progesterone facilitation and inhibition likely involve direct actions of progesterone on the pePOA neurons, and are consistent with a role for the pePOA neurons in transducing steroid effects on LHRH neurons.

Short term effect of steroids on catecholamine secretion from bovine adrenal medulla chromaffin cells

Bovine chromaffin cells were used to examine neuronal modulation, as their function is similar to sympathetic post-ganglionic neurons. The effect of steroids on evoked catecholamine secretion from primary culture of bovine adrenal medullary cells was investigated. A wide range of progestins, androgens and estrogens was found to have a significant effect on catecholamine secretion induced by the natural neurotransmitter acetylcholine (ACh). The androgens (especially androstandione and androsterone), as a class were the most effective in inhibition of stimulated secretion, while the estrogens had little, to no, effect. Among all steroids tested, progesterone had the most significant effect, other progestins were less potent. Progesterone inhibited catecholamine secretion evoked by ACh, nicotine and oxotremorine-M in a dose-dependent manner with similar IC 50 values in the μM range. It also blocked the secretion evoked by high potassium concentration (59 nM) or veratradine (100 μM), but no effect was seen on the secretion evoked by the calcium ionophore A-23187 (10 μM). Progesterone inhibition of ACh or oxotremorine-M stimulation was immediate and sustained. These results suggest that progesterone and other steroids might have a membrane effect probably acting through blockade of calcium influx necessary for the secretory response.

Glucocorticoids and anabolic/androgenic steroids inhibit the synthesis of GABAergic steroids in rat cortex.

Cerebral side effects of therapy with glucocorticoids include mental alterations and behavioral disturbances such as nervousness, insomnia, changes in mood or psychological state and psychopathies of manic-depressive or schizophrenic type. We have investigated the effects of glucocorticoids, anabolic/androgenic steroids and the 5-alpha-reductase inhibitor N,N-bis (1-methyl)-3-oxo-4-aza-5-alpha-androstane-17-beta-carboxamide (1-MAC) on the synthesis of the GABAergic steroids 5-alpha-pregnane-3.20-dione (5-alpha-dihydroprogesterone) and 5-alpha-pregnane-3-alpha-ol-20-one (5-alpha-tetrahydroprogesterone) in rat cortex in vitro. We found potent inhibition of progesterone-3-alpha-hydroxysteroid dehydrogenase (3-alpha-HSDH) by prednisone, prednisolone, dexamethasone and fluocortolone. Inhibition of progesterone 5-alpha-reductase by glucocorticoids was not found. In addition, inhibition of 3-alpha-HSDH and 5-alpha-reductase by androgenic/anabolic steroids and inhibition of 5-alpha-reductase by 1-MAC could be demonstrated. The inhibition of progesterone metabolism in the cerebral cortex by exogenous steroids might lead to altered neuronal activity. We conclude that this mechanism could induce the cerebral side effects, such as mental modifications and behavioral disturbances, of the drugs investigated.

Incomplete androgen and progesterone suppression following midluteal GnRHa prior to controlled ovarian hyperstimulation for IVF-ET.

We aimed to determine if midluteal GnRH agonist (GnRHa) use prior to controlled ovarian hyperstimulation (COH) results in uniform progesterone and androgen suppression and whether elevations of these hormones occurring early in follicular development may adversely effect the outcome of IVF-ET. Forty-four COH cycles using midluteal GnRHa were evaluated. Serum gonadotropins (LH and FSH) and gonadal steroids (E2, A, P4, and T) were measured after 10 days of GnRHa administration [cycle day 31 (CD 31)] and again on the day of hCG administration, following COH. Cycle outcomes evaluated were the number of oocytes retrieved, morphologic grade, fertilization, implantation, pregnancy, and spontaneous abortion rates. Endogenous serum FSH was uniformly suppressed (6.32 +/- 0.47 IU/L) on CD 31, however, LH was not (23.76 +/- 0.76 IU/L). Five and four tenths percent of cycles demonstrated low-level P4 elevations (> or = 0.9 ng/ml), 24.4% demonstrated serum androstenedione levels > or = 600 pg/ml, and 39% of cycles were characterized by serum T levels > or = 200 pg/ml despite evidence of E2 suppression (< or = 30 pg/ml) and the absence of follicular growth by sonography. LH levels were not predictive of incomplete P4 or androgen suppression. Elevations of either P4, A, or T occurring early in the follicular phase were not found to correlate with an impairment in clinical cycle outcome. Midluteal GnRHa use prior to COH may result in incomplete suppression of circulating progresterone and androgens. However, these relative elevations, occurring early in the development of the follicular cohort, did not appear to affect IVF cycle outcome adversely.

Progesterone inhibition of water permeability in Bufo arenarum oocytes and urinary bladder.

The ovarian oocytes from Bufo arenarum (BAO) but not those from Xenopus laevis (XLO) would have water channels (WC). We now report that the injection of the mRNA from BAO into the oocytes from XLO increased their water osmotic permeability (Pi) (reduced by 0.3 mM HgCl2 and reversed by 5 mM beta-mercaptoethanol). A 30-min challenge with progesterone induced, 18 h later, a reduction of the mercury-sensitive fraction of Pf in the BAO (but not in XLO). The mRNA from BAO pretreated with progesterone lost its capacity to induce WC in the XLO, but the hormone did not affect the expression of the WC in XLO previously injected with the mRNA from BAO. Pf was also measured in urinary bladders of BAO. Eighteen hours after a challenge with progesterone, a reduction in the hydrosmotic response to oxytocin was observed. Finally, the mRNA from the urinary bladder of BAO was injected into XLO. An increase in Pf was observed. This was not the case if, before the mRNA extraction, the bladders were treated with progesterone. We conclude that the BAO WC share progesterone sensitivity with the oxytocin-regulated water channel present in the toad urinary bladder.

Research on New Methods of Emergency Contraception

The ideal emergency contraceptive would be highly effective (surpassing the 75% effectiveness rate of the Yuzpe method), free of side effects, non-disruptive of the menstrual cycle, easily administered, interceptive, and affordable. Prevention of fertilization rather than ovulation would be an important advance since it would increase the window of time after unprotected intercourse that a drug can be administered. Recent research in this area has focused on levonorgestrel alone, danazol, anti-estrogens, gonadotropin-releasing hormone antagonists, and progesterone inhibition. Crucial to further advances is basic research on embryonic development and signaling before implantation, tubal transport and milieu, endometrial development before implantation, and the inter-relationships between the embryo and uterus during implantation. Until more knowledge is amassed about these physiological mechanisms, no new methods are likely to emerge. In the interim, an emphasis is being placed on the evaluation of anti-progestogens such as RU-486 that are easier to administer and associated with fewer side effects than the Yuzpe method.

Estradiol up-regulation of pituitary progesterone binding is required for progesterone inhibition of luteinizing hormone release

This study examines the regulation of progesterone receptor (PR) in the inhibition of pituitary luteinizing hormone (LH) secretion. Ovariectomized ewes underwent hypothalamic-pituitary disconnection, were pulsed with gonadotropin-releasing hormone (GnRH) and received 1 of 4 treatments: estradiol alone (E), estradiol priming before progesterone (E+P), E removed and replaced with P (E-P), or no steroids (C). P treatment for 24 h, with E or following E-priming, reduced LH pulse amplitude by 55% (p<0.05). E alone did not affect LH release. E increased pituitary cytosolic P binding capacity fourfold over controls (p<0.01) and P further increased binding to eight times controls (p<0.01). Pituitary PR mRNA increased to 149 and 171% of C in E and E+P groups, respectively (p<0.05), but E removal resulted in PR mRNA levels not different from controls. Pituitary receptors for GnRH were tripled by E alone compared to C (p<0.01), whereas P alone or with E had no effect. These data suggest an E-induced, direct pituitary inhibition of LH secretion by P and that this effect of P is associated with E-enhanced binding of P in the pituitary. Additionally, the direct pituitary effects of P on LH secretion cannot be accounted for by influences on GnRH receptor numbers.

Oxytocin affects the release of steroids, insulin-like growth factor-I, prostaglandin F2alpha and cyclic nucleotides by human granulosa cells in vitro.

The aim of the present experiments was to examine the effects of oxytocin (1-10 000 ng/ml) on hormone and cyclic nucleotide secretion by human granulosa cells cultured in a serum-supplemented medium. The release of progesterone, oestradiol, insulin-like growth factor-I, prostaglandin F2alpha, cAMP and cGMP into the incubation medium was analysed by radioimmunoassay. An inhibition of progesterone, but not of oestradiol release was observed. Oxytocin also stimulated insulin-like growth factor-I, prostaglandin F2alpha, cAMP and cGMP output. The results suggest an involvement of oxytocin in the autocrine/paracrine regulation of steroid, insulin-like growth factor, prostaglandin and cyclic nucleotide release by human ovarian cells.

Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium.

Unlike most normal adult tissues, cyclic growth and tissue remodeling occur within the uterine endometrium throughout the reproductive years. The matrix metalloproteinases (MMPs), a family of structurally related enzymes that degrade specific components of the extracellular matrix are thought to be the physiologically relevant mediators of extracellular matrix composition and turnover. Our laboratory has identified MMPs of the stromelysin family in the cycling human endometrium, implicating these enzymes in mediating the extensive remodeling that occurs in this tissue. While the stromelysins are expressed in vivo during proliferation-associated remodeling and menstruation-associated endometrial breakdown, none of the stromelysins are expressed during the progesterone-dominated secretory phase of the cycle. Our in vitro studies of isolated cell types have confirmed progesterone suppression of stromal MMPs, but a stromal-derived paracrine factor was found necessary for suppression of the epithelial-specific MMP matrilysin. In this report, we demonstrate that transforming growth factor beta (TGF-beta) is produced by endometrial stroma in response to progesterone and can suppress expression of epithelial matrilysin independent of progesterone. Additionally, we find that an antibody directed against the mammalian isoforms of TGF-beta abolishes progesterone suppression of matrilysin in stromal-epithelial cocultures, implicating TGF-beta as the principal mediator of matrilysin suppression in the human endometrium.

Progesterone inhibition of the development of the luteolytic signal in cows

Mated cows that subsequently undergo luteolysis have significantly lower plasma concentrations of progesterone than do cows in which pregnancy is maintained. Two groups of ovariectomized cows were treated with progesterone and oestradiol to recreate luteal phases exhibiting the extremes of plasma concentrations of progesterone (6.0 +/- 0.4 and 12.4 +/- 0.8 ng ml-1) normally seen in intact cows. The effect of this difference in progesterone on the development of the luteolytic signal was determined by monitoring the basal and oxytocin-stimulated plasma concentrations of the principal metabolite of prostaglandin F2 alpha' PGFM on days 12-16 of the simulated luteal phases. Basal PGFM concentrations were similar in the low and high progesterone groups on all days. The mean plasma concentration of PGFM showed a larger increase following oxytocin in the low progesterone group on days 13 and 14 (P < 0.05) and on days 15 and 16 (P < 0.1). These results demonstrate that a low plasma concentration of progesterone results in the development of a stronger luteolytic signal, and provide an explanation for the fact that cows with lower plasma concentrations of progesterone are more prone to embryo loss.

Progesterone suppression of glutamic acid decarboxylase (GAD67) mRNA levels in the preoptic area: correlation to the luteinizing hormone surge.

The progesterone-induced LH surge in the estrogen-primed rat is thought to be mediated through interneurons since LHRH neurons reportedly lack steroid receptors. Along these lines, glutamate and gamma-aminobutyric acid (GABA) neurons are considered to be two of the major interneurons in the hypothalamus involved in the control of LHRH and LH secretion. Glutamate is a major excitatory amino acid neurotransmitter while GABA is a major inhibitory amino acid neurotransmitter in the control of LH secretion. Glutamate is converted in the brain to GABA by the rate-limiting enzyme glutamic acid decarboxylase (GAD). Regulation of GAD by steroid hormones would therefore appear to be a logical mechanism for the control of LHRH and LH secretion based on the changes in glutamate and GABA production which would result from such regulation. Therefore, the purpose of the present study was to determine whether the steroid hormone, progesterone, regulates the GAD enzyme in the hypothalamus at a time when it induces an LH surge. To accomplish this aim, northern and dot blot analysis were used to measure GAD65 and GAD67 mRNA levels in the preoptic area (POA) and medial basal hypothalamus (MBH) in ovariectomized (OVX) immature rats, OVX rats primed with 2 mu g of estradiol (E2) for 2 days, and OVX rats primed with 2 mu g of E2 for 2 days and administered progesterone (1 mg/kg, 09.00 h) to induce an LH surge. The results of the study show that GAD67 mRNA levels were suppressed by progesterone in the POA at 12.00 h (at the start of the surge) (24.4% suppression vs. E2 only; p <0.01) in the first experiment. This suppression was confirmed in a second experiment as progesterone suppressed GAD67 mRNA levels in the POA at 12.00 h (31.8% vs. E2 only; p <0.05) and also at 14.00 h (40% vs. E2 only; p <0.05) which correlates to the peak of the LH surge. The changes in GAD67 by progesterone were restricted to the POA, as GAD67 mRNA levels in the MBH were unaffected by the steroid treatment. Furthermore, the effect of progesterone on POA GAD67 mRNA levels was specific as it was blocked by prior treatment with the antiprogestin, RU486 (400 mu g). In the MBH, both GAD67 and GAD65 mRNA levels were increased by estradiol treatment at 16.00 h and this increase was prevented by progesterone administration in the presence of estrogen priming. In conclusion, these studies demonstrate that the mRNA for the key enzyme responsible for the conversion of the major excitatory amino acid neurotransmitter in the hypothalamus (glutamate) to the major inhibitory amino acid neurotransmitter (GABA), is regulated by the steroid hormone progesterone. Progesterone suppression of GAD67 mRNA levels in the POA, where LHRH neurons reside, and at a time immediately preceding and during the LH surge, could provide a mechanism for greater stimulation of LHRH neurons through enhanced excitatory glutamate production concomitant with deceased inhibitory GABA production in the POA.

Rat ovarian granulosa cell as a site of endothelin reception and action: attenuation of gonadotropin-stimulated steroidogenesis via perturbation of the A-kinase signaling pathway.

Endothelins (ETs) are a family of vasoactive peptides that may be involved in granulosa cell (GC) luteinization or follicular maturation. However, the precise role of ET in ovarian physiology remains unknown. We have investigated whether the rat GC is a site of ET reception and have characterized the antigonadotropic effect of ET in cultured GC from immature rats. Two major ET binding species (52 and 30 kDa) were observed after cross-linking of GC membranes with radiolabeled ET-1, although the smaller protein may represent a degradative product. Unlabeled ET-1, ET-2, or ET-3 were equipotent in displacing radiolabeled ET-1 from these putative ET receptors, with EC50s of 0.3-0.7 x 10(-9) M. Similarly, ET-1, ET-2, and ET-3 were equipotent (EC50s of about 10(-10) to 10(-9) M) in inhibiting the FSH-supported accumulation of progesterone. ET-1 (10(-7) M) also inhibited (> 90%) FSH-supported estrogen accumulation. Maximum progesterone inhibition (> 90%) by ET-1 (10(-7) M) was achieved throughout the range of FSH does and cell densities tested and by 48 h or 72 h of culture. ET-1 was not cytotoxic in the dose range tested. Forskolin-stimulated progesterone accumulation was similarly inhibited by ET-1, suggesting that ET-1 inhibits cAMP-mediated (e.g., FSH or forskolin-stimulated) progesterone accumulation. ET-1 inhibited (74%) the FSH-stimulated accumulation of cAMP, suggesting that it acts at sites related to cAMP generation or degradation. In addition, ET-1 inhibited 8-bromo-cAMP-generated progesterone accumulation (60%), suggesting that it also acts at sites distal to cAMP generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of uterine collagenase gene expression: interactions between serotonin and progesterone

This report seeks to further define the requirements for the previously established induction of collagenase gene expression by serotonin and inhibition by progesterone in primary cultures of rat uterine smooth muscle cells. Detectable increases in collagenase production were observed after as little as 3 h exposure of cells to 5 μM serotonin, with maximal induction occurring after approximately 8 h of exposure. The apparent half-life of collagenase mRNA upon removal of serotonin was estimated to be approximately 12 h, and was not dependent on the duration of induction. Inhibition by either cycloheximide or progesterone showed similar half lives for collagenase mRNA, however a much shorter half-life (6 h) was obtained in the presence of actinomycin D. These experiments suggest that neither serotonin induction nor progesterone inhibition of collagenase synthesis represents a primary effect on collagenase gene transcription. Rather they appear to be secondary to changes that occur at one or more primary intermediate genes whose induction or decay must occur prior to changes in collagenase transcription. The progesterone receptor antagonist, RU-486, abrogates the ability of progesterone to inhibit serotonin-induced collagenase gene expression, indicating that the effects of progestins in this system likely are receptor-mediated. Finally, the present studies demonstrate that pretreatment of cells for times as long as 5 days with medroxyprogesterone in the absence of serotonin is unable to prevent subsequent serotonin-induced collagenase mRNA increases. These data suggest the possibility of a unique interaction between the molecular pathways of inducer and inhibitor, one in which serotonin may help mediate the progesteronedependent repression of the levels of collagenase mRNA.

Mode of action of RU 486.

RU 486 is a 19-norsteroid which has a specific high affinity binding to the progesterone and glucocorticoid receptor. It is generally accepted that RU 486 acts as a pure progesterone antagonist almost without agonistic activity. RU 486 acts mainly directly on the target organ, such as the endometrium, but also to some extent indirectly through an effect on the pituitary gonadotrophin secretion. The effect of RU 486 during the menstrual cycle is dependent on time of treatment. Treatment before ovulation will result in a prolongation of the proliferative phase of the menstrual cycle, while treatment during the mid- and late luteal phase will invariably induce bleeding, often followed by a second bleeding episode at the expected time of menstruation. The only treatment period which does not influence the menstrual cycle is treatment immediately following ovulation. Treatment during the proliferative phase has no effect on endometrial morphology but inhibits follicular development and delays oestrogen and LH surge. Treatment on the first days following ovulation has no effect on ovarian steroid concentration, but will significantly delay endometrial development, cause a change in the concentration of oestrogen and progesterone receptor concentration enzyme activity and production of substances thought to be progesterone dependent. The change in endometrial development is sufficient to prevent implantation. In mid- and late luteal phase, treatment with RU 486 will result in endometrial shedding in spite of normal progesterone levels. Post-ovulatory treatment with RU 486 will also significantly change uterine contractility. In early pregnancy, withdrawal of progesterone inhibition will result in uterine contractility and a significant increase in the sensitivity of the myometrium to prostaglandin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of basic fibroblast growth factor, transforming growth factor-beta and nerve growth factor on the secretory function of the bovine corpus luteum in vitro.

The effects were investigated of basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta) and nerve growth factor (NGF) on the release of progesterone and oxytocin from the bovine corpus luteum (CL) at different stages of the oestrous cycle. A microdialysis system (MDS) of CL and a cell culture system with a reduced number of endothelial cells were used. In the MDS of CL from the mid-luteal stage (days 8-12 of the oestrous cycle), infusion with bFGF (0.1, 1, 10 and 100 ng/ml), TGF-beta (0.1, 1 and 10 ng/ml) and NGF (0.1, 1, 10 and 100 ng/ml) for 30 min induced significant acute effects on the release of progesterone. Both bFGF and NGF stimulated the release of progesterone during peptide infusion, TGF-beta and also bFGF in the period thereafter. This stimulation was dose-dependent during and after the infusion only for bFGF. This response pattern was observed at all luteal stages for the three growth factors, but bFGF was more stimulatory at the early (days 5-7) and mid-luteal stages during and after peptide infusion. The release of oxytocin was stimulated by bFGF in a dose-dependent manner. At the highest dose, bFGF, TGF-beta and NGF stimulated the release of oxytocin throughout all three luteal stages. When luteal cells were cultured with growth factors, only TGF-beta showed a dose-dependent inhibition of both basal and LH-stimulated progesterone as well as oxytocin release (measured between 48 and 52 h of culture). NGF had an inhibitory effect only on the basal release of oxytocin. bFGF had no effect on the release of either hormone under continuous stimulation in cell culture. The results indicate that bFGF, TGF-beta and NGF act directly and acutely on the secretory function of bovine CL in the MDS but also have long-term effects as shown in cell culture. bFGF appears to be an important autocrine/paracrine regulator of CL function, since local expression of its mRNA, peptide synthesis and its mitogenic and non-mitogenic actions have now been confirmed. Endothelial cells from the CL have been identified as target cells for bFGF. Differences observed between the two systems might thus be attributed to the presence or absence of cell-to-cell contact and a reduced number of endothelial cells, as well as to the duration of peptide stimulation and medium changes every 24 h compared with the flow-through conditions in the MDS.

Progesterone inhibits the estrogen-induced gonadotropin surge in the rhesus monkey independent of endogenous opiates.

Administration of an estrogen challenge during the luteal phase, a time when progesterone concentrations are elevated, fails to elicit a gonadotropin-positive feedback response. The purpose of the present study was to determine if endogenous opiates are involved in the mechanism by which progesterone blocks the estrogen-induced gonadotropin surge in monkeys. To this end, rhesus monkeys in the luteal phase were pretreated with either saline or various regimens of nalmefene, a long-acting opiate antagonist, before being given an estrogen challenge. Three groups of animals were given nalmefene (10 mg, iv) every 12 h beginning 24, 48, or 96 h before an estrogen challenge and continued until 48 h after the start of the estrogen challenge. A fourth group received a continuous sc infusion of nalmefene (20 mg/day) via osmotic minipumps beginning 48 h in advance of the estrogen challenge. In a second experiment, monkeys in the follicular phase received progesterone implants at the time of an estrogen challenge and iv injections of nalmefene every 12 h for 48 h. Gonadotropin and steroid levels were monitored in both experiments by collecting blood samples by saphenous venipuncture at intervals of 6-12 h. The majority of luteal phase animals that were pretreated with saline were unresponsive to the estrogen challenge. Only 2 of 16 (12.5%) had an increase in LH concentrations that could be classified as a surge. Animals pretreated with iv nalmefene every 12 h beginning 48 h before the estrogen challenge exhibited a higher incidence of positive feedback responses (8 of 12 or 66.7%). A concomitant FSH surge was observed in 3 of these instances. However, when progesterone concentrations, which declined before the estrogen challenge in the nalmefene-treated group, were supplemented with exogenous progesterone, nalmefene failed to evoke any LH surges. Six of 8 animals that received nalmefene by sc infusion exhibited LH responses. However, the amplitude and duration of these LH responses were diminished, and no FSH responses were observed. Monkeys pretreated with nalmefene for either shorter (24 h) or longer (96 h) periods before the challenge were less responsive (0 responses out of 6 trials and 1 response out of 4 trials, respectively). Nalmefene was equally ineffective in preventing progesterone inhibition of the estradiol-induced LH surge in follicular phase animals (0 of 15 animals had LH surge). These results indicate that nalmefene antagonism of endogenous opiates does not enable estrogen to exert positive feedback effects on LH release when progesterone levels are high, such as during the luteal phase or after progesterone administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that MEHP inhibits rat granulosa cell function by a protein kinase C-independent mechanism

We have recently shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the reproductive toxicant di-(ethylhexyl) phthalate (DEHP), inhibited FSH-but not forskolin-, isoproterenol-, or cholera toxin-stimulated granulosa cell cAMP accumulation in vitro. In addition, MEHP also inhibited FSH-stimulated progesterone production, a cAMP-dependent process. Similar to MEHP, the protein kinase C (PKC) activator, 12-0-tetradecanoyl-phorbol 13-acetate (TPA) has been shown to inhibit rat granulosa cell cAMP accumulation in a FSH-specific manner, and decrease FSH-stimulated progesterone production. Due to the similarity with respect to inhibition of cAMP accumulation, we conducted studies to determine if the inhibitory actions of MEHP on granulosa cell function are mediated via activation of PKC. Treatment of granulosa cells for 48 h with 100 μM MEHP produced no effect on forskolin- or isoproterenol-stimulated progesterone production, indicating that MEHP does not have a post-cyclic AMP site of action with respect to progesterone inhibition. Unlike the FSH-specific effect seen with MEHP, treatment with 10 nM TPA inhibited FSH-, forskolin-, and isoproterenol-stimulated progesterone production. In addition, maximally inhibitory concentrations of TPA and MEHP caused significantly greater inhibition of FSH-stimulated cAMP accumulation than either compound alone. Finally, addition of the progesterone precursor, pregnenolone, reversed the FSH-stimulated progesterone production inhibition by MEHP, but not that by TPA. Taken together, these data indicate that the inhibitory effects of MEHP on granulosa cell function are independent of phorbol ester-sensitive PKC activation.

Evidence for modulation of progesterone secretion by calcium and protein kinase C activators in ovine chorionic cells

The hypothesis that calcium-dependent mechanisms may be involved in regulating ovine placental steroidogenesis was investigated using chorionic cells isolated by enzymatic digestion. Treatment of the cells with the calmodulin antagonist trifluoperazine (TFP) or pimozide caused a dose-related inhibition of progesterone (P 4) production by 80 per cent (P < 0. 001) at 40 μM TFP and 56 per cent (P < 0.001) at 10 μM pimozide. Moreover, the conversion of25 hydroxycholesterol (25 OH Chol.) to P 4 was impaired in the presence of these compounds. These experiments suggest the involvement of a calcium-calmodulin system in the regulation of ovine placental P 4 synthesis. Interestingly, calcium ionophoreA23187 caused a gradual decline in P 4 secretion and completely blocked it at I μM (P < 0.001) and remains absent even in the presence of 25 OH Chol. In contrast, EGTA increased P 4 secretion (P < 0.01). Further, in the presence of 3 mm EGTA the inhibitory effect of 1,μM A23187 was fully reversed. Taken together these results suggest that extracellular calcium could play a role of negative modulation of P 4 secretion in these cells. The possible involvement of protein kinase C (PKC) was tested using tumorpromoting phorbol ester (PMA) or permeant diacylglycerols (OAG or DOG). These compounds were unable to modify basal P 4 secretion but reduced 25 OH Chol stimulated secretion to basal level. The phorbol ester that was unable to activate PKC had no effect on the metabolism of 25 OH Chol. Thus, PMA and diacylglycerol effects are probably mediated by PKC These data support the hypothesis that PKC activation plays a role in the modulation ofcholesterol side-chain cleavage activity in ovine chorionic cells. These results show that calcium-dependent processes are involved in both positive and negative control of P 4 secretion by ovine placenta. Our results also suggest a role for calmodulin and PKC pathways in modulating this secretion.