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.

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.

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.

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.

Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls.

BACKGROUND: Our purpose was to evaluate the effect of the GnRH agonist (GnRHa), leuprolide acetate (LA), and the GnRH antagonist (GnRHant), Antide, on apoptosis and expression of apoptosis-related proteins in endometrial epithelial cell (EEC) cultures from patients with endometriosis and controls (infertile women without endometriosis). METHODS: Biopsy specimens of eutopic endometrium were obtained from 22 patients with endometriosis and from 14 women that served as controls. Apoptosis was examined in EEC after incubation with LA and Antide. Bax, Bcl-2, Fas and FasL expression was evaluated after exposure to LA, Antide or a combination of both. The percentage of apoptotic cells (%ApC) was assessed by the acridine orange-ethidium bromide technique, and protein expression was evaluated by western blot and immunocytochemistry. RESULTS: LA 100 and 1000 ng/ml increased the %ApC in EEC from patients with endometriosis (both P < 0.05) and controls (p < 0.05 and P < 0.01, respectively). Antide 10(-5) M increased the %ApC in EEC from patients with endometriosis and controls (P < 0.01). In EEC from women with endometriosis, Bax expression increased after treatment with LA, Antide and LA + Antide (P < 0.05, P < 0.001 and P < 0.001), whereas Bcl-2 expression decreased after exposure to LA and Antide (P < 0.001 and P < 0.01). FasL expression increased after LA, Antide and LA + Antide treatments (P < 0.01, P < 0.001 and P < 0.01). No significant changes were observed on Fas expression. CONCLUSIONS: GnRH analogues enhanced apoptosis in EEC, and this was accompanied by an increase in expression of the pro-apoptotic proteins Bax and FasL and a decrease in expression of the anti-apoptotic protein Bcl-2.

Gonadotropin-releasing hormone agonist affects rat ovarian follicle development by interfering with FSH and growth factors on the prevention of apoptosis.

Apoptosis is the biological process by which follicular cells are eliminated in atretic follicles. The aim of the present study was to examine the in vitro effect of a GnRH-a (leuprolide acetate, LA) and its interactions with FSH, dibutyryl cAMP, and growth factors (IGF-I, EGF, and FGF) on follicular apoptosis in early antral ovarian follicles obtained from prepubertal DES- treated rats. Follicles cultured 24 hr in the absence of hormones showed spontaneous onset of apoptotic DNA fragmentation. The presence of FSH suppressed the spontaneous onset of apoptotic DNA fragmentation (75-85%). Quantitative estimation of DNA cleavage from ovarian follicles revealed no significant changes in DNA fragmentation after in vitro LA treatment (1-100 ng/ml). However, coincubation with LA interfered partially with the effects of FSH on apoptosis suppression. This apoptosis suppression was also obtained by treatment with dibutyryl cAMP (80%), and was partially prevented by the presence of LA in the cultures. Follicles were cultured 24 hr with FGF, EGF, or IGF-I, and these factors suppressed DNA fragmentation (70, 60, and 70% respectively), while the presence of LA (100 ng/ml) in the culture medium prevented this effect. In conclusion, we show that the rescue from apoptotic DNA fragmentation produced in early antral follicles by FSH, cAMP, and growth factors, is prevented by coincubation with LA. This GnRH analog would thus interfere in the pathway of FSH, cAMP and/or growth factors by an as yet unknown mechanism.

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.

Apoptosis y expresión de Bcl-2 y Bax en el endometrio eutópico de pacientes con endometriosis / Apoptosis and Bcl-2 and Bax in normal endometrium of patients with endometriosis

El objetivo de este trabajo es investigar una posible predisposición de las células endometriales de pacientes con endometriosis (EDT), a ser resistentes a la muerte celular programada. Se evaluó apoptosis y expresión de las proteínas Bcl-2 y Bax en 30 cortes de tejido de endometrio eutópico, 14 de mujeres con EDT y 16 de controles (C). Para la determinación de apoptosis, se utilizó el kit Apoptag-Plus basado en la localización y tinción de los extremos 3-OH de los fragmentos de ADN. Los resultados se expresan como nº de células apoptóticas (CA)/campo a 630x de aumento. Se detectaron CA sólo en el epitelio glandular. Se observó menor cantidad de CA en tejido endometrial proveniente de pacientes con EDT: 2,26 ñ 0,53 vs 9,37 ñ 1,69 en los C (p<0,001). Esta disminución s

Apoptosis y expresión de Bcl-2 y Bax en el endometrio eutópico de pacientes con endometriosis / Apoptosis and Bcl-2 and Bax in normal endometrium of patients with endometriosis

El objetivo de este trabajo es investigar una posible predisposición de las células endometriales de pacientes con endometriosis (EDT), a ser resistentes a la muerte celular programada. Se evaluó apoptosis y expresión de las proteínas Bcl-2 y Bax en 30 cortes de tejido de endometrio eutópico, 14 de mujeres con EDT y 16 de controles (C). Para la determinación de apoptosis, se utilizó el kit Apoptag-Plus basado en la localización y tinción de los extremos 3'-OH de los fragmentos de ADN. Los resultados se expresan como nº de células apoptóticas (CA)/campo a 630x de aumento. Se detectaron CA sólo en el epitelio glandular. Se observó menor cantidad de CA en tejido endometrial proveniente de pacientes con EDT: 2,26 ñ 0,53 vs 9,37 ñ 1,69 en los C (p<0,001). Esta disminución se conservó a lo largo del ciclo menstrual. En la fase proliferativa tardía, los resultados fueron de: 7,08 ñ 0,92 vs 1,9 ñ 0,73 (p<0,05), para las muestras provenientes de mujeres C y pacientes con EDT respectivamente; mientras que en el endometrio secretorio los niveles de apoptosis detectados fueron de 11,6 ñ 1,74 en los C vs 2,7 ñ 0,82 en EDT (p<0,001). No se observaron diferencias significativas de apoptosis en endometrio de acuerdo al grado de la enfermedad. La evolución de los productos de protooncógenes, bcl-2 y bax se realizó utilizando metodologías de inmunohistoquímica. Se halló incrementada la expresión de la proteína antiapoptótica Bcl-2 en el endometrio proliferativo proveniente de pacientes con EDT comparado con el C. La expresión del antagonista de Bcl-2, Bax, aumentó durante la fase secretoria tanto en muestras provenientes de pacientes como de C, y se halló ausente durante la fase proliferativa. De los resultados se desprende que las células endometriales de pacientes con EDT poseen características apoptóticas alteradas que las harían más susceptibles a crecer en un sitio ectópico. Estas no se ven modificadas a lo largo del ciclo menstrual. La proteína Bcl-2 estaría implicada en la protección de la apoptosis de las células endometriales eutópicas de pacientes con EDT durante la fase proliferativa del ciclo menstrual. La proteína Bax estaría involucrada en la regulación de la muerte celular programada que se produce en el endometrio eutópico previo a la menstruación. La resistencia a la muerte celular programada que posee el endometrio eutópico de pacientes con EDT, estaría relacionada con la etiología y/o fisiopatología de la enfermedad

Similar embryotoxic effects of sera from infertile patients and exogenous interferon-gamma on long-term in-vitro development of mouse embryos.

Circulating embryotoxic factors could be responsible for reproductive failures observed in patients suffering from recurrent spontaneous abortions (RSA) and endometriosis. The mouse bioassay has been widely used to detect such factors, since sera from these patients inhibit early embryonic development. This bioassay consists in the in-vitro culture of two-cell mouse embryos in the presence of different sera up to the blastocyst stage (72 h of culture). In the present study experiments were performed over long culture times (3-7 days), from two-cell to spreading stages, to determine the in-vitro effect of sera obtained from RSA or endometriosis patients, as well as the effect of interferon (INF)-gamma on embryo development. An embryotoxicity cut-off value of 45% blastocyst formation was established using control sera. When development to the blastocyst stage was considered only 25% of RSA and 20% of endometriosis sera were embryotoxic. However, all RSA sera significantly inhibited hatching (P < 0.05) and spreading stages (P < 0.01). IFN-gamma (10 micrograms/ml) (P < 0.001) did not impair early embryo development, but significantly inhibited blastocyst spreading. These observations suggest that culture to advanced embryonic stages increases the sensitivity of the bioassay and that IFN-gamma alters in-vitro peri-implantation mouse embryo development.

Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: relationship between steroidogenesis and apoptosis.

The purpose of this study was to evaluate the effects of GnRH-analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)-superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase-dispersed ovarian cell cultures, though estradiol (E2) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH-stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side-chain cleavage (P450SCC) levels in corpora lutea from LA-treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end-labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins.

Regulation of ovarian follicle differentiation in gonadotrophin-stimulated rats.

The aim of the present study was to investigate the regulation of the in vitro DNA synthesis of ovarian cells recovered from prepubertal rats 48 h after administration of pregnant mare's serum gonadotrophin alone (granulosa cells) or followed by human chorionic gonadotrophin (luteal cells). Isolated granulosa cells were cultured in serum-free medium, different stimuli added for periods of 48 h, and 3H-thymidine incorporation was measured. Both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) inhibited 3H-thymidine incorporation by cultured granulosa cells in a dose-dependent manner (FSH: 10, 100, 200 ng/mL = 26, 41, 49% inhibition, respectively; LH: 0.1, 1, 10 ng/mL = 11, 37, 75% inhibition, respectively). On the other hand, estradiol was found to stimulate 3H-thymidine incorporation in granulosa cells (Estradiol: 5, 50, 500 ng/mL = 17, 37, 76% stimulation, respectively). In luteal cells, the rate of basal 3H-thymidine incorporation was very low (granulosa cells: 2560 +/- 310; luteal cells: 661 +/- 92 cpm/100,000 cells) and not modified by any stimulus. To determine the possible production of an inhibitory growth factor by the early corpus luteum, 3H-thymidine incorporation by granulosa cells was assessed in the presence of 10% conditioned media (CM) recovered from luteal cell cultures. A marked inhibition both in basal and estradiol-stimulated 3H-thymidine incorporation was observed (74 and 76% of inhibition, respectively). Results suggest that an inhibitory growth factor produced by luteal cells after luteinizing gonadotrophin stimulus could be involved in the differentiation of growing follicles to corpus luteum.

GnRH receptors and GnRH endocrine effects on luteoma cells.

An ovary implanted into the spleen of an ovariectomized rat develops into a luteinized tumor, growing in response to gonadotrophins. Previously, it was shown that in vivo Buserelin, a gonadotrophin-releasing hormone (GnRH) analog, inhibited tumor growth. To determine if GnRH had a direct effect on tumor cells, the presence of GnRH receptors as well as the endocrine effects of buserelin were studied on tumoral tissue. GnRH receptors were present in luteoma in similar concentrations and dissociation constant (Kd) to control estrous ovaries. In vivo treatment with buserelin did not modify luteoma GnRH receptors. In organ incubations, luteoma secreted significantly higher estradiol and lower progesterone than estrous ovaries; addition of buserelin did not modify steroid secretion. The same difference in basal steroid secretion between luteoma cells and luteal cells superovulated prepubertal ovaries was observed in cell cultures. Although luteinizing-hormone (LH)-stimulated progesterone in both kinds of cells, buserelin significantly inhibited LH-stimulated progesterone only in luteoma cells. These results describe clear differences in basal steroid secretion between tumoral and normal tissue. Furthermore, they show that luteoma possess GnRH receptors similar to those in normal ovarian tissue, and that GnRH analogs have endocrine effects on these cells. Therefore, a direct effect of buserelin on luteoma cells can be postulated.

[Regulation of follicular luteinization by an agonist of gonadotropin-releasing hormone (GnRH)]. / Regulación de la luteinización folicular por un análogo de la hormona liberadora de gonadotrofinas (GnRH).

Long treatments with GnRH agonist are used in patients to suppress the endogenous secretion of gonadotropins, however; these analogs have a direct effect on the ovary. The aim of this work was to study the in vivo effect of the GnRH analogue, leuprolide acetate (LA) on ovarian steroidogenesis and apoptosis mechanisms. LA (1 microgram/rat/day) was injected to PMSG/hCG superovulated rats. Corpora lutea were isolated by microdissection and incubated (4/0.5 ml) during 3 h with LH (10 ng/ml) or dibutyryl cAMP (dcAMP 1 mM). Progesterone production was measured observing in LA treated rats a decrease in basal and LH stimulated values (Basal = Control C: 96.6 +/- 9.6; LA: 22.9 +/- 2.8; LH = C: 145.7 +/- 4.9; LA: 23.6 +/- 2.0 ng/ml, p < 0.001). In contrast, dcAMP stimulated significantly both groups (C: 153.9 +/- 11.8; LA: 83.15 +/- 8.2). cAMP production was lower in LA corpora lutea and LH was not able to stimulate them (Basal = C: 7.29 +/- 1.6; LA: 1.17 +/- 0.6; LH = C: 13.2 +/- 0.4; LA: 2.5 +/- 0.4 ng/ml, p < 0.01). Corpus luteum of both groups showed similar protein content. On the other hand, taking into account that in ovarian histological slides of LA treated rats we observed more atresic follicles and less corpora lutea, we determined the amount of apoptotic cells. The criterion used was the presence of apoptotic bodies and nuclear chromatin aggregated in dense masses beneath the nuclear envelope. An increase of apoptotic cells in the LA group ovaries was detected. This result was confirmed by immunohistochemical technics (TUNEL). It was concluded that LA treatment produces in ovarian cells a failure in the LH receptor-adenilate cyclase system and an increase in cellular apoptosis.

Effect of streptozotocin-induced diabetes on phosphodiesterase activity in rat luteal cells.

The present studies were carried out to characterize the cAMP-phosphodiesterase enzyme (PDE) in luteal cells recovered from pseudopregnant rats with streptozotocin-induced diabetes. A significant increase in the specific activity of the enzyme was detected in luteal cells from diabetic rats (Group D) with respect to control rats (Group C). This increase could not be prevented by insulin therapy (Group I). Luteal cells from Groups C and D rats responded in vitro to insulin by increasing their PDE activity (% of stimulus of specific activity: C = 75%, D = 110%). However, in cells isolated from Group I, the hormone caused an inhibition of PDE activity (% of inhibition of specific activity: 48%). When cytosolic fractions from Groups C, D and I were submitted to ion exchange chromatography, two PDE activity peaks could be observed and the activity of the different fractions was increased in the presence of Ca2+ and calmodulin. Nevertheless, the Ca(2+)-calmodulin effect was much lower in the extracts from Groups D and I than for controls. Kinetic studies of luteal PDE showed nonlinear Lineweaver-Burk graphs with two apparent ATP hydrolysis sites. Similar K(m) values were found for PDE from groups C, D, and I, whereas the Vmax2 for the enzyme was higher in Groups D and I. The endogenous concentration of cAMP, measured by RIA, showed no significant differences among Groups C, D, and I. On the basis of these results, we conclude that the specific activity of PDE is significantly increased in luteal cells from streptozotocin-induced diabetic animals, which could explain the previously described reduction in LH-stimulated progesterone production by luteal cells in diabetic rats.

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.

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.

Anterior pituitary estradiol receptors associated with the reinstatement of ovulatory cycles after lactation interruption in the rat.

The participation of adenohypophyseal estradiol receptors in the reinstatement of ovulatory cycles after lactation interruption was investigated. In rats whose pups were removed on day 13 postpartum (LRX), prolactin levels fell as from 1600 h on the same day, estradiol peaked on the morning of day 15 and gonadotropins and prolactin (PRL) surged on the afternoon of day 15. No significant changes in gonadotropins or estradiol levels were observed in rats which remained with their litters (LRP); in these rats daily afternoon surges of PRL were detected. No significant variations in anterior pituitary nuclear or cytosolic estradiol receptors were determined in LRP rats. In the nuclear fraction of LRX rats, an important increase (430.8 +/- 124.9%) in receptor titers was observed on day 15. In these animals a significant increase (34.8 +/- 1.3%) in cytosolic estradiol receptors was observed on day 14, followed by a fall on day 15 (-31.6 +/- 6.6%) in comparison to day 13 levels. The receptor variations observed on day 15 closely resemble estrous cyclic changes determined in adult females. However, an observation which does not resemble those cycle variations is the increase in cytosolic receptors observed on day 14 in LRX rats. This increase may be the consequence of a decrease in dopamine levels induced by pup removal. To our knowledge this is the first time that the involvement of pituitary estradiol receptors in the reinstatement of ovulatory cycles after lactation interruption has been described.