Effect of lithium chloride on the luteal steroidogenesis in gonadotropin-stimulated rat

Main function of corpus luteum is progesterone synthesis that is significantly accompanied with an increase in levels of mRNA encoding of steroidogenic enzymes known as luteal markers. This study was designed to evaluate effects of lithium chloride on the release of steroid hormones and steroidogenic enzymes in gonadotropin-stimulated rats. Immature 23 days old Wistar rats were divided into 10 groups; each group comprised of 8 rats, and induced with single injection of pregnant mare's serum gonadotrophin [PMSG] and followed by single injection of human chorionic gonadotropin [hCG]. Then, rats were given lithium chloride [LiCl] or saline at 12 hours post-hCG injection. Ovaries were collected in 4-hour interval from 8-24 hour post-hCG injection. Expression pattern of steroidogenic acute regulatory protein [StAR], side-chain cleavage cytochrome P450 [P450scc] and 3beta-hydroxysteroid dehydrogenase [3beta-HSD] genes were determined by semi-quantitative RT-PCR. In addition, serum levels of progesterone and 17beta-estradiol were measured by ELISA. Our results showed that hCG stimulation of progesterone was markedly diminished and transcript levels of key steroidogenic enzymes were altered in the hormone-stimulated rats following LiCl treatment. These results suggest that critical steps in the function of corpus luteum are disrupted by lithium. It is concluded that LiCl is an effective factor for suppressing of steroid genes expression

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 microm) were isolated by micro-dissection and cultured in 0 (control), 10(-3), 10(-5), 10(-7), and 10(-9) M SNP. To examine the reversible effect of SNP, PFs were cultured with 10(-5) M SNP + 1 mM Nomega-nitro-L-arginine methyl ester (L-NAME) or 1.0 microg hemoglobin (Hb). The results showed that greater concentrations of SNP (10(-3), 10(-5), 10(-7) M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10(-9) M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.

Expression of 3b-Hydroxysteroid dehydrogenase and P450 side chain cleavage enzyme in the human uterine endometrium

The enzyme complex 3b-hydroxysteroid dehydrogenase/delta(5)-delta(4)-isomerase (3beta-HSD) is involved in the biosynthesis of all classes of active steroids. The expression of 3beta-HSD in human uterine endometrium during the menstrual cycle and decidua was examined in an effort to understand its role during ova implantation. 3beta-HSD was weakly expressed in the glandular epithelium of the proliferative phase and moderately expressed in the glandular epithelium of secretory phase of the endometrium. In the decidua of the ectopic pregnancy, 3beta-HSD was strongly expressed. The human uterine endometrial 3beta-HSD was identified as being the same type as the placental 3beta-HSD by RT-PCR and sequence analysis. In addition to the expression of 3beta-HSD, P450scc was expressed in the decidua of the ectopic pregnancy. These results suggest that pregnenolone might be synthesized from cholesterol by P450scc de novo and then, it is converted to progesterone by 3beta-HSD in the uterine endometrium. The data implies that the endometrial 3beta-HSD can use not only the out-coming pregnenolone from the adrenal gland but also the self- made pregnenolone to produce progesterone. The de novo synthesis of progesterone in the endometrium might be a crucial factor for implantation and maintenance of pregnancy.

Effects of IL 6 on steroidogenesis of human granulosa cells in vitro

Recent studies suggest that in addition to gonadotropins, immunological factors, such as cytokines play an important role in production of steroid hormones. The purpose of this study was to examine effects of IL-6 on basal and FSH stimulated secretions of estradiol and progesterone in the presence of androstendione by human granulosa cells [GC] in vitro. Graunlosa cells were harvested at the time of follicular aspiration after ovarian hyperstimulation according to standard protocols with hMG from patients undergoing IVFET. The cells [2 X 10[4] viable cells per well] were cultured with HAM and # 101; s F-10 without any supplements [control] or increasing concentrations of recombinant human [rh] IL-6, [8,16,32,64,128 pg/ml] added in the absence or presence of FSH [96 IU/ml]. Media were collected after 24,48,72 and 96 hours at a 24h interval and estradiol and progesterone levels were measured by an enzyme immunoassay [EIA] with automated system. Results of this study showed that leuteinized GC in the absence of FSH and the presence of androgen was able to produce estradiol and progesterone in vitro. This production was significantly increased in the presence of FSH. Basal and FSH stimulated productions of estradiol were significantly [P < 0.05] inhibited by increasing amounts of IL-6. Although this inhibitory effect on basal production of progesterone was not significant. IL-6 in a dose-dependent manner significantly [P < 0.05] inhibited FSH stimulated production of progesterone by GC. These results suggest that IL-6 may play an important role in the production of estradiol and progesterone and any disorders in level of IL-6 may cause estradiol and progesterone release disturbances

Human granulosa cells in vitro: characteristics of growth, morphology and influence of some cytokines on steroidogenesis.

Granulosa cells (GCs) were characterised morphologically by light and electron microscopy. The steroidogenic capability of GCs in vitro was estimated by radioimmunoassay (RIA): oestradiol (E2), progesterone (P) and androstenedione (A) secreted into the culture medium were measured. The influence of several culture media and anchorage of the cell either to plastic vessels (monolayer) or to collagen fibrils (in gel) were studied. As the various culture media were assayed with regard to their suitability for IVF, it was found that Ham's F10 is quite satisfactory (in agreement with other observations on embryo cultures). A chemically defined medium BM 86 was found to be inadequate. In addition to the two cell types which are known, a third cell type which can perform efficient aromatisation (E2 production) in vitro is characterised here. The influence of cytokines/growth factors (GF) like insulin-like GF (IGF-1), epidermal GF (EGF), platelet-derived GF (PDGF) and fibroblast GF (FGF) on steroidogenesis was tested either alone or with human chorionic gonadotrophin (hCG). Except for oestradiol (E2) from early GCs, hCG generally stimulated progesterone (P) and E2 secretion. EGF by itself enhanced the secretion of P but not of E2. EGF did not affect hCG stimulation of P, but reduced that of E2. In contrast, in pre-ovulatory GCs IGF-I reduced the stimulatory effect of hCG on both E2 and P. In early GCs IGF-I potentiated hCG stimulation of P. In early GCs, neither hCG nor IGF-I nor a combination of IGF-I with hCG had any effect on E2 production.

Anti-steroidogenic activity of floral extract of Thespesia populnea Corr. in mouse ovary.

Anti-steroidogenic activity of various extracts of T. populnea was screened in female albino mice. The weight of the uterus and ovaries were reduced significantly and the cholesterol and ascorbic acid content in ovaries were significantly elevated due to the treatment with extract of T. populnea. The significant inhibition of delta 5, 3 beta hydroxy steroid dehydrogenase and glucose-6-phosphate dehydrogenase, the two key enzymes involved in ovarian steroidogenesis were also observed in mouse ovaries after 15 days of treatment.

Effect of insulin-like growth factor-1 on steroidogenesis in cultured carp ovarian follicles: interactions with estradiol.

The biological action of insulin like growth factor-1 (IGF-1) on follicular steroidogenesis during follicular development in common carp was examined. Studies were carried out by culturing small (1-2 mm diam.) and large (> 2 mm diam.) follicles. IGF-1 (0.3-100 ng/ml) had no effect on progesterone accumulation or aromatase activity during 48 hr culture of small follicles. Progesterone accumulation by large follicles was also unaffected by IGF-1 over the same period, although aromatase activity was stimulated in a dose dependent manner (8-fold increase over basal levels with a maximum stimulatory dose of 30 ng IGF-1/ml). In contrast, small and large follicles responded to IGF-1 in terms of both progesterone accumulation and aromatase activity after longer periods of culture (4 days for progesterone and 6 days for aromatase). Concurrent treatment of small follicles with estradiol (10(-7) M) enhanced the action of IGF-1 on both indices of steroidogenesis and advanced the time at which IGF-1 stimulated activity was first detectable. The effect of estradiol on follicular IGF-1 responsiveness were independent of cell number. In summary, these results demonstrate varied actions of IGF-1 carp ovarian follicular steroidogenesis in vitro. The results indicate that carp follicles acquire responsiveness to IGF-1 in terms of aromatase activity during follicular development in vivo and that estradiol can induce the response in vitro. The results also suggest that estrogen and progesterone biosynthesis by cultured carp ovarian follicles is differentially regulated by IGF-1. Together, these results provide new insights into the biological actions of IGF-1 in fish ovary.

Rol del oxido nítrico en la síntesis de prostaglandina F2alpha y progesterona durante la luteolisis en la rata / Role of nitric oxide in the synthesis of prostaglandin F2alpha and progesterone during luteolysis in the rat

En el cuerpo lúteo (CL), la prostaglandina F2alpha (PGF2alpha) es un agente luteolítico. El óxido (NO) es una molécula mensajera capaz de regular diversos procesos patofisiológicos, algunos de ellos relacionados com el tracto rerpoductivo femenino. El objetivo del presente estudio fue investigar el rol del NO ovárico en la producción de PGF2alpha y progesterona (Pg) durante la regresión del CL en la rata. Se utilizó para ello el modelo de la rata pseudopreñada, obteniéndose un cuerpo lúteo funcional por 9 + 1 días. Fueron inyectados en bursa ovárica dos inhibidores competitivos de la óxido nítrico sintasa (Nos), NG-monometil-L-arginina (L-NMMA), 1 mg/kg); NW-nitro-L-arginina metil éster (L-NAME, 3 mg/kg) así como también un generador de NO como el nitroprusiato sódico (SNP, 0.05 mg/kg). Los resultados obtenidos indican que el NO, producido en el ovario durante la fase final del desarrollo del CL (días 8 y 9), actuaría aimentando la producción de PGF2alpha ovárica y disimuyendo la progesterona sérica desencadenando la regresión luteal. Se há propuesto un mecanismo de feedback positivo entre la PGF2alpha y el NO hacia la fase final del desarrollo del Cl, para asegurar la luteólisis. Esto fue evaluado mediante la medición de la actividad de la Nos, luego de haber inyectado una dosis luteolítica de PGF2alpha (3mug/kg) a ratas en estadio medio (día 5) y tardío (día 9) del desarrollo luteal. Los resultados confirmaron nuestra hipótesis; no se observó un efecto en el estadio medio del desarrollo del Cl, pero en la fase final se encontró un aumento en la actividad de la enzima Nos en aquellos animales que habían recibido la dosis mencionada de PGF2alpha.

Stud male protection of implantation in food-deprived mice: evaluation of status of ovarian hormones

A falha de implantaçäo em camundongos recém-inseminados induzida por privaçäo alimentar de 48 horas, iniciada às 9 horas no quarto dia após o coito, é evitada pela exposiçäo aos machos. Na presente investigaçäo, estudou-se o efeito da privaçäo alimentar, bem como a influência da presença dos machos durante a privaçäo alimentar nos esteróides ovarianos. Estudos radioimunológicos revelaram que estradiol e testosterona näo foram alterados significativamente nas fêmeas privadas de alimento, independente da presença ou ausência do macho, o que foi comparável ao visto em fêmeas sem privaçäo alimentar. No entanto, o nível sérico de progesterona nas fêmeas privadas foi significativamente reduzido. Por outro lado, näo houve reduçäo no nível de progesterona nas fêmeas privadas de alimento quando em presença dos machos, como ocorreu nas fêmeas controles, i.e., sem jejum. Os resultados suportam a hipótese de que a liberaçäo de prolactina hipofisária, com conseqüente diminuiçäo no desenvolvimento de corpo lúteo funcional, seja o fator endócrino primário na falha de implantaçäo em camundongos com estresse nutricional. Além disso, estes resultados indicam que a presença dos machos contrapöe o fator acima nas fêmeas privadas de alimento.

Neuroendocrine mechanisms of lactational infertility in women

The current knowledge on the mechanisms of lactational infertility, discussed during a symposium of investigators in this subject, is reviewed. Three periods of lactation are examined: the first weeks postpartum, the period of extended lactational amenorrhea and the recovery of ovarian function. In the first postpartum weeks the inhibition of ovarian function is accounted by diminished pituitary response to GnRH, since exogenous GnRH fails to elicit a LH increase. Suckling can extend the period of ovarian inhibition for weeks, months or years, although it does not fully suppress pulsatile secretion of LH beyond the first weeks. Extended lactational amenorrhea is associated with low LH plasma levels, a great PRL increase in response to suckling, low basal E2 levels and a suppression of estrogen positive feedback. Decreased immunoreactive LH levels may result from partial suppression of the LH pulse generator and a smaller mass of GnRH released in each burst. The role of neurotransmitters, PRL and ovarian factors is discussed. After the recovery of ovulatory cycles suckling still has a residual infertility effect, associated to inadequate luteal function. The sources of variation among women and populations were recognized. Areas in which research is needed to improve the understanding of the mechanisms that sustain lactational amenorrhea are suggested

Las hormonas esteroides y la etiopatogénesis de padecimientos hepáticos de naturaleza fibrótica / Fibrotic liver disease and steroid hormones

Existen hipótesis en el sentido de que el estatus hormonal está involucrado en la distinta susceptibilidad entre el hombre y la mujer en el desarrollo de enfermedad hepatobiliar, ya que el hígado es órgano blanco para hormonas esteroides. En el desarrollo del fenómeno fibrogénico durante la cirrosis hepática de diversa etiología están involucradas las células de Ito, los miofibroblastos y diversas citocinas como factor transformador de crecimiento-ß, interleucina-6 y factor de necrosis tumoral-alfa. Se sabe que en el organismo existen asas de regulación mutua entre citocinas, glucocorticoides y esteroides sexuales; en el hígado, esta interacción pudiera afectar el proceso fibrogénico a través de diferenciación de células de Ito. El conocimiento del papel preciso de los glucocorticoides y las hormonas esteroides sexuales sobre los mecanismos fibrogénicos, permitiría sustentar racionalmente la viabilidad de la manipulación hormonal en el tratamiento de padecimiento hepático de natulareza fibrótica

Factor quimiotáctico para espermatozoides: una nueva función biológica de la progesterona / Chemiotactic factor for spermatozoa: a new biological function of progesterone

Nuestro grupo describió recientemente la existencia de un factor quimiotáctico para espermatozoides (FQE) conteniendo en el líquido de folículos maduros. Simultáneamente, fue posible desarrollar un nuevo método que permite valorar la capacidad quimiotáctica de los espermatozoides y que por su simplicidad hace posible el estudio sistemático de las características del FQE. En este estudio se abordó la caracterización molecular del FQE de líquido folicular (LF). Se estudiaron LF de mujeres dentro de un programa de fertilización asistida y que fueron calificados como maduros de acuerdo a diferentes criterios. Los LF fueron fraccionados con diferentes técnicas que permitieron separar una fracción activa con características fisicoquímicas de lípido. La cromatografía en capa fina reveló la presencia de diferentes esteroides, que fueron ensayados individualmente para actividad quimiotáctica. Solamente la progesterona mostró la actividad buscada y su efecto mostró una curva dosis-respuesta dentro de valores fisiológicos. Nuestro estudio permitió identificar a la progesterona como el FQE previamente descrito. Esta función del esteroide es completamente novedosa y su mecanismo de acción es objeto de estudio en nuestro laboratorio

Capacidad endócrina del blastocito de mamífero: II biosíntesis y metabolismo estrogénico / Endocrine capacity of blastocyst (mammal) II Biosynthesis and estrogenic, metabolism

De los estudios realizados en embriones preimplantados de mamíferos, llama la atención su capacidad endócrina para sintetizar hormonas esteroides: progesterona, progestinas y diversos estrógenos, los cuales efectuan localmente las propiedades del oviducto y del endometrio para crear un entorno apropiado para su nutrición, migración y desarrollo que permita su implantación en el útero materno. Entre los esteroides secretados por el embrión, los estrógenos son de un interés especial debido a su importancia potencial en los eventos bioquímicos asociados con el proceso de implantación. El propósito de este artículo es contribuir el conocimiento de la biosíntesis y metabolismo estrogénico por el embrión preimplantado.

Suppression of steroidogenesis in mice granulosa cells by a synthetic nonapeptide fragment of human seminal plasma inhibin.

A synthetic nonapeptide, which is C-terminal sequence of 94-amino acid of prostatic inhibin peptide was tested for progesterone and estrogen secretion by mouse granulosa cell cultures. Nonapeptide suppressed the progesterone and estrogen synthesis, the magnitude of suppression was highest at 5 ng dose level for progesterone and 50 ng dose level for estradiol. The study suggests that, nonapeptide exerts its effect by impairing the binding of FSH to granulosa cell receptors.

Efecto de diferentes fracciones del líquido folicular sobre la esteroidogenesis y proliferación de las células de granulosa in vitro / Effect of different fractions of follicular fluid on the steroidogenesis and proliferation of granulosa cells in vitro

Se determinó el de las fracciones obtenidas por cromatografía en cromatografía en SEPHACRYL S-300 del líquido de foliculo ováricos bovinos, en diferentes estadios de desarrollo, sobre la esteroidogénesis y proliferación de células de granulosa de la misma especie, pero en etapas muy tempranas de desarrollo independientemente del estadio de maduración, el líquido folicular está constituído por cuatro grupos de proteínas de diferentes pesos moleculares. En la fracción 1 se encontró un efecto inhibitorio significativo (p<0,05) sobre la síntesis de progesterona. El efecto de las diferentes fracciones sobre la esteroidogénesis varía tanto en relación con el estadio de maduración como con el tiempo de cultivo. No se encontró un efecto marcadamente significativo de las fracciones sobre la proliferación celular

In vitro effect of human placental lactogen on steroidogenesis in human placental tissue from early pregnancy.

Studies were carried out with human placental lactogen (hPL) to elucidate its role in regulation of steroidogenesis (progesterone and estrogens) during early pregnancy in humans. Our in vitro studies with early pregnancy placenta under different doses of hPL demonstrated that this hormone could stimulate the synthesis of progesterone as well as estrogens (estrone and estradiol) from their respective precursors.