Induction of polyphosphoinositide breakdown in rat corpus luteum by prostaglandin F2 alpha.

The present study examines the possibility that, in the rat corpus luteum, an initial action of prostaglandin F2 alpha (PGF2 alpha) is to induce a ligand-stimulated breakdown of membrane inositol phospholipids. Luteal cells in primary culture were prepared from immature rats after PMSG and human CG priming. In 32P-prelabeled cells, PGF2 alpha caused a rapid decrease in the level of radiolabel found in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, as early as 20 sec after addition of the hormones. At 1 and 2.5 min, the effect of 10(-6) M PGF2 alpha on phosphatidylinositol 4,5-bisphosphate was significantly greater than that caused by 10(-6) M LHRH in identical cell cultures. By contrast, the levels of the 32P-prelabeled phosphatidylinositol and phosphatidic acid were increased at 5 min by PGF2 alpha or LHRH. Concomitant with the alterations in cellular levels of 32P-prelabeled phospholipids, PGF2 alpha markedly enhanced the accumulation of 3H-labeled inositol phosphates, i.e. inositol 1-phosphate, inositol diphosphate, and inositol triphosphate, during a 5-min incubation. A significant increase of radiolabeled inositol diphosphate was seen as early as 1 min after the addition of either PGF2 alpha or LHRH; PGF2 alpha was more effective than LHRH in this regard. The stimulatory effect of LHRH on inositol phosphate accumulation could be blocked completely by the concomitant presence of a potent LHRH antagonist, and at the concentration used (10(-6) M) the effects of PGF2 alpha and LHRH were not additive. Interestingly, the addition of an exogenous phospholipase C also caused a similar enhancement of inositol phosphate accumulation in identical cell cultures. For the first time, these data suggest that, at the level of the corpus luteum, hydrolysis of phosphoinositides may immediately follow PGF2 alpha (and to a lesser extent LHRH) receptor binding, and this in turn may lead to the generation of 1,2-diacylglycerol and inositol phosphates, resynthesis of phosphatidic acid and phosphatidylinositol, and mobilization of Ca2+.

Sclerosing stromal tumor of the ovary.

This case study of a sclerosing stromal tumor of the ovary in a 29-year-old woman demonstrates that the tumor was a functioning lesion that secreted estradiol, progesterone, and testosterone. Clinically, there was anovulation with resultant infertility, which was resistant to therapy with clomiphene citrate. Removal of the tumor was followed by reversal of these abnormalities.

Applications of monoclonal antibodies in solid-phase immunoassays of human luteinizing hormone.

Monoclonal antibodies to human luteinizing hormone (hLH) were generated by a modified hybridoma technique. Out of forty hybrid cell lines that were shown to secrete antibodies reacting with hLH, LH35 and LH40 were further characterized biochemically and immunologically. LH35 was found to secrete immunoglobulin G1 antibody specific to the alpha-subunit of LH, whereas that of LH40 was beta-subunit specific. Association constants between the antibodies and LH were determined to be 2 X 10(8) and 1 X 10(9) M-1, respectively, by using competitive radioimmunoassay and Scatchard plots. Monoclonal antibodies from LH35 and LH40 were purified from the respective ascites fluids by ammonium sulfate fractionations and DEAE ion-exchange chromatography. The purified alpha-subunit-specific antibody of LH35 was immobilized on polystyrene balls (6 mm in diameter), whereas purified LH40 antibody was conjugated with horseradish peroxidase or labeled with iodine-125. Solid-phase radio- and enzyme immunoassays were designed to measure relatively low concentrations of LH (2-100 mIU/ml). The LH surge during the midcycles of women with normal menstrual cycles could easily be detected from daily urine or serum specimens by a 1-h assay procedure. It is proposed that this new LH immunoassay procedure can be routinely used for predicting ovulation of women with normal menstrual cycles.

Prolactin in human milk: the influence of nursing and the duration of postpartum lactation.

The concentrations of prolactin in the milk of nine postpartum lactating mothers were determined by radioimmunoassay between days 3 to 280 of the puerperium (n = 324 samples). In addition, prolactin in milk was also determined at the beginning and the end of suckling in three of the same women, who provided 21 paired samples of foremilk and hindmilk between days 7 and 88 of the puerperium. Prolactin content was highest at 43.1 +/- 4 ng/ml (mean +/- SEM) in the early transition milk immediately after the colostrum phase during the first postpartum week, decreasing to 11.0 +/- 1.4 ng/ml in the mature milk (p less than 0.01) when weaning occurred in those mothers who breastfed for up to 40 weeks post partum. During suckling, the foremilk contained significantly more prolactin as compared with the hindmilk (29.5 +/- 2.7 versus 21.0 +/- 3.2 ng/ml; p less than 0.01). These findings, taken together with the known biologic potency of prolactin in breast milk, the osmoregulatory influence of the hormone in mammary and intestinal function, and its absorption by the newborn experimental animal, suggest that the presence of prolactin in milk may play some role in both lactation and the intestinal absorptive function of the suckling newborn.

Relationship of prolactin and estradiol to human chorionic gonadotropin following molar gestation.

Serial plasma concentrations of the beta subunit of human chorionic gonadotropin (beta-hCG), prolactin, and estradiol were examined and correlated in 22 women after evacuation of a molar pregnancy for up to 15 weeks into the postmolar phase. The mean levels of prolactin were lower, estradiol levels were higher, and beta-hCG levels were slightly lower but comparable to those reported for women in the first postpartum week. Mean concentrations of all three hormones then declined during the postmolar period; however, beta-hCG remained detectable (greater than or equal to 5 mIU/ml) in some women for up to the fifteenth week, while the nadirs in mean prolactin and mean estradiol levels occurred at 14 and 13 weeks, respectively. The plasma concentrations of beta-hCG correlated with those of prolactin (r = 0.93) and estradiol (r = 0.94), while the levels of the latter two hormones also correlated (r = 0.84).

High glucose concentration and phosphoenolpyruvate carboxykinase activity in human and rat fetal liver cultures.

In cultures of human and rat fetal liver, phosphoenolpyruvate carboxykinase activity increases during the first 24 hr of culturing. This increase can be suppressed by adding cycloheximide to the culture medium or by adding a high glucose concentration. This, however, applies only to human fetal liver and to fetal liver from rats obtained just before term. In younger rat fetal liver, glucose, on the contrary, increases the activity of phosphoenolpyruvate carboxykinase. A high glucose concentration in the medium also leads to higher citrate cleavage enzyme activity and to lower alpha-glycerolphosphate dehydrogenase (cytoplasmic) activity in rat fetal liver cultures.

Studies of monoclonal antibodies against human chorionic gonadotropin. II. Applications of human chorionic gonadotropin monoclonal antibodies on immunoassays.

Monoclonal antibodies generated against human chorionic gonadotropin (hCG) were utilized in radio- and enzyme-immunoassays of this reproductive hormone in biological fluids. One of the monoclonal antibodies, beta-4D6, was shown to have extremely high affinity (Ka = 8 X 10(10) M-1)) and high specificity (less than or equal to 0.6% LH cross-reactivity) to beta-subunit of hCG and whole hCG. It was used in competitive radioimmunoassays (RIA) for the determination of low levels of serum hCG. An excellent correlation was obtained concerning the assay results between the monoclonal antibody-based system and others using conventional polyclonal anti-sera. In combination with another discrete hCG monoclonal antibody, solid-phase sandwich radiometric and enzyme immunoassays were established. These immunoassays could easily be performed, and they offer sensitivity and efficacy of hCG determination comparable to those of conventional ones.

Assessment of maximal fertilization rates with intracytoplasmic sperm injection.

PURPOSE: Maximal fertilization rates following ICSI were assessed using two essential steps: immobilization of sperm and aspiration of oocyte cytoplasm. METHODS: ICSI procedure was performed for couples (N = 42) in whom the male suffered severe infertility or failure of fertilization in previous IVF cycle using different oocyte cytoplasmic aspiration and sperm immobilization methods. Outcome in four patient groups was measured by oocyte damage, fertilization rate, and pregnancy rate. RESULTS: Maximal fertilization (90%) were achieved from the group which used immobilization of sperm by hard-touching the tail with a pipette and optimal aspiration of oocyte cytoplasm. CONCLUSIONS: The results suggest that if the immobilization of sperm and aspiration of oocyte cytoplasm are handled right during ICSI, this procedure can be expected to yield a 90% fertilization rate.

Role of androgens in menstrual disorders of nonhirsute and hirsute women, and the effect of glucocorticoid therapy on androgen levels in hirsute hyperandrogenic women.

The plasma concentrations of total testosterone, free testosterone index, androstenedione, 17 beta-estradiol, luteinizing hormone, follicle-stimulating hormone, prolactin, and urinary 17-ketosteroid and 17-ketogenic steroid excretion were measured in 48 nonhirsute and 119 hirsute patients. Hormone data were compared within and between groups according to whether the menstrual cycles were eumenorrheic, amenorrheic, or oligomenorrheic. Eleven hirsute women treated with prednisone were followed for 6 months. It was concluded that: (1) androstenedione, testosterone, free testosterone index, and adrenal androgens alone or in combination play a role in the pathogenesis of the hirsutism observed in eumenorrheic women and in the amenorrhea and oligomenorrhea of both hirsute and nonhirsute women; (2) body weight correlated with adrenal adrogens (17-ketosteroids) in nonhirsute women and with androstenedione in hirsute women; (3) prednisone significantly suppressed androstenedione and 17-ketosteroids (p less than 0.05), with a decline of testosterone to 65% and luteinizing hormone to 51% of pretreatment values, with favorable clinical effects on the hirsutism, menstrual dysfunction, and infertility; (4) concentrations of 17 beta-estradiol were lower in amenorrheic than in eumenorrheic and oligomenorrheic women of both groups.

Relationship of oral contraceptives and the intrauterine contraceptive devices to the regression of concentrations of the beta subunit of human chorionic gonadotropin and invasive complications after molar pregnancy.

One hundred ninety-four patients with pathologically confirmed molar pregnancy and intact uteri were studied prospectively. Group A included 177 patients in whom the beta subunit of human chorionic gonadotropin (hCG-beta) declined to normal (less than 5 mlU/ml) without chemotherapy, whereas group B included 17 patients with invasive complications in the postmolar phase which necessitated the use of chemotherapy. Only women with intact uteri were included in the study. In group A, there were no significant differences in the human chorionic gonadotropin (hCG) positive interval between women who used intrauterine contraceptive devices, barrier and other methods, and those who used oral contraceptives. Differences in the proportions of women in groups A and B who used the oral contraceptives and intrauterine contraceptive devices were not observed. However, the mean dosage of estrogen and the proportion of women who ingested more than 50 micrograms of estrogen were higher in group B. These data suggest that (1) the oral contraceptives with less than 50 micrograms of estrogen and the intrauterine contraceptive devices do not prolong the hCG-beta positive interval nor increase the risk of invasive complications in the postmolar phase which requires the use of chemotherapy; and (2) the dose of estrogen (in formulations that contain more than 50 micrograms) rather than the oral contraceptives per se may influence the risk of these postmolar complications.

Effect of prolactin in an experimental model of the ovarian hyperstimulation syndrome.

In the rabbit model of the ovarian hyperstimulation syndrome, animals given ovine prolactin with human menopausal gonadotropins (hMGs), as compared to animals receiving hMGs alone, demonstrated an increase in the formation of ascitic fluid, a decrease in mean plasma estradiol, and an increase in the mean plasma progesterone concentrations. The ovarian estradiol and progesterone content reflected that of the peripheral blood. These data suggest that, under the conditions of these experiments, prolactin may play a role in the pathogenesis of ascites formation but not the ovarian enlargement observed in this syndrome. Although the plasma estradiol levels were lower and the progesterone levels were higher in the animals treated with prolactin and gonadotropins, this did not prevent the occurrence of ascites, a cardinal clinical sign of this gonadotropin-induced syndrome.

Successful induction of ovulation and conception with pulsatile intravenous administration of human menopausal gonadotropins in anovulatory infertile women resistant to clomiphene and pulsatile gonadotropin-releasing hormone therapy.

Gonadotropins are released in a pulsatile fashion at a frequency of between 1 and 2 hours in the follicular phase of the menstrual cycle. Human menopausal gonadotropins are usually administered intramuscularly. We evaluated the gonadal response to intravenous human menopausal gonadotropins administered in a pulsatile fashion over nine treatment cycles in three anovulatory infertile women. Human menopausal gonadotropin pulses in doses up to 12 IU follicle-stimulating hormone at frequencies between 2 to 3 hours over 3 to 17 days resulted in ovulation in five cycles with one pregnancy being conceived. In the ovulatory cycles (5,000 to 10,000 IU of human chorionic gonadotropin was used to induce ovulation), the 17 beta-plasma estradiol level was 961 +/- 128 versus 326 +/- 95 pg/ml (mean +/- SEM) in the anovulatory cycles (p = 0.015). The dose of human menopausal gonadotropins (in ampules of Pergonal, 75 IU of follicle-stimulating hormone and 75 IU of luteinizing hormone) in the intravenous cycles needed to induce ovulation was 12.3 +/- 1.4 versus 20.4 +/- 0.9 for intramuscular cycles (n = 80 in 23 women, p = 0.008). Treatment was well tolerated and without complications. We are continuing to explore the use of this apparently more efficient mode of administering human menopausal gonadotropins to anovulatory patients resistant to other techniques of ovulation induction therapy.

Clinical, endocrinologic, and intraovarian prostaglandin F responses to H-1 receptor blockade in the ovarian hyperstimulation syndrome: studies in the rabbit model.

The effects of chlorpheniramine maleate, an H-1 receptor blocker, on clinical and endocrinologic features and intraovarian prostaglandin F (PGF) content were assessed in the rabbit model of the ovarian hyperstimulation syndrome. H-1 receptor blockade prevented ascites, attenuated ovarian enlargement (2.68 +/- 0.37 gm versus 4.15 +/- 0.056 gm; p less than 0.05), and augmented intraovarian PGF content (8.4 +/- 0.84 versus 3.95 +/- 1.12 pg/mg protein; p less than 0.05). Steroidogenesis was unaffected. In the control group, in which weights remained stable, animals with minimal ascites (scores less than or equal to 2; n = 4) were compared to other control animals with a greater accumulation of fluid (scores greater than or equal to 3; n = 4). The former also exhibited lower ovarian weights (2.94 +/- 0.41 versus 5.35 +/- 0.59 gm; p less than 0.05) and higher PGF ovarian content (6.05 +/- 1.56 versus 1.8 +/- 0.75 pg/mg of protein; p less than 0.05). This triad of minimal ascites, lower ovarian weights, and elevated intraovarian PGF seen both in treated animals and in inherently more resistant control animals did not appear to depend on changes in body weight. The conclusion reached was that H-1 receptor blockade prevented ascites, reduced ovarian enlargement, and augmented PGF content but did not affect steroidogenesis. This protective effect of chlorpheniramine may be mediated at least in part by prostaglandins.

Effects of clomiphene citrate on ovarian function in hypophysectomized rats.

On Days 28-30 of age, hypophysectomized rats were treated with oestradiol-17 beta (0.1 mg/day) and/or clomiphene citrate (0.1 mg/day). Subsequent treatment with PMSG (10 i.u., on Day 31) and hCG (10 i.u., on Day 33) was identical for all animals. Rats were killed on Day 34. Treatment with oestradiol-17 beta alone resulted in ovulations of 45.1 +/- 5.5 oocytes/rat (mean +/- s.e.m.). There were no ovulations among animals treated with clomiphene citrate alone but treatment with oestradiol-17 beta and clomiphene citrate resulted in a significant (P less than 0.05) reduction (23.1 +/- 7.6 oocytes/rat) in ovulatory response. Similarly, ovarian weights and serum progesterone concentrations were highest in the oestradiol-17 beta-treated rats, intermediate in those given oestradiol plus clomiphene citrate and the lowest in rats receiving clomiphene citrate alone. We suggest that clomiphene citrate exerts direct ovarian antiovulatory and oestrogen-antagonist actions.

X-chromosome inactivation (XCI) patterns in placental tissues of a paternally derived bal t(X;20) case.

Non-random X-chromosome inactivation (XCI) is often seen in female carriers of balanced X-autosome translocations and is generally attributed to a selective growth of cells that inactivate the normal X chromosome. However, little is known concerning when in development the selection acts, and thus whether skewed XCI would also be seen in placental tissues. Furthermore, as males with X-autosome translocations are normally infertile, all translocations studied to date for XCI-skewing have been either maternal or de novo in origin. We now present an analysis of XCI status in cord blood, umbilical cord and four different extraembryonic tissues from a female carrier of a paternally derived balanced (X;20) translocation. Using methylation based assays to determine XCI status, we found preferential inactivation of the non-translocated X in cord blood, umbilical cord and amnion samples of the propositus. Remarkably, random XCI was evident in several placental tissues analyzed (chorion, and chorionic villi trophoblast and mesenchyme). While these findings support the hypothesis of strong selection against cells with an inactive translocated X-chromosome in most embryonic/fetal tissues, they also suggest weaker selective forces taking place during placental development. Additionally, the finding of normal placental development in the present case, rules out the possibility of a parental bias to XCI in human extraembryonic tissues as a requisite for normal development. The finding of hypomethylation in extraembryonic tissues for two out of three markers used in the study is consistent with previous findings demonstrating low levels of methylation in these tissues.

An association between sex chromosomal aneuploidy in sperm and an abortus with 45,X of paternal origin: possible transmission of chromosomal abnormalities through ICSI.

BACKGROUND: Although it has been speculated that the increased de-novo chromosomal abnormalities in ICSI pregnancies may be associated with an increase of aneuploidy in sperm from infertile men, little direct evidence exists to support this claim. We studied sperm from an infertile man with an abortus from ICSI to determine if increased sex chromosomal aneuploidy in the sperm could have contributed to the karyotype of the abortus. METHODS: The couple underwent ICSI due to severe oligozoospermia. Spontaneous aborted material was subjected to cytogenetic and molecular tests to ascertain the existence, type and origin of a chromosomal abnormality. Sperm from the man were analysed by multi-coloured fluorescent in-situ hybridization (FISH) with probes specific for chromosomes X, Y and 18. RESULTS: At 8+ weeks after embryo replacement, the patient spontaneously miscarried. Both cytogenetic and comparative genomic hybridization analysis of aborted material showed a 45,X karyotype. Origin of the abnormality was established as a loss of the paternal X chromosome. FISH analysis of sperm revealed 19.6% (1990/10,164) nullisomy for a sex chromosome and 18.6% (1886/10,164) with XY disomy, which is significantly increased when compared to controls with 0.3% (58/20,429) and 0.1% (20/20,429) respectively (P<0.0001). CONCLUSIONS: This study indicates that the paternal origin of the 45,X abortus was likely the result of a high level of nullisomy in the sperm and provides evidence for the transmission of chromosomal abnormality from sperm to the conceptus through ICSI.

Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle-stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action.

Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha-dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity.

Adrenal and sex steroid hormone production by a virilizing adrenal adenoma and its diagnosis with computerized tomography.

The presence of an androgen-secreting tumor in a 29-year-old woman was confirmed and its location was determined by computerized axial tomographic (CAT) scanning. The hormone production from this virilizing adrenal adenoma was studied in vivo and in vitro. The major secretory products of the tumor (as compared to normal adrenal tissue) were testosterone (24-fold) and 17 beta-estradiol (five-fold). Although the adenoma produced lesser amounts of dehydroepiandrosterone sulfate (DHEAS), the demonstration of elevated serum testosterone and DHEAS in serial samples was a better marker for an androgen-secreting adrenal tumor than were the urinary 17-ketosteroids, which remained in the upper limit of normal. The hormone production from the tumor depended neither on adrenocorticotropic hormone nor on human chorionic gonadotropin. The conclusions were that: (1) on the basis of serial measurements of serum testosterone and DHEAS, virilizing adrenal adenomas may be suspected when the concentrations of these hormones reach or exceed 200 ng/dl and 6,600 ng/ml, respectively; (2) the high-resolution CAT scanner can accurately localize these tumors; (3) cosmetic and menstrual dysfunction regressed after resection of the tumor; and (4) virilizing adrenal adenomas may produce both androgens and estrogens.

Prolactin, estradiol, and thyroid hormones in umbilical cord blood of neonates with and without hyaline membrane disease: a study of 405 neonates from midpregnancy to term.

Concentrations of prolactin (PRL), estradiol (E2), thyroxine (T4), triiodothyronine (T3), and reverse T3 in umbilical cord blood were measured by radioimmunoassay in neonates with (n = 60) and in a control group without (n = 345) hyaline membrane disease. Mean levels of all hormones assayed were not significantly different between the two groups at various stages of gestation. In the control group, gestational age correlated positively with PRL and inversely with reverse T3, whereas birth weight correlated positively with PRL, T4, and T3, but inversely with reverse T3 levels. Thus, larger, more mature neonates tended to have higher cord levels of PRL, T4, T3 and lower concentrations of reverse T3. The data also suggest that, in the premature neonate, various obstetric complications and exposure in utero to beta-mimetic drugs and glucocorticoids may be important determinants of the concentrations of E2 and thyroid hormone in cord blood.

A possible role for prolactin in the control of human chorionic gonadotropin and estrogen secretion by the fetoplacental unit.

The effects of prolactin (PRL) on fetoplacental function were studied by measuring the beta subunit of human chorionic gonadotropin (hCG-beta), estradiol (E2), progesterone (P), and PRL concentrations throughout seven pregnancies in a control group (N = 6 women) and in three pregnant women with prolactinomas, who were receiving bromocriptine. In one of the latter, estriol (E3) was also assayed. Long-term suppression of PRL was associated with augmentation of hCG-beta at the first-treimester peak and in late pregnancy. Concomitant augmentation of E3 (in late gestation) and possibly E2, but not P, levels was also observed. This effect on hCG appeared dependent on PRL rather than the dopaminergic effect of bromocriptine. Short-term drug induced alterations in PRL (over 3 hours) during early pregnancy did not result in significant changes in hCG-beta or steroid concentrations. In each control pregnancy, a significant negative correlation (p less than 0.05) was observed between hCG-beta and PRL, while a significant positive correlation between the latter and E2 concentrations in these women was also demonstrated. Apart from its effect on lactation, osmoregulation, and gonadal and adrenal function, a further role for PRL during reproduction appears to be in the control of hCG and estrogen secretion in the fetoplacental unit.