Placental luteinizing hormone-releasing factor and its synthesis.

The synthesis of a placental luteinizing hormone-releasing factor (pLRF), which is immunologically, physiochemically, and biologically indistinguishable from synthetic LRF, was demonstrated. The incorporation of 3H-labeled leucine by human placental tissue in vitro into pLRF was determined by purification on carboxymethyl-cellulose and specific immunoprecipitation of the 3H-labeled pLRF. The specific activity of the pLRF released into the medium increased 100-fold from day 1 to day 2 of culture and attained a concentration of 2.84 microcuries per microgram. These data indicate that the pLRF that was released initially was endogenous, whereas that released subsequently reflected synthesis.

Ectopic pituitary transplants stimulate synthesis and release of follicle-stimulating hormone in golden hamsters.

It was previously reported that PRL can stimulate FSH release in immature female rats and PRL-deficient male dwarf mice. We have examined the effects of PRL-producing ectopic pituitary grafts on plasma FSH and LH levels in adult male golden hamsters during exposure to both long (14 h of light, 10 h of darkness) and short (5 h of flight, 19 h of darkness) photoperiods. In both groups, the presence of one pituitary graft resulted in a significant elevation of pituitary and plasma FSH levels. Although there were no statistically significant changes in the concentration of LH in the pituitary or plasma of either group. LH values were more variable than FSH values, and these findings do not rule out an effect on LH. To determine whether the ability of ectopic pituitary grafts to stimulate FSH release depends on changes in testicular or adrenal function, additional experiments were done in castrated male hamsters, in castrated males given testosterone propionate, and in castrated adrenalectomized hamsters maintained with injections of cortisol. The results indicate that the increase in FSH release in engrafted animals does not depend on the testes or the adrenals, but can be prevented by exogenous cortisol. In adult female hamsters, pituitary grafts increased plasma LH levels above the concentrations observed in diestrous, proestrous, or pregnant controls, but did not alter plasma FSH levels. In contrast, in ovariectomized and ovariectomized-estradiol benzoate-treated animals, pituitary grafts increased plasma FSH levels without significantly affecting plasma LH. It is concluded that PRL-producing ectopic pituitary homografts significantly stimulate FSH synthesis and release in male golden hamsters, and that this effect is probably not mediated through the changes in gonadal or adrenal function.

Does prolactin modify testosterone feedback in the hamster? Pituitary grafts alter the ability of testosterone to suppress luteinizing hormone and follicle-stimulating hormone release in castrated male hamsters.

Adult male golden hamsters maintained in a long photoperiod (14 h of light and 10 h of darkness) or in a short photoperiod (5 h of light and 19 h of darkness for 7 weeks) were castrated and either given one anterior pituitary transplant under the kidney capsule or sham-operated. Additional animals were castrated and grafted or sham-grafted at the time of transfer to the short photoperiod. Starting 2 weeks after castration, all animals were injected three times a week with 20 micrograms testosterone propionate (TP). After 3 weeks, the dose of TP was increased to 80 micrograms and, after an additional 2 weeks, to 320 micrograms per injection. Blood samples were collected 2 weeks after castration and 1 day after the last injection of 20, 80, and 320 micrograms TP. Short photoperiod reduced and pituitary grafts increased plasma PRL levels. Plasma testosterone levels were related to the dose of injected TP, but were not influenced by photoperiod or pituitary transplants. Before the onset of TP injections, plasma LH and FSH levels in grafted and sham-grafted hamsters did not differ. In each of the three photoperiod conditions, injections of TP were consistently less effective in suppressing plasma gonadotropin levels in pituitary-grafted animals than in sham-grafted controls. These results indicate that PRL modulates the effects of exogenous testosterone on LH and FSH release in adult castrated male golden hamsters, this effect of PRL is due to reducing the sensitivity of the hypothalamic-pituitary system to feedback inhibition by testosterone, and suppression of pituitary PRL release in short photoperiod may be partially responsible for the concomitant increase in the sensitivity of LH and FSH release to inhibition by testosterone.

Action of chicken II GnRH on the human placenta.

The stability, receptor binding, bioactivity, and production of chicken II GnRH and its analogs in the human placenta were studied. Both chicken II and mammalian GnRH are rapidly degraded by placental enzymes, yet the chicken II isoform is six times more stable. Analogs of chicken II GnRH were synthesized, and their stability in the presence of placental enzymes was tested. The D-Arg(6)-chicken II GnRH-aza-Gly(10)-NH(2) analog was found to be resistant to enzymatic degradation. The placental receptor binding activity of the chicken II GnRH analogs and chicken II GnRH was compared with that of mammalian GnRH and its analogs. Because D-Arg(6)-chicken II GnRH-aza-Gly(10)-NH(2) had the highest affinity for the placental receptor and was stable in placental extracts, the bioactivity of this analog on the regulation of placental human CG (hCG) release was compared with that for mammalian and chicken II GnRH using a human term placental explant culture system. This chicken II GnRH analog effected a stimulation of hCG at the lowest concentration studied (250 nM). With extended exposure and/or higher concentrations of this chicken II GnRH analog, the release of hCG from human placental explants was inhibited. Using a placental explant perifusion system and a highly specific RIA for chicken II GnRH, the pulsatile release of chicken II GnRH from the early human placenta was demonstrated. These studies are the first to demonstrate bioactivity of a second form of GnRH in a human tissue and to identify the pulsatile release of chicken II GnRH from a human tissue. These data led us to propose that chicken II GnRH and its synthetic analogs may be potent ligands for hormone regulation during pregnancy.

Ovarian refractoriness to gonadotropins in cases of inappropriate lactation: restoration of ovarian function with bromocryptine.

In ten patients with amenorrhea-galactorrhea who had hyperprolactinemia, ovulation could not be induced clomiphene citrate or exogenous gonadotropins. Treatment with bromocryptine in eight of these patients resulted in suppression of PRL in all, cessation of galactorrhea and ovulation in seven and conception in five.

Effects of porcine follicular fluid on gonadotropin concentrations in rhesus monkeys.

The administration of steroid-free charcoal-treated porcine follicular fluid to long term castrated female rhesus monkeys lowered basal serum concentrations of FSH and had almost no effect on serum LH. Treatment with porcine follicular fluid before the administration of exogenous LRH inhibited the release of FSH, but also affected the release of LH. This inhibition was especially striking on the suppression of the peak of release of both FSH or LH at 20 min. These findings suggest that an inhibin-like material present in follicular fluid could play an important role in the secretion of FSH and LH in rhesus monkeys.

Failure of indomethacin to block the estrogen-induced luteinizing hormone surge in the ovariectomized rhesus monkey.

Prostaglandins have been shown to be stimulatory to LH release in several species, although it is not known, in the primate, whether this effect is mediated through the hypothalamic-pituitary axis or by an indirect effect mediated through the ovary. In the present experiments, indomethacin, an inhibitor of prostaglandin synthesis, did not significantly alter basal LH levels or the positive and negative feedback effects of estrogen on LH release in the ovariectomized rhesus monkey. Therefore, it appears that the previously demonstrated effect of indomethacin on LH release in the intact monkey was mediated through ovarian mechanisms.

Luteolytic effect of D-Trp6-luteinizing hormone-releasing hormone in the rhesus monkey (Macaca mulatta).

A potent agonist of luteinizing hormone, D-Trp6-LRH, was administered at different stages during the luteal phase of rhesus monkeys (Macaca mulatta). Luteolysis, evidenced by short luteal phases and decreased serum progesterone concentrations, was consistently induced when the analog was injected on days 3 or 5 post ovulation; however, no effect was observed when it was given on day 7 post ovulation. Increasing doses of hCG administered from days 6-10 post ovulation prevented the luteolytic effect of D-Trp6-LRH. The possible mechanisms of action and the potential uses of LRH analogs as contraceptive agents are discussed.

Effects of delta 9-tetrahydrocannabinol during the follicular phase of the rhesus monkey (Macaca mulatta).

It is well documented in the literature that delta 9-tetrahydrocannabinol (THC) decreases serum concentrations of pituitary gonadotropins in several species. To study its effects in the menstrual cycle of regularly cycling rhesus monkeys, 2.5 mg/kg THC were administered to five animals from days 1-18 of the cycle [ovulation day in our colony, 15 +/- 1 day (mean +/- SD)]. Controls received vehicle (Tween 80 and saline) in an identical protocol. Animals were bled daily or every other day, and serum total estrogens, LH, PRL, and progesterone were determined by RIA. Serial laparoscopies were performed to visualize ovulation. Whereas animals treated with vehicle presented normal cycle lengths (26, 26, 29, 30, and 34 days), those treated with THC presented abnormal lengths (145, 76, 22, 94, and 59 days). All vehicle-treated cycles were ovulatory, while four of five THC cycles were anovulatory (P < 0.02). Five THC-treated animals were anovulatory in the posttreatment cycle. To determine the site of action of THC-induced anovulation, five animals received THC, human menopausal gonadotropin, and hCG simultaneously. All ovulated normally, as determined by laparoscopic visualization of stigma. Normal luteal phases were evidenced by normal luteal phase lengths and serum progesterone concentrations. These findings are of clinical relevance, since they were achieved with doses of THC that produce blood concentrations similar to those found in heavy marijuana users.

Overnight plasma profiles of melatonin and certain adenohypophyseal hormones in men.

Melatonin levels exhibited a day-night rhythm with highest levels at night. Nocturnal plasma melatonin concentrations were unrelated to sleep stages, whereas secretion of GH was temporally related to slow wave sleep. Levels of corticotropin rose in the later sleep cycles. We found no relationship between endogenous nocturnal melatonin and adenohypophyseal hormone levels. The results indicate that in young men nocturnal levels of melatonin are controlled separately from those of LH, PRL, corticotropin, and GH.

Effect of exogenous arachidonic acid and enzyme inhibitors on placental prostanoid production.

Prostanoids play an important role throughout pregnancy and during the initiation and progress of labor. The human placenta, at term, produces large quantities of prostanoids, yet little is known of the rate-limiting steps regulating their biosynthesis. In these studies, the effect of exogenous arachidonic acid and of enzyme inhibitors on the release of placental prostanoids was investigated. Basal prostaglandin E (PGE), prostaglandin F (PGF), thromboxane (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) increased from the fifth hour in culture, yet the increased PGF release did not result in further 13,14-dihydro-15-keto-PGF2 alpha (PGFM) production. Addition of varying doses of arachidonic acid (0.2-10 micrograms/ml) had no significant effect on PGFM, TxB2 or 6-keto-PGF1 alpha, although the endogenous arachidonate was similar to or much less than the doses studied. Only at the 10 micrograms/ml dose was a delayed increase of PGE and PGF observed. Incubation with indomethacin resulted in an immediate inhibition of PGE, PGF, TxB2 and 6-keto-PGF1 alpha, with a delayed inhibition of PGFM. The phospholipase A2 inhibitor, quinacrine (10 microM), had no significant effect on PGE, PGFM, TxB2 or 6-keto-PGF1 alpha. PGF was inhibited within the first hour of quinacrine exposure, but no significant inhibition was observed thereafter. Ca2+ chelator, EDTA, effected an inhibition of only 6-keto-PGF1 alpha. Nordihydroguaiaretic acid, a leukotriene inhibitor, reduced PGE release as well as 6-keto-PGF1 alpha. These data demonstrate the high biosynthetic competence of the human term placenta to produce prostanoids. This capacity does not appear to be rate-limited by arachidonic acid availability. However, the metabolism of PGF to PGFM appears to be saturated. In addition, the production of placenta prostacyclin may be affected by Ca2+ levels, as chelating agent inhibited the release of 6-keto-PGF1 alpha.

Chorionic peptidase inactivates GnRH as a post-proline peptidase.

Recently, we have described a chorionic peptidase (C-ase-1) which inactivates gonadotropin releasing hormone (GnRH), oxytocin, angiotensin II and thyrotropin releasing hormone. Since all these hormones contain a proline residue, we proposed that C-ase-1 may act as a post-proline peptidase. Using HPLC and amino acid analyses, we have defined the products which resulted from enzymatic inactivation of GnRH by C-ase-1. The N-terminal nonapeptide of GnRH was isolated by HPLC and confirmed by amino acid composition analyses. Thus, it was demonstrated that C-ase-1 acts as a post-proline peptidase when inactivating GnRH, yielding the nonapeptide, i.e., des-Gly10-NH2-GnRH, and Gly-NH2. The levels of intrauterine GnRH, angiotensin II, oxytocin and thyrotropin releasing hormone may be affected and integrated by this enzyme. Thus, C-ase-1 may play an important role in the regulation of the paracrine and endocrine function during pregnancy.

Placental prostanoid release in severe intrauterine growth retardation.

Our objective was to evaluate prostanoid release from the placentae of pregnancies complicated by severe intrauterine growth retardation (IUGR) and without hypertension, compared with placentae from normal, uncomplicated term pregnancies. A perifusion system was utilized to study the release of prostanoids 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), thromboxane B2(TxB2), prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) from human placentae from pregnancies complicated by normotensive severe IUGR (n = 9, five at term and four preterm) and normal control pregnancies (n = 6). For each placenta, triplicate chambers of tissue were perifused at a rate of 6 ml/h, and samples were collected from hours 5-10. Prostanoids were measured using specific and sensitive radioimmunoassays. In the IUGR group, the basal placental production of the vasoconstrictor thromboxane was not increased, nor was the ratio of cumulative TxB2 to 6-keto-PGF1 alpha elevated compared with normal term controls. In three term IUGR placentae, the ratio was significantly decreased compared with controls. The basal placental production of the vasoconstrictor PGF2 alpha was likewise not increased compared with controls, nor was the ratio of PGF2 alpha to PGE2 elevated. Two of the placentae in the term IUGR group demonstrated significant elevations of PGE2 and 6-keto-PGF1 alpha. Overall, the IUGR placentae released normal or low normal levels of the prostanoids studied. The pattern of placental prostanoid release over time was similar to that of the normal term placentae. The term and preterm placentae of pregnancies complicated by severe IUGR did not exhibit an excess production of vasoconstrictor prostanoids. Therefore, strategies designed to reduce thromboxane production in severe IUGR without hypertension may be unjustified.

Gestational age-related inhibition of placental hCG, alpha hCG and steroid hormone release in vitro by a GnRH antagonist.

Human placental tissues have been shown to contain gonadotrophin-releasing hormone-(GnRH)-like activity. Thus, the effect of a potent GnRH antagonist (N-Ac-Pro1,D-p-Cl-Phe2,D-Nal(2)3,6-GnRH, obtained from Syntex Laboratories) on placental hormonal release was studied. Explant cultures of placentae of 6 to 15 weeks' gestation were studied. This GnRH antagonist did not inhibit the alpha human chorionic gonadotrophin (alpha hCG), human chorionic gonadotrophin (hCG), oestrone or oestradiol release from the six- and nine-week placental cultures, but greatly suppressed the release of these hormones in the placental cultures from 13- and 15-week gestations. Synthetic GnRH partially reversed the action of this antagonist on the hormonal releases in the 15-week placental cultures. These data demonstrate a gestational age-related action of this antagonist on placental hormonal release. Thus, a role for the endogenous GnRH-like activity of the placenta in the control of placental hormonogenesis is indicated.

Characterization and purification of a placental protein that inactivates GnRH, TRH and angiotensin II.

A protein that inactivates the immunoreactivity of GnRH, TRH and angiotensin II has been isolated from human term placentae. Only in the presence of DTT, a sulphydryl agent, are OXY and SRIF also inactivated by this protein. However, it is without effect on CRF, hCS, or hCG. It also inhibits the biological activity of GnRH, i.e. its ability to stimulate pituitary LH and FSH. The ability of this protein to inactivate GnRH, TRH or angiotensin II can be inhibited by various peptidase inhibitors. Thus, we have postulated that it is a chorionic peptidase, specific for these peptides, and herein called chorionic peptidase-1 (C-ase-1). Isolation of this protein, C-ase-1, has been effected using permeation, ion exchange and affinity chromatography. As estimated by SDS-PAGE and HPLC analyses, C-ase-1 has an apparent molecular weight of 58,000. It is proposed that C-ase-1 may be an important chorionic regulator of GnRH, TRH and angiotensin II levels during pregnancy.

Nonthyroidal control of metabolism after burn injury: possible role of glucagon.

The endocrine basis for control of metabolism in nonthyroidal illness is not yet understood. Burn injury is associated with reduced serum concentrations of thyroid hormones and with resting hypermetabolism. One index of severity is total burn size (TBS, % body surface). After overnight fasting and recumbency, resting metabolic rate (MR, O2 consumption) was measured weekly and plasma was sampled for determination of glucose, total cholesterol, triglycerides, insulin, glucagon, somatostatin, growth hormone, norepinephrine, epinephrine, and cortisol in 28 burned men, 17-23 years old, TBS 2%-85%, including 8 controls with minimal injury (TBS less than or equal to 7.5%). MR was elevated in proportion to burn size mainly in the first week then declined toward normal. Growth hormone was not changed. Two multiple regression analyses (validated by random partitioning of data) determined which plasma variables independently reflected residual variation in MR: without TBS entered as a variable, high MR was associated with elevated glucose, cortisol, and glucagon, and low cholesterol (cumulative r2 = 0.79); with TBS entered, high MR was associated with greater TBS, elevated norepinephrine, and again high glucagon and low cholesterol (r2 = 0.81). Resting metabolism after burn injury is controlled not by the thyroid but may be controlled by a set of antiinsulin hormones that does not include growth hormone, but possibly includes glucagon.

Studies in human fetal endocrinology: II. LH and FSH content and concentration in the pituitary.

The luteinizing hormone (LH) and follicle-stimulating hormone (FSH) content and concentration in 67 whole fetal pituitary glands have been determined by specific radioimmunoassay. A distinct pattern was observed for LH and FSH related to fetal age and sex. In the female, a mid-gestation peak of LH and FSH was found, followed by a rapid and significant decline in both gonadotropins. There was a secondary increase in the content but not the concentration from 24 weeks' gestation to term. In the male, LH and FSH content continued to increase throughout gestation. However, the rise after 20 weeks' gestation is related to the increasing pituitary weight. These data are discussed in relation to the endocrine status of the fetal gonad and hypothalamus at comparable gestational ages.

Maternal serum corticotropin-releasing hormone at midgestation in Hispanic and white women.

OBJECTIVE: Maternal circulating corticotropin-releasing hormone analysis at midgestation has been proposed as a parameter for the prediction of preterm birth. However, one recent study has reported that corticotropin-releasing hormone concentrations at midgestation differ in the black and white populations. These findings led us to investigate whether other populations have differing concentrations of maternal circulating corticotropin-releasing hormone that may require reference to specific population-based medians for optimal midgestational screening. METHODS: In this study we have defined the mean and median concentrations of maternal circulating corticotropin-releasing hormone in Hispanic and white populations at each gestational week from 14 to 18 weeks of pregnancy, using a sensitive and specific radioimmunoassay. RESULTS: Corticotropin-releasing hormone concentrations were found to be significantly lower in the Hispanic population as compared with whites at 16, 17, and 18 weeks' gestation. The distribution of corticotropin-releasing hormone, expressed as multiples of the median (MoM) using the appropriate ethnicity-related median, was estimated for each gestational week and for each population. No differences were observed in the distribution of the ethnicity-adjusted MoM for Hispanics and whites. CONCLUSION: These data demonstrate that ethnicity is a significant factor affecting corticotropin-releasing hormone concentrations at midgestation in the Hispanic and white populations. The use of ethnicity-specific medians to estimate the ethnicity-specific MoM for the corticotropin-releasing hormone concentrations may enhance the predictive value of midgestational maternal corticotropin-releasing hormone as a screening parameter for the prediction of preterm birth.

Luteolytic effect of azastene in the nonhuman primate.

The ability to block steroidogenesis with 4,4,17-alpha-trimethylandrost-5-eno[2,3,-d]isoxazol-17-ol (azastene) was studied in 3 different models. Oral administration of 500 mg to rhesus monkeys on different days of their luteal phase induced marked depression of circulating progesterone concentrations, and in some cases early onset of menses. Simultaneous administration of human chorionic gonadotropin (hCG) during the midluteal phase did not overcome the luteolytic effect of azastene. Concentrations of 50 micrograms/ml of azastene inhibited testosterone secretion by decapsulated mice testes in vitro in response to hCG [controls, 1165 +/- 196 ng/ml, azastene, 306 +/- 40 ng/ml (P less than .01)]. Production of progesterone by dispersed luteal cells from rhesus monkey corpora lutea was markedly inhibited by the presence of 25 and 50 micrograms/ml azastene in the incubation media (P less than .05 and less than .01, respectively). The availability of a compound that blocks in vivo and in vitro gonadal steroidogenesis opens a new approach to postcoital contraception in primates because of its luteolytic and interceptive activity. The possible mechanisms of action of azastene are discussed.

Effects of danazol on gonadotropin levels in castrated rhesus monkeys.

Danazol is widely used in the management of endometriosis and mammary dysplasia. However, its mechanism of action is still obscure because of the few studies done and the controversial results obtained. Antigonadotropic activity has been postulated by some investigators, whereas others have observed no effect on the gonads. In the present study, three castrated female rhesus monkeys received 400 mg of danazol daily, by gavage for 19 days, while 2 controls received 400 mg of lactose daily. Blood samples were drawn every other day from 2 weeks prior to 3 weeks after the administration of the drug. Plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) decreased rapidly and then were maintained until danazol was discontinued. Prompt return to pretreatment levels occurred 1-2 days after the discontinuation of therapy. Prolactin levels were normal during therapy. Luteinizing hormone releasing factor (LH-RF), 100 microgram, administered intravenously during danazol therapy, caused a normal, prompt release of gonadotropins, demonstrating an intact pituitary response. The authors conclude that 1) danazol is a potent antigonadotropic agent, 2) its effect is abolished promptly after discontinuation, and 3) its probable biological action is at the hypothalamic level.