RESUMEN
The aim of the present study was to evaluate the signaling pathways involved in the follicle-stimulating hormone (FSH) regulated mitogenic activity. For this purpose, 18-d-old chick embryo testis cells were dissociated and cultured for 60 h on polycarbonate membranes. The culture medium was Dulbecco modified Eagle's medium with or without high pure human FSH (hFSH), human recombinant FSH, or different regulators of tyrosine kinase activity as herbimycin A and genistein, or serine/threonine kinases [cyclic adenosine monophosphate (cAMP)-dependent protein serine kinase and protein kinase C] as cAMP, phorbol myristate, and forskolin. In some experiments the regulators were added simultaneously with hFSH. The [3H]-thymidine incorporation was used as an indicator of DNA synthesis. In addition, fragments of chick embryo testis were cultured in the presence or absence of FSH or herbimycin A, and 5-bromo-2'-deoxy-uridine was added to identify the proliferating cell subpopulations. The effect of hFSH on [3H]-thymidine incorporation began at 24 h, and the increment was significant at 36 and 60 h of culture. The hFSH as well as human recombinant FSH significantly stimulated [3H]-thymidine incorporation to testicular cells. The 5-bromo-2'-deoxy-uridine technique showed a high signal in pericordonal and interstitial cells of the hFSH-treated groups, confirming the results obtained using [3H]-thymidine uptake. The treatment with the tyrosine kinase inhibitor herbimycin A increased the [3H]-thymidine uptake, but genistein did not. Regulators of PKA such as cAMP and forskolin, as well as PKC regulators and the phorbol ester phorbol myristate, did not influence cell proliferation. In summary, an inhibitor of tyrosine kinase, herbimycin A, induced per se an increment in chick embryo testis cell proliferation, a fact that strongly suggests that tyrosine kinase signaling pathway functions by inhibiting the proliferation of these cells. On the other hand, the cAMP-PKA pathway had no significant role during the embryonic stage of chick embryo testis. Our results also showed that the effect of FSH on chick embryo cell proliferation occurs mainly in pericordonal and interstitial testis cells.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Hormonas/farmacología , Testículo/citología , Testículo/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Pollos , MasculinoRESUMEN
The development of the chick embryo gonads is influenced by gonadotropins [follicle-stimulating hormone (FSH), luteinizing hormone, human chorionic gonadotropin (hCG)]. We have previously shown that insulin enhanced the production of androgens in the testis of the newly hatched chicken and increased the proliferation of chick embryo testis cells. In the present paper, we have studied the effect of insulin on embryonic chick embryo ovarian cells and compared them with those of human FSH and hCG. The ovaries of 18-d-old chick embryos were dissociated and cultured for different periods in Dulbecco's modified Eagle's medium in the presence and absence of insulin, human FSH, hCG, and combinations of them. 3H-thymidine incorporation was used as an indicator of cell proliferation; steroids were measured by radioimmunoassay. Results showed that insulin enhanced the proliferation of ovarian cells in a dose- and time-dependent manner. Gonadotropins did not affect significantly the ovarian cell proliferation. Insulin did not change 17beta-estradiol production. The combination of insulin and FSH or insulin and hCG decreased the stimulation of estrogen secretion caused by the addition of the gonadotropins. In some experiments, ovarian cells were cultured with or without insulin, and subpopulations were identified. The results showed that insulin but not human FSH or hCG increased the proliferation of germinal cells after 60 h in culture. Insulin and human FSH did stimulate the other 2 subpopulations. In summary, present results suggest that insulin is an important hormone in the development of the chick embryo ovary.
Asunto(s)
Estradiol/biosíntesis , Insulina/farmacología , Ovario/citología , Ovario/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Femenino , Ovario/metabolismoRESUMEN
The present study evaluated the follicle-stimulating hormone (FSH) effect on cell proliferation and steroid production by chick embryo testis. Dissociated cells from 18-d-old embryos were cultured on polycarbonate membranes in defined media. In some experiments, cells were further separated by a metrizamide gradient, and 5 cellular subpopulations were recovered and cultured. [3H]thymidine was added to the culture media. When necessary, 17beta-estradiol, human FSH (hFSH), recombinant human FSH (rhFSH), or human chorionic gonadotropin (hCG) was added to the medium at the beginning of the culture. The total number of cells and the incorporation of [3H]thymidine increased when hFSH or rhFSH was added. No changes were produced by the addition of hCG or 17beta-estradiol. The dose-response curve to hFSH resulted in an ED50 of 0.25 IU/mL. The stimulatory effect of hFSH on total number of cells and on [3H]thymidine incorporation was significant at 36 h of culture and was maintained up to 60 h. Testosterone production increased with the addition of FSH or rhFSH, meanwhile estradiol production was below the limit of detection of RIA. The hFSH proliferative effect measured as [3H]thymidine incorporation was observed only in the F3, F4, and F5 fractions of the density gradient. Present results show that hFSH and rhFSH, but not hCG or estradiol, stimulate testis cell proliferation in a time- and dose-dependent manner. The combination of [3H]thymidine incorporation and testosterone production in fractions obtained from the metrizamide density gradients suggests that the cell fractions of the chick embryo testis show a differential response to FSH.
Asunto(s)
División Celular/efectos de los fármacos , Embrión de Pollo , Hormona Folículo Estimulante/farmacología , Esteroides/biosíntesis , Testículo/citología , Testículo/embriología , Animales , Células Cultivadas , Pollos , Gonadotropina Coriónica/farmacología , Relación Dosis-Respuesta a Droga , Estradiol/biosíntesis , Estradiol/farmacología , Humanos , Masculino , Microscopía Electrónica , Proteínas Recombinantes , Testosterona/biosíntesis , Timidina/metabolismo , TritioRESUMEN
This investigation addresses the potential regulation of enzymes involved in the biosynthesis of steroid hormones during early stages of gonadal development by follicle-stimulating hormone (FSH). Gonadal cells of 10-day-old chick embryo and cells of the left ovary of 18-day-old chick embryo were cultured for 60 h in a defined medium with or without the addition of FSH (2.0 IU/ml). At the end of the culture, cells were recovered and evaluated by biotransformation of tritiated steroid precursors and mRNA levels were evaluated by RT-PCR. The production of estrone from androstenedione was increased in the FSH-treated cells, both human FSH (hFSH) and recombinant human FSH (rhFSH), indicating a stimulatory effect on aromatase (P450arom). Similarly, the intensity of the band corresponding to P450arom mRNA was higher in hFSH and rhFSH than in control and chorionic gonadotropin (hCG) groups. The P450arom stimulation was observed in the ovary of 10- and 18-day-old chick embryo. The transformation of dehydroepiandrosterone to androstenedione was taken as evidence of 3beta-hydroxysteroid dehydrogenase function. This enzyme was stimulated in the cultured ovarian cells of 18-day-old chick embryos treated with hFSH and rhFSH compared with controls. The production of pregnenolone in the mitocondrial fraction of 18-day-old chick embryo ovary was increased when cultured with hFSH and rhFSH. This observation together with the increase in the band intensity corresponding to mRNA of P450 cholesterol side-chain cleavage indicates stimulation by FSH treatment; hCG produced a similar effect. Somatic cells of the medullary cords are proposed to be FSH target cells in the ovary of the chick embryo.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Ovario/embriología , Ovario/enzimología , Esteroides/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Androstenodiona/biosíntesis , Androstenodiona/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Células Cultivadas , Embrión de Pollo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Gonadotropina Coriónica/farmacología , Deshidroepiandrosterona/metabolismo , Estrona/biosíntesis , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Ovario/efectos de los fármacos , Pregnenolona/biosíntesis , Progesterona/metabolismo , ARN Mensajero/análisis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testosterona/metabolismo , Factores de Tiempo , TritioRESUMEN
The aim of this study was to evaluate the in vitro effect of human follicle-stimulating hormone (hFSH) on cellular proliferation and steroid hormone secretion in the left ovary, the right ovary, and the testis of the chick embryo. Gonads from 8- to 18-day-old chick embryo were cultured in a defined medium during 60 h under basal and hFSH-stimulated conditions (0.5 IU/ml of culture medium). At the end of the culture, the incorporation of ¿(3)Hthymidine and the total number of cells were measured to estimate gonadal cell proliferation. The secretion of 17beta-estradiol and testosterone in the culture medium was radioimmunologically assayed in order to evaluate the steroidogenic function of the cultured gonadal cells. The response to hFSH stimulation was observed in the left ovary, the right ovary, and the testis from the 8-day-old chick embryo. In the left ovary, cellular proliferation was not augmented by hFSH in the 8-, 10-, and 13-day-old chick embryo; meanwhile, the proliferative stimulus of hFSH was observed in the 15- and 18-day-old embryos. In the same ovary, 17beta-estradiol and testosterone secretion were stimulated after hFSH treatment at all evaluated stages (8-18 days of chick embryo development). In the right ovary, an increment in proliferation and steroidogenesis was induced by hFSH in the 8-, 10-, and 13-day-old chick embryo. Afterward, the right gonad did not respond to hFSH. Testis cells displayed hFSH response as an increment in cell proliferation at all embryonic ages (8-18 days of chick embryo development). There was a transient lack of response to hFSH in testosterone secretion at 10 and 13 days of development. The in vitro effect of hFSH on cell proliferation and steroid hormone secretion changed in the ovary and the testis according to the age of the embryo. These changes could be attributed to the growth of the left ovary and the testis and the regression of the right ovary. Probably, paracrine factors modulated the gonadotropin effect on the target cells during embryonic development of chick embryo gonads.
Asunto(s)
División Celular/efectos de los fármacos , Hormona Folículo Estimulante/farmacología , Ovario/embriología , Esteroides/biosíntesis , Testículo/embriología , Animales , Recuento de Células , Embrión de Pollo , Medios de Cultivo Condicionados , Técnicas de Cultivo , ADN/biosíntesis , Estradiol/biosíntesis , Estradiol/metabolismo , Femenino , Humanos , Masculino , Ovario/citología , Ovario/metabolismo , Testículo/citología , Testículo/metabolismo , Testosterona/biosíntesis , Testosterona/metabolismo , Factores de TiempoRESUMEN
The capability of granulosa and theca interna cells, from preovulatory follicles of the domestic hen, to metabolize steroid precursors was evaluated. Granulosa and theca interna cells were isolated from ovarian preovulatory follicles at three different developmental stages: F1, F3 and F5. Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4) and testosterone (T) were employed as precursors and their metabolic products were evaluated. The major metabolite of P5 by granulosa cells was P4, but we also observed low amounts of 5beta-pregnandione. DHEA metabolism by granulosa cells yielded mainly A4, and minute quantities of 5beta-androstan-3,17-dione (5beta-dione) were detected. The only significant metabolite obtained in granulosa cells from A4 was 5beta-dione, whereas T was only transformed into A4. On the other hand, P5 metabolism by theca interna cells yielded A4 as the main product, also P4, 17alpha-OHP4, 17alpha-OHP5, 5beta-pregnandione, and DHEA, were found. When DHEA was the precursor A4 was produced in higher amounts than 5beta-dione. A4 was mainly transformed into 5beta-dione. In similar conditions, T was transformed into A4. These results show that granulosa cells have enzymatic activities of 3beta-hydroxysteroid dehydrogenase/5-4 isomerase (3beta-HSD from P5 and DHEA), 17beta-hydroxysteroid dehydrogenase (17beta-HSD from T) and 5beta-reductase (from P5, DHEA and A4). Whereas theca interna cells have enzymatic activities of cytochrome P450c17 (from P5 and P4), 3beta-HSD (from P5 and DHEA), 17beta-HSD (from T) and 5beta-reductase (from P4, DHEA and A4). These data support the concept that theca interna cells have the ability to synthesize androgens from progestins produced in granulosa cells. In addition, since theca interna cells did not show the capacity to aromatize androgens suggests that interaction between theca interna and theca externa cells occurs in vivo, thus confirming the three cell model for estrogen production. Furthermore, the fact that other metabolites were produced both in granulosa and theca interna cells, but in a different extent, suggests that complex mechanisms are participating in the regulation of steroid synthesis in avian ovary follicles.
Asunto(s)
Pollos/metabolismo , Folículo Ovárico/metabolismo , Esteroides/metabolismo , Androstenodiona/biosíntesis , Androstenodiona/metabolismo , Animales , Cromatografía en Capa Delgada/veterinaria , Deshidroepiandrosterona/metabolismo , Femenino , Células de la Granulosa/metabolismo , Pregnenolona/metabolismo , Progesterona/biosíntesis , Progesterona/metabolismo , Cintigrafía/veterinaria , Esteroides/biosíntesis , Testosterona/metabolismo , Células Tecales/metabolismoRESUMEN
The purpose of the present study was to evaluate the effect of human follicle-stimulating hormone (hFSH) on cellular proliferation in the chick embryo ovary. Left ovaries from 18-day-old chick embryos (Babcock B300) were dissociated by trypsin (0.25%) treatment. In some experiments, dissociated ovarian cells were further separated by a continuous metrizamide gradient (0-20%). Four cellular subpopulations were recovered from the density gradient: (a) typical steroidogenic cells (F1, density 1.026 g/ml), (b) primary oocytes (F2, 1.048 g/ml), (c) pregranulosa cells, and (d) poorly differentiated epithelial cells (both cellular types were found in F3, 1.059 g/ml, and F4, 1.071 g/ml). Samples (5 x 10(5) cells) of dissociated cells of the whole ovary and of the four cellular subpopulations obtained from the density gradient were cultured on polycarbonate membranes in a defined medium with 0.1 microCi of [3H]thymidine for 60 hr. When necessary, 17beta-estradiol, hFSH, recombinant human FSH, and hCG were added to the medium at the begining of the culture. The total number of cells and the incorporation of [3H]thymidine to the ovarian cell aggregate at the end of the culture increased when hFSH and recombinant hFSH (0.5 IU/ml) were added. No changes were produced with hCG (2.0 IU/ml) and 17beta-estradiol (200 ng/ml) treatments. The secretion of 17beta-estradiol by cultured ovarian cells was stimulated in the hFSH-, recombinant hFSH-, and hCG-treated groups. The dose-response curve to hFSH resulted in an ED50 of 0.03 IU/ml. In the temporal curve, the stimulatory effect of hFSH on the total number of cells and the [3H]thymidine incorporation were observed at 36 hr of culture and maintained up to 60 hr. The proliferative effect of hFSH measured as [3H]thymidine incorporation was observed only in the F4 fraction of the density gradient, which includes poorly differentiated epithelial cells and pregranulosa cells from the ovarian medulla and cortex, respectively. Present results demonstrate a dose-dependent stimulation of DNA synthesis by hFSH in the inmature ovary of the 18-day-old chick embryo. Either pregranulosa cells or poorly differentiated epithelial cells or both subpopulations would be the target cells for the proliferative effect of the gonadotropin.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Ovario/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Embrión de Pollo , Gonadotropina Coriónica/farmacología , Relación Dosis-Respuesta a Droga , Estradiol/análisis , Estradiol/farmacología , Femenino , Humanos , Microscopía Electrónica , Ovario/citología , Ovario/metabolismo , Ovario/ultraestructura , Proteínas Recombinantes/farmacología , Timidina/metabolismo , Factores de Tiempo , TritioRESUMEN
The purpose of this study was to evaluate the ability of theca externa cells from preovulatory follicles of the domestic hen to metabolize tritiated steroid precursors. Theca externa cells were isolated from ovarian preovulatory follicles at three different developmental stages; F1 (35 mm), F3 (26 mm), and F5 (13 mm). Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T) were employed as precursors and their metabolic products were evaluated after separation by thin-layer chromatography. The major metabolite of P5 by theca externa cells was 5-pregnen-3 beta, 20 beta-diol (F5, 47%; F3, 39%; and F1, 24%), but minimal quantities of P4 were detected. Progesterone metabolism yielded mainly 4-pregnen-20 beta-ol-3-one (F5, 52%; F3, 34%; and F1, 49%). When DHEA was used as precursor, A4 was produced in higher amounts (F5, 29%; F3, 23%; and F1, 11%) than estrone (E1) (F5, 1.5%; F3, 0.9%; and F1, 0.4%). Androstenedione was mainly transformed into E1 (F5, 11.9%; F3, 12.2%; and F1, 0.2%) but lower quantities of T and 17 beta-estradiol (E2) were found. Testosterone was actively transformed into A4 (F5, 50%; F3, 50%; and F1, 30%), but a low transformation to E2 (F5, 1.9%; F3, 1.7%; and F1, 1.4%) and E1 (F5, 2%; F3, 1%; and F1, 0.5%) was found. These results show that theca externa cells from preovulatory follicles of hen have enzymatic activities of 20 beta-reductase (from P5 and P4), 3 beta-hydroxysteroid dehydrogenase/5-4 isomerase (from P5 and DHEA), 17 beta-hydroxysteroid dehydrogenase (from A4 and T), and aromatase (from A4 and T). Furthermore, the enzyme activities decrease with follicular maturation, except for 20 beta-reductase which is constant. These data support the concept that theca externa cells have the ability to synthesize different steroids than reported in theca externa cells. In addition, since theca externa cells did not show the capacity to produce androgens but these steroids were aromatized to estrogens by these cells, it was suggested that the interaction between theca interna cells and theca externa cells occurs in vivo, thus supporting the multicellular theory for estrogen production.
Asunto(s)
Pollos/metabolismo , Folículo Ovárico/metabolismo , Ovulación/fisiología , Esteroides/metabolismo , Células Tecales/metabolismo , Androstenodiona/metabolismo , Animales , Cromatografía en Capa Delgada , Deshidroepiandrosterona/metabolismo , Femenino , Folículo Ovárico/citología , Pregnenolona/metabolismo , Progesterona/metabolismo , Testosterona/metabolismoRESUMEN
EEG activity was recorded from right and left parietal cortex in adult female rats daily during 6 days. Immediately after EEG recording vaginal smears were taken and were microscopically analyzed to determine the estral stage. Absolute and relative powers and interhemispheric correlation of EEG activity were calculated and compared between estral stages. Interhemispheric correlation was significantly lower during diestrous as compared to proestrous and estrous. Absolute and relative powers did not show significant differences between estral stages. Absolute powers of alpha1, alpha2, beta1 and beta2 bands were significantly higher at the right parietal cortex. Comparisons of the same EEG records with estral stages randomly grouped showed no significant differences for any of the EEG parameters. EEG activity is a sensitive tool to study functional changes related to the estral cycle.
Asunto(s)
Electroencefalografía , Estro/fisiología , Animales , Femenino , Lóbulo Parietal/fisiología , Ratas , Ratas WistarRESUMEN
We worked with neuronal cells of chick embryo cultures. The cells were obtained from 7 days-old embryos and cultured for 12 days. The experimental group was submitted to a single dose of corticosterone during the first 48 hours of culture. We measured the membrane resting potential at different external potassium concentrations, as well as the passive electrical properties of the membrane, resistance, time constant and capacitance. We found that the control cells develop progressively their potassium permeability and the electrical properties of the membrane until reaching values in this parameters that were similar to those found in adult cells. Corticosterone application induced a significant enhancement in these parameters in 3 and 6 days-old cultures as compared with the control cultures. These results suggest that glucocorticoids accelerate the differentiation process of the neurons in culture when applied at very early stages of development. We discuss the possible mechanisms involved in this action.
Asunto(s)
Corticosterona/fisiología , Conductividad Eléctrica/fisiología , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Embrión de Pollo , Corticosterona/análisis , Medios de Cultivo/química , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Potasio/farmacocinéticaRESUMEN
Corticosterone (1 microgram/g) was administered to the pregnant rat mother at 17, 18 and 19 days of gestation. The pups were killed at birth or at 6 or 12 days of age and the morphological and biochemical development of the brain, with special emphasis in the cerebellum was studied. The brain and cerebellar weight was slightly diminished in the corticosterone treated animals. Corticosterone produces changes in the pattern of development of the cerebellar layers, causing an accelerated decrement of the external granular layer at 12 days of life. The total protein content of the cerebellum was increased in the hormone treated pups at birth, 6 and 12 postnatal days. Newborn corticosterone treated animals showed a decreased DNA content, but this phenomenon was completely reversed at 12 days. It was concluded that corticosterone given to the pregnant mother influences the time pattern of the development of the brain and the cerebellum.
Asunto(s)
Cerebelo/crecimiento & desarrollo , Corticosterona/administración & dosificación , Efectos Tardíos de la Exposición Prenatal , Animales , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Cerebelo/efectos de los fármacos , Cerebelo/embriología , Femenino , Edad Gestacional , Masculino , Proteínas del Tejido Nervioso/metabolismo , Tamaño de los Órganos , Embarazo , RatasRESUMEN
Progressive increase in membrane resting potential (MRP) values in cultured chick embryo brain neurons were recorded from days 2 to 10 of development. Application of corticosterone or prednisone in 24 hr old cultured cells caused a significant increment in MRP recorded at day 2 of culture with respect to control. When ouabain was added to the corticosterone treated cells its effect on the MRP was impaired. An increment in protein synthesis and a reduction in sodium concentration were also observed in corticosterone pretreated cells. These results show that corticoids accelerate the differentiation process in cultured cells particularly the MRP, stimulating perhaps the Na(+)-K(+) ATPase pump.