Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

STUDY QUESTION: Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ovarian hyperstimulation syndrome (OHSS) altered and in part responsible for the high vascular permeability observed in these patients. STUDY ANSWER: FF S1P levels are lower in FF from patients at risk of OHSS and treatment with S1P may reduce vascular permeability in these patients. WHAT IS KNOWN ALREADY: Although advances have been made in the diagnosis, and management of OHSS and in basic knowledge of its development, complete prevention has proven difficult. STUDY DESIGN, SIZE, DURATION: A total of 40 FF aspirates were collected from patients undergoing ART. The women (aged 25-39 years old) were classified into a control group (n = 20) or a group at risk of OHSS (n = 20). The EA.hy926 endothelial cell line was used to assess the efffects of FF from patients at risk of OHSS with or without the addition of S1P. An animal model that develops OHSS in immature Sprague-Dawley rats were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Migration assays, confocal microscopy analysis of actin filaments, immunoblotting and quail chorioallantoic membrane (CAM) assays of in-vivo angiogenesis were performed and statistical comparisons between groups were made. MAIN RESULTS AND THE ROLE OF CHANCE: The S1P concentration was significantly lower in FF from patients at risk of OHSS (P = 0.03). The addition of S1P to this FF decreased cell migration (P < 0.05) and prevented VE-cadherin phosphorylation in endothelial cells (P < 0.05). S1P in the FF from patients at risk of OHSS increased the levels of VE-cadherin (P < 0.05), N-cadherin (P < 0.05) and ß-catenin (P < 0.05), and partially reversed actin redistribution in endothelial cells. The addition of S1P in FF from patients at risk of OHSS also decreased the levels of vascular endothelial growth factor (VEGF121; P < 0.01) and S1P lyase (SPL; P < 0.05) and increased the levels of S1PR1 (P < 0.05) in endothelial cells. In CAMs incubated with FF from patients at risk of OHSS with S1P, the number of vessel branch points decreased while the periendothelial cell coverage increased. Additionally, in a rat OHSS model, we demonstrated that vascular permeability and VEGF121 and its receptor KDR expression were increased in the OHSS group compared to the control group and that S1P administration decreased these parameters. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The results of this study were generated from an in-vitro system. This model reflects the microvasculature in vivo. Even though the ideal model would be the use of human endothelial cells from the ovary, it is obviously not possible to carry out this kind of approach in ovaries of patients from ART. More studies will be necessary to delineate the effects of S1P in the pathogenesis of OHSS. Hence, clinical studies are needed in order to choose the most appropriate method of prevention and management. WIDER IMPLICATIONS OF THE FINDINGS: The use of bioactive sphingolipid metabolites may contribute to finding better and safer therapeutic strategies for the treatment of OHSS and other human diseases that display aberrant vascular leakage. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants ANPCyT (PICT 2012-897), CONICET (PIP 5471), Roemmers and Baron Foundation, Argentina. The authors declare no conflict of interest.

A link between Notch and progesterone maintains the functionality of the rat corpus luteum.

In this study, we investigated the interaction between the Notch pathway and progesterone to maintain the functionality of the corpus luteum (CL). When Notch signaling is activated, the γ-secretase complex releases the active intracellular domains (NICD) of their receptors, which exert survival effects. We designed studies to analyze whether the in vitro inhibition of Notch affects progesterone production, steroidogenic regulators, apoptotic parameters, and signaling transduction pathways in the cultures of CL isolated from pregnant and superovulated rats. We detected a decrease in progesterone production when corpora lutea (CL) were incubated with N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor. This effect could be in part due to the decrease detected in the CL protein levels of P450scc because STAR and 3ß-hydroxysteroid dehydrogenase were not affected by Notch inhibition. Besides, the addition of aminoglutethimide to the CL culture medium decreased NICD of NOTCH1. We observed an increase in the expression of active CASPASE3 (CASP3) after inhibition by Notch, which was reversed by the presence of progesterone. The BAX:BCLXL ratio was increased in CL treated with DAPT and the presence of progesterone reversed this effect. In addition, phosphorylation of AKT was inhibited in CL treated with DAPT, but had no effect on ERK activation. To demonstrate that the action of DAPT is specifically related with the inhibition of Notch, CLs were incubated with DLL4 antibody and a decrease in progesterone production was detected. These results suggest the existence of a novel link between progesterone and the Notch signaling pathway to maintain the functionality of the CL.

Effects of a selective cyclooxygenase-2 inhibitor on endometrial epithelial cells from patients with endometriosis.

BACKGROUND: Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, also has anti-proliferative properties and pro-apoptotic effects on different in vivo and in vitro models, two actions that may be efficacious in therapy for endometriosis. We evaluated the effects of celecoxib on apoptosis and proliferation, and vascular endothelial growth factor (VEGF) production and COX-2 expression and activity in endometrial epithelial cells (EECs). METHODS AND RESULTS: Thirty-two endometriosis and 13 control women were included in the study. EECs from eutopic endometrium and control biopsies were cultured with different doses of celecoxib. Celecoxib at 50, 75 and 100 microM (versus vehicle control) inhibited EEC proliferation in cultures from controls (P < 0.05, P < 0.01 and P < 0.01, respectively) and patients with endometriosis (P < 0.05, P < 0.01 and P < 0.01), as assessed by (3)H-thymidine uptake. Celecoxib at 50, 75 and 100 microM induced apoptosis in EEC from controls (P < 0.05, P < 0.001 and P < 0.001) and patients with endometriosis (P < 0.001, P < 0.001 and P < 0.01), as revealed by the Acridine Orange-Ethidium Bromide technique. Western blot analysis showed that celecoxib was effective at increasing COX-2 protein at 100 microM in EEC from endometriosis patients (P < 0.05). In EEC from endometriosis patients, celecoxib at 25, 50 and 100 microM was also effective in reducing COX-2 activity, reflected in the reduction of prostaglandin E(2) (PGE(2)) synthesis (P < 0.001), and VEGF secretion (P < 0.001; P < 0.05 and P < 0.001), assessed by enzyme-linked immunosorbent assay. Exogenous PGE(2) did not reverse celecoxib-induced growth inhibition. CONCLUSIONS: This study suggests a direct effect of celecoxib on reduction of endometrial growth and supports further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.

Gamma secretase inhibitor impairs epithelial-to-mesenchymal transition induced by TGF-ß in ovarian tumor cell lines.

Ovarian cancer is characterized by being highly metastatic, a feature that represents the main cause of failure of the treatment. This study investigated the effects of γ-secretase inhibition on the TGF-ß-induced epithelial-mesenchymal transition (EMT) process in ovarian cancer cell lines. SKOV3 cells incubated in the presence of TGF-ß showed morphological and biochemical changes related to EMT, which were blocked by co-stimulation with TGF-ß and the γ-secretase inhibitor DAPT. In SKOV3 and IGROV1 cells, the co-stimulation blocked the cadherin switch and the increase in the transcription factors Snail, Slug, Twist and Zeb1 induced by TGF-ß. DAPT impaired the translocation of phospho-ß-catenin to the inner cell compartment observed in TGF-ß-treated cells, but was not able to block the induction at protein level induced by TGF-ß. Moreover, the inhibitor blocked the increased cell migration and invasiveness ability of both cell lines induced by TGF-ß. Notch target genes (Hes1 and Hey1) were induced by TGF-ß, decreased by DAPT treatment and remained low in the presence of both stimuli. However, DAPT alone caused no effects on most of the parameters analyzed. These results demonstrate that the γ-secretase inhibitor used in this study exerted a blockade on TGF-ß-induced EMT in ovarian cancer cells.

Peripheral and Central Mechanisms Involved in the Hormonal Control of Male and Female Reproduction.

Reproduction involves the integration of hormonal signals acting across multiple systems to generate a synchronised physiological output. A critical component of reproduction is the luteinising hormone (LH) surge, which is mediated by oestradiol (E2 ) and neuroprogesterone interacting to stimulate kisspeptin release in the rostral periventricular nucleus of the third ventricle in rats. Recent evidence indicates the involvement of both classical and membrane E2 and progesterone signalling in this pathway. A metabolite of gonadotrophin-releasing hormone (GnRH), GnRH-(1-5), has been shown to stimulate GnRH expression and secretion, and has a role in the regulation of lordosis. Additionally, gonadotrophin release-inhibitory hormone (GnIH) projects to and influences the activity of GnRH neurones in birds. Stress-induced changes in GnIH have been shown to alter breeding behaviour in birds, demonstrating another mechanism for the molecular control of reproduction. Peripherally, paracrine and autocrine actions within the gonad have been suggested as therapeutic targets for infertility in both males and females. Dysfunction of testicular prostaglandin synthesis is a possible cause of idiopathic male infertility. Indeed, local production of melatonin and corticotrophin-releasing hormone could influence spermatogenesis via immune pathways in the gonad. In females, vascular endothelial growth factor A has been implicated in an angiogenic process that mediates development of the corpus luteum and thus fertility via the Notch signalling pathway. Age-induced decreases in fertility involve ovarian kisspeptin and its regulation of ovarian sympathetic innervation. Finally, morphological changes in the arcuate nucleus of the hypothalamus influence female sexual receptivity in rats. The processes mediating these morphological changes have been shown to involve the rapid effects of E2 controlling synaptogenesis in this hypothalamic nucleus. In summary, this review highlights new research in these areas, focusing on recent findings concerning the molecular mechanisms involved in the central and peripheral hormonal control of reproduction.

Ovarian follicle-stimulating hormone binding changes associated with the reinstatement of ovulatory cycles after lactation interruption in the rat.

The aim of this work was to study the endocrine changes that occur during the reinstatement of the ovulatory cycles after lactational infertility in the rat. Hormonal patterns and specific binding of [125I]FSH to ovaries of lactating rats that kept their pups (LRP) or were separated from their pups on day 13 postpartum (LRX) were studied on days 13-16 postpartum. In LRP rats gonadotropin levels remained low and unvarying throughout the experiment; PRL levels were high in the morning, low at 1300 h, and then surged in the afternoon. Estradiol levels were very low in LRP rats in serum as well as in ovarian homogenates, and progesterone levels decreased gradually from days 13 to 16. No changes in either receptor number or dissociation constants (Kd) were observed in [125I]FSH binding to ovaries of LRP rats. In LRX rats, LH peaked on the afternoon of day 15 (P less than 0.05). FSH decreased from morning levels on day 13 to morning levels on day 15, and then peaked at 1600 h on day 15 (p less than 0.05). PRL decreased rapidly (day 13 1600 h levels significantly lower than day 13 1100 h levels), then remained low and peaked on the afternoon of day 15 (P less than 0.05). IN LRX rats progesterone levels decreased more markedly than in LRP rats and then surged in the afternoon of day 15. Serum estradiol levels rose significantly in the morning of day 15, while ovarian homogenate estradiol titers had already risen on the morning of day 14. Significant increases in number of [125I]FSH-binding sites and Kd values were observed in LRX rats on day 15 postpartum. These results clearly show that litter removal at midlactation (day 13) induces the reinstatement of hormonal cyclicity, and this is accompanied by changes in ovarian FSH receptors.

Insulin action and characterization of insulin receptors in rat luteal cells.

The effect of insulin (Ins) on luteal cell function was assayed and Ins binding was characterized in isolated collagenase-dispersed rat luteal cells. Ins stimulated progesterone production at concentrations over 10(-8) M, whereas human CG (hCG) produced maximal stimulation at 10(-11) M. Ins had an additive effect when it was added to the luteal cells in the presence of hCG in concentrations that produced submaximal stimulation. At 10(-9) M Ins the additive effect on hCG response became apparent (80% of maximal progesterone production). The maximal hCG response cannot be exceeded, even in the presence of high amounts of both hormones. hCG maximally stimulated cAMP production at 10(-10) M, whereas Ins neither stimulated cAMP production nor enhanced hCG response. Ins binding to luteal cells attained highest values after 30 min of incubation at 20 C. A curvilinear Scatchard plot was obtained and it was analyzed using a two-binding site model. The affinity constant values were 2.1 X 10(8) M-1 and 5.2 X 10(6) M-1 and the number of binding sites were 35,000 and 900,000 per cell, respectively. Bound Ins was not displaceable by hCG, human GH, human LH, epidermal growth factor, or ovine PRL. A protein moiety was essential for the receptor activity, since after trypsin digestion marked loss of binding was verified. Subcellular fractionation was performed and the highest binding was observed in the 105,000 X g pellet fraction. This study demonstrates that Ins binds specifically to rat luteal cells and stimulates steroidogenesis, adding support for the hypothesis that insulin acts directly upon the ovary.

Comparison between bioactive and immunoactive luteinizing hormone (LH) in ovariectomized streptozotocin-induced diabetic rats: response to LH-releasing hormone.

The effect of streptozotocin-induced diabetes on circulating levels of immunoactive LH (I-LH) and bioactive LH (B-LH) was investigated. LH was measured in adult ovariectomized (OVX) rats before and after acute LHRH administration, with or without estradiol benzoate (Eb) treatment (10 micrograms, 48 and 24 h before experiments). I-LH and B-LH were measured in the same samples by RIA and the rat interstitial cell testosterone assay, respectively. OVX diabetic animals showed a significant reduction in both I-LH (63%) and B-LH (73%). Treatment with Eb induced a decrease in basal I-LH and B-LH levels in all experimental groups (50%). These values were normalized after insulin therapy. No alterations in the pituitary responsiveness to LHRH were detected when I-LH levels were determined. However, B-LH levels assayed after LHRH stimulation were significantly decreased in diabetic animals. Insulin treatment was unable to restore this response. The effect of Eb treatment on these parameters was also tested. In these conditions LHRH injections induced similar increases in serum I-LH and B-LH in both diabetic and control rats. These results indicate that, in diabetic OVX rats, basal and LHRH-induced LH has a reduced bioactivity, but this reduction is reversed by Eb treatment. This might indicate that the major defect lies in the ovary rather than at the pituitary level, supporting the notion of an important role of the steroid milieu on the B-LH modulation.

Alterations in the prolactin secretion in streptozotocin-induced diabetic rats. Correlation with pituitary and hypothalamus estradiol receptors.

The present study examined the effects of streptozotocin-induced diabetes on prolactin (Prl) secretion and its correlation with estrogen receptor levels in the anterior pituitary and hypothalamus. Prl was measured in adult ovariectomized rats and after estradiol treatment (10 micrograms estradiol benzoate (Eb) 48, 24 and 1 h before experiments) or acute TRH administration (4 micrograms/kg body weight). Substantial decreases in estradiol- and TRH-induced Prl release were observed in diabetic rats. Insulin therapy was able to restore this response. Measurement of nuclear estradiol receptors by exchange assay in the pituitary of Eb-treated rats revealed a significant reduction in receptor levels in the diabetic group and a restoration to normal values in insulin-treated diabetic rats. Similar results were obtained by measuring total pituitary receptor content (cytosolic plus nuclear receptors). No significant changes were observed in nuclear hypothalamic estradiol receptors. However, the number of total hypothalamic estradiol receptors was diminished in diabetic rats although the translocation was proportionally greater in these animals. These results indicate that the disrupted reproductive functions described in streptozotocin diabetic rats may be due, at least in part, to deficiencies in Prl secretion and pituitary estradiol action.

Effect of prolactin on the steroidogenic response of rat luteal cells.

The role of prolactin on some ovarian functions was studied in collagenase-dispersed luteal cells obtained from PMSG/hCG-primed rats. The in vitro effect of ovine prolactin (oPrl) on luteal cell function was assayed. This hormone produced a dose-dependent increase of progesterone production and an additive effect on hCG stimulation. oPrl had no effect on cAMP production. Chronic effects of prolactin were studied in sulpiride (S), bromocriptine (Br) and oPrl-treated rats. Serum levels of prolactin were significantly higher in S-treated animals whereas Br administration rendered undetectable values. Serum progesterone was reduced in Br-treated animals and LH levels were similar in all groups studied. In vitro studies demonstrated a marked reduction of hCG stimulation of progesterone and cAMP production by luteal cells from hypoprolactinemic animals, while a significant increase was observed in hyperprolactinemic states. oPrl and S treatment significantly increased ovarian LH binding sites while a reduction was observed in Br-treated rats. These data suggest that luteal cell function is regulated by circulating levels of prolactin and that this hormone has some direct effect on the steroidogenic process.

Effect of a NADPH generating system on the steroidogenic response in rat luteal cells.

The effect of a NADPH generating system (NADPH-GS) on the function of rat luteal cells was studied. Cells were obtained from pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) primed immature rats and further incubated with a NADPH-GS. This system produced an increase in progesterone production and maximal stimulation was achieved at 1 mM NADP+ (10- to 15-fold). This effect was enhanced by addition of luteinizing hormone (LH 0.25 nM) to the incubation medium. On the contrary, insulin (2 nM) inhibited the effect observed with the NADPH-GS. The conversion of progesterone into 20 alpha-hydroxy-progesterone was not responsible for the changes observed. To analyze the site of NADPH action, pregnenolone and progesterone were measured using two inhibitors of steroid biosynthesis; aminoglutethimide and cyanoketone. The results confirm the specific site of action of NADPH at the mitochondrial conversion of cholesterol to pregnenolone. The effect of NADPH-GS was also observed in cultured purified luteal cells suggesting that the action of NADPH could be mediated by a free entry of the cofactor across the luteal cell plasma membrane. It can be concluded that the addition of NADPH improves the luteal cell incubation conditions and contributes to understanding the regulatory action of LH and insulin on the ovarian steroidogenic process.

In vivo regulation of the steroidogenic activity of rat luteal cells by insulin.

The aim of the present study was to determine the long-term effects of insulin treatment on luteal cell function. For this purpose, superovulated prepubertal rats were treated with insulin (group I) or vehicle (group C) for 9 days. Serum progesterone (P4) levels were increased in the insulin-treated group (55 +/- 10 vs 134 +/- 31 ng/ml, P < 0.05). Isolated luteal cells were incubated 3 h, and P4 and 20 alpha-hydroxy-progesterone (20 alpha-OH-P) were measured in the incubation media. A decrease in P4 levels and an increase in 20 alpha-OH-P values [P4 (ng/ml): C = 26.6 +/- 0.3; I = 20 +/- 2; 20 alpha-OH-P (ng/ml): C = 62 +/- 2; I: 120 +/- 7; P < 0.01] were observed in group I. In addition, progestagen (P4 + 20 alpha-OH-P) levels were higher in group I (C = 88 +/- 2; I = 140 +/- 9 ng/ml; P < 0.001). When cytochrome P450scc contents were measured by immunoblotting, a marked increase was observed in luteal cells obtained from group I. LH receptor numbers were decreased in luteal cells isolated from group I (C = 388,834 +/- 14,146; I = 303,057 +/- 13,392 sites/cell; P < 0.001) with a concomitantly diminished LH responsiveness. It is concluded that in vivo treatment of superovulated rats with insulin increases luteal progestagen production by increasing the content of cytochrome P450scc.

Regulation of the steroidogenic response of cultured human granulosa cells: effects of serum and 25-hydroxycholesterol.

OBJECTIVE: To examine the effect of serum and 25-hydroxycholesterol on steroidogenesis in cultured human granulosa cells from women undergoing assisted fertilization. DESIGN: Retrospective. SETTING: Private Fertility Clinic and National Research Institute. PATIENTS: Women undergoing IVF-ET or GIFT programs. RESULTS: In serum-free medium P production decreased significantly with culture time (2, 4, 6, and 8 days: 566 +/- 128, 161 +/- 50, 71 +/- 16, and 36 +/- 7 ng/mL P, respectively; conversion factor to SI unit, 3.180; mean +/- SEM). The addition of 25-hydroxycholesterol (10 micrograms/mL), a substrate for steroidogenesis, did not prevent the decrease in P levels. However, P production was greater in the presence of this substrate at all times. The presence of fetal bovine serum (10% FBS) in the cultures allowed the maintenance of 75% of P production with respect to the initial time considered (at which maximal P values are detected). Cultured granulosa cells treated with 10 ng/mL LH in the presence of FBS showed an increase in the percentage of stimulation with culture time (2, 4, and 7 days: 2.4%; 54.8%, and 55.1%, respectively). This effect was not observed when 25-hydroxycholesterol was added to the cultures. Similar results to that obtained by LH were attained when steroidogenesis was stimulated with 0.1 mM dibutyryl cyclic adenosine 3':5' monophosphate (cAMP). In addition, cAMP production in response to 100 ng/mL LH in the presence of 0.1 mM methyl-isobutyl-xanthine decreased with culture time, showing a time dependency similar to that observed for P. CONCLUSION: Our results demonstrate that the decrease in granulosa cell steroidogenic activity with culture time is inhibited by serum but not by 25-hydroxycholesterol, suggesting that other factors despite LH and cholesterol are necessary to support the luteal function.

Effect of a gonadotropin-releasing hormone agonist on luteinizing hormone receptors and steroidogenesis in ovarian cells.

OBJECTIVE: To examine the effect of a gonadotropin-releasing hormone agonist (GnRH-a), leuprolide acetate (LA), on human chorionic gonadotropin/luteinizing hormone (LH) receptors content and progesterone (P) and estradiol (E2) production in cultured granulosa or luteal cells. DESIGN: Prospective. SETTING: Private Fertility Clinic and National Research Institute. PATIENTS: Twenty patients undergoing in vitro fertilization or gamete intrafallopian transfer programs. RESULTS: Human chorionic gonadotropin/LH receptors in human granulosa cells increased after 48 hours of culture, and LA inhibited such effect. Leuprolide acetate, 1 ng/mL, in the cultures produced an increase in P production. On the contrary, LA inhibited E2 production. Additionally, the in vivo effect of LA (2 micrograms/rat per 7 days) was studied in corpus luteum of superovulated rats. Luteal cells from LA-treated rats in culture produced lower P than the controls but showed an increase in aromatase activity. Luteal LH receptors declined after 48 hours of culture with LA. CONCLUSION: The high doses of gonadotropin necessary to induce ovarian hyperstimulation when GnRH-a is administered could be related with an inhibitory effect of these agonists on LH receptors and aromatase activity.

Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis.

OBJECTIVE: To evaluate and compare spontaneous apoptosis and Bcl-2 and Bax expression in eutopic endometrium from women with and without endometriosis. DESIGN: Apoptosis and Bcl-2 and Bax expression were examined in eutopic endometrium from women with and without endometriosis. SETTING: Instituto de Biología y Medicina Experimental-CONICET, Department of Gynecology and Department of Gynecological Pathology, Clínicas University Hospital, Buenos Aires, Argentina. PATIENT(S): Women with untreated endometriosis (n = 14) and controls (n = 16). INTERVENTION(S): Collection of endometrial samples during diagnostic or therapeutic laparoscopy. MAIN OUTCOME MEASURE(S): Apoptotic cells were detected with use of the dUTP nick-end labeling (TUNEL) assay; Bcl-2 and Bax expressions were assessed with use of immunohistochemical techniques. RESULT(S): Spontaneous apoptosis was significantly lower in eutopic endometrium from patients with endometriosis, compared with healthy controls (2.26 +/- 0.53 and 9.37 +/- 1.69 apoptotic cells/field, respectively) and was independent of cycle phase. An increased expression of Bcl-2 protein was found in proliferative eutopic endometrium from patients with endometriosis. Bax expression was absent in proliferative endometrium, whereas there was an increase in its expression in secretory endometrium from both patients and controls. CONCLUSION(S): Women with endometriosis show decreased number of apoptotic cells in eutopic endometrium. The abnormal survival of endometrial cells may result in their continuing growth into ectopic locations.

Progesterone production in cultured human granulosa cells: correlation with follicular fluid hormone levels.

STUDY OBJECTIVE: To examine the progesterone (P) production by cultured granulosa cells and the hormonal content in the follicular fluid (FF) of ovarian-hyperstimulated women. DESIGN: Retrospective. SETTING: Private Fertility Clinic and National Research Institute. PATIENTS: Eighteen patients undergoing in vitro fertilization or gamete intrafallopian transfer programs. RESULTS: Progesterone levels Measured in the culture medium of granulosa cells decreased sixfold with culture time. Human luteinizing hormone (LH) increased P production only when basal P production was less than 1 microgram/mL. Granulosa cell P production in culture was negatively correlated with FF LH-human chorionic gonadotropin (hCG) levels. Follicular fluid follicle-stimulating hormone (FSH) levels were positively correlated with FF P and 17 beta-estradiol (E2) concentrations. Similar results were found between FF LH (hCG) and E2 levels, but there was no relationship between FF LH (hCG) and FF P values. CONCLUSION: The high dose of hCG administered during gonadotropin treatment could induce a decrease in the in vitro granulosa cell P production.

Cellular mechanisms of acute estrogen negative feedback on LH secretion: norepinephrine, dopamine and 5-hydroxytryptamine metabolism in discrete regions of the rat brain.

Rats on diestrous day 1 were ovariectomized (OVX) and killed 10 days later. LH was measured by RIA and the metabolism of NE, DA and 5-HT were assayed concurrently in the suprachiasmatic (SCN), medial preoptic (MPO), dorsomedial (DMN), rostral (ANr) and caudal (ANc) arcuate nuclei as well as the median eminence (ME) utilizing HPLC with electrochemical detection. Serum LH increased 10-12 fold 10 days following OVX compared to diestrous controls. The injection of estradiol benzoate (Eb, 20 micrograms in corn oil/rat, SC) did not affect LH concentrations at 30 minutes but decreased serum LH both 60 and 180 min following its administration. OVX caused an increased NE metabolism (estimated by the concentration of the NE metabolite, 3-methoxy-4-hydroxyphenylethylene glycol) in the SCN, MPO, ME, and DMN and a decreased NE metabolism in the ANc compared to diestrous control values. All of these changes were reversed or attenuated 180 minutes following Eb treatment. Observed changes in the DA and 5-HT neuronal systems were more restricted and less dramatic with the largest effects on DA metabolism occurring in the DMN and ME and the clearest changes in 5-HT metabolism occurring in the MPO, ANr, and ANc. The results demonstrate that the inhibition of LH secretion following the injection of Eb to OVX rats is accompanied by changes in metabolism in NE neurons in preoptic (SCN and MPO) and medial (ME, DMN, and ANc) hypothalamic areas, as well as in DA neurons in the DMN and ME, and in 5-HT neurons in the MPO, ANr, and ANc.

Steroid-monoamine feedback interactions in discrete brain regions using as a model the monosodium glutamate (MSG)-lesioned rat.

The present studies examine the effects of neonatal treatment with monosodium glutamate (MSG) on dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) metabolism in discrete brain regions and correlate them with steroid receptor kinetics in the anterior pituitary (PIT), preoptic hypothalamus (POA) and caudal hypothalamus (HYP), and with steroid negative and positive feedback effects on luteinizing hormone (LH) secretion. Substantial decreases in the neuronal activity of all three amines in the arcuate nucleus, decreased DA and 5-HT metabolism in the suprachiasmatic nucleus and, surprisingly, increased metabolism of 5-HT and NE in the median eminence was observed in adult ovariectomized (OVX), MSG-treated versus OVX, vehicle-treated litter mate controls. Measurement of estradiol receptors in the nuclear and cytosolic fractions of the POA, HYP and PIT from MSG- and vehicle-treated rats killed during diestrus or 2 weeks after OVX revealed no differences. Similarly, no differences in cytosolic progestin receptors between control and MSG unprimed or estradiol-primed, OVX rats or on progestin receptor translocation induced by progesterone in Eb-primed rats were observed. Negative and positive feedback effects of estradiol or the positive feedback of progesterone on LH secretion were not significantly impaired in MSG rats, and indeed, MSG animals actually were hyper-responsive to the administration of the steroids or of luteinizing hormone-releasing hormone. These results indicate that the MSG-induced damage to DA, 5-HT and NE elements observed within several preoptic and hypothalamic nuclei does not impair estrogen and progestin receptor kinetics, nor does it prevent adequate negative or positive steroid feedback responses, if appropriate steroid regimens are employed, and that the impaired gonadal function reported in these animals does not result primarily from inadequate steroid feedback mechanisms.

High correlation between prolactinemia, 125-I hLH binding and progesterone secretion by an experimental luteoma.

Autoimplantation of an ovary, containing fresh corpora lutea, into the spleen of an ovariectomized rat is followed by strong luteinization and size increase of the grafted gonad. Thus, large amounts of luteal tissue for biochemical studies, and their histological controls are available. Furthermore, progesterone secretion can be easily determined in samples collected from the portal vein. Since prolactin has been implicated in the control of luteal tissue, the role of this hormone on hLH binding and progesterone secretion was determined. Different levels of endogenous serum prolactin were achieved by pharmacological treatments with neurotropic agents. Scatchard plots of 125-I hLH binding data derived from luteoma particulate fractions revealed the presence of one type of binding site with high affinity. At the same time as binding increased, prolactinemia augmented, with a high correlation (R:0.99) between prolactinemia and LH binding. Moreover, progesterone secreted by the luteoma increased as LH binding sites augmented (R:0,97). It is concluded that a high correlation between prolactinemia and LH binding, as well as between this last parameter and progesterone output exists in the experimental luteoma.

Cellular mechanisms of acute estrogen negative feedback on LH secretion: pituitary responsiveness to LHRH and estradiol receptor kinetics in the pituitary, preoptic hypothalamic area, and the caudal hypothalamic area of the rat brain.

In order to examine the cellular mechanisms by which estradiol (E2) exerts its acute negative feedback upon luteinizing hormone (LH) secretion, a temporal correlation was made among serum LH concentrations, pituitary responsiveness to luteinizing hormone-releasing hormone (LHRH), and the translocation of E2 to nuclear (NER) receptors of the pituitary (PIT), preoptic hypothalamic area (POA), and the caudal hypothalamic area (HYP). Rats on diestrous day 1 were ovariectomized (OVX) and killed 10 days later. LH and LHRH were measured by RIA. NER was measured by an exchange assay. Serum LH increased 10-12-fold 10 days following OVX as compared to diestrous controls. The injection of estradiol benzoate (Eb, 20 microgram in corn oil/rat, sc) did not affect LH concentrations at 30 min but decreased serum LH at both 60 and 180 min following its administration. Pituitary responsiveness to LHRH (measured as the increase in LH 10 min after iv injections of 100 ng LHRH/rat) was not altered at 60 min but was significantly decreased 180 min following Eb injection. Thus, serum LH decreased prior to a detectable alteration in pituitary responsiveness to LHRH. Translocation of E2 receptors to NER of the HYP and PIT began 60 min following Eb injection and was maximal at 180 min. In contrast, translocation of E2 receptors in the POA was maximal at 60 min, and had recovered to control values 180 min following Eb administration.(ABSTRACT TRUNCATED AT 250 WORDS)