RESUMEN
The aim of the present study was to determine the endocrine activity of cultured early antral follicles (EAF) isolated from prepubertal diethylstilbestrol-treated rats. The effect of steroidogenic substrates and FSH on steroid, inhibin A and B, Pro-alphaC and activin A production was evaluated. Androsterone was the predominant steroid produced by EAF. The addition of androstenedione, androstenedione+FSH and progesterone stimulated oestradiol production, whereas 25-hydroxycholesterol (25-OH-Chol) increased progesterone production. Inhibin A, B, Pro-alphaC, and activin A were produced under basal conditions. The predominance of inhibin B over inhibin A was not affected by the addition of androstenedione or progesterone. Inhibin A and activin A production was stimulated by FSH. 25-OH-Chol increased Inha, Inhba and Inhbb mRNA expression and the production of the three molecular forms of inhibins but decreased activin A production. These results show that FSH and the steroid follicular microenvironment differentially modulate the gene expression of inhibin/activin subunits, their assembly and secretion.
Asunto(s)
Activinas/metabolismo , Inhibinas/metabolismo , Folículo Ovárico/metabolismo , Precursores de Proteínas/metabolismo , Activinas/biosíntesis , Activinas/genética , Aminoglutetimida/farmacología , Animales , Células Cultivadas , Femenino , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidroxicolesteroles/farmacología , Inhibinas/biosíntesis , Inhibinas/genética , Folículo Ovárico/efectos de los fármacos , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Esteroides/biosíntesisRESUMEN
Mammary gland morphogenesis and differentiation are mediated through the combined activities of systemic hormones and locally synthesized growth factors. Activin, a member of the transforming growth factor (TGF)-beta superfamily, is known to regulate the growth and differentiation of several cell types. In the present study, we investigated the role of activin in rat mammary gland on different stages of development. We found that activin A in vitro inhibits the proliferation of isolated acini, and this effect increases with the development of the gland. This factor also produces in vitro an inhibition of the final differentiation of acini obtained from 19th day pregnant rats. We also report the expression of activin receptors IIA and IIB mRNA in whole rat mammary gland and acini, with decreased levels of expression of type IIA (in both compartments) and IIB (in acini) during pregnancy and lactogenesis. In addition, we show that activin betaB-subunit mRNA decreases throughout pregnancy, and that the mRNA levels of follistatin (Fst) (its ligand protein) are high in cycling rats and at the beginning of pregnancy and diminish thereafter, having the acini higher levels of expression. Our data show that activin betaB-subunit, follistatin and ActRIIA and IIB transcripts are expressed in rat mammary gland at appropriate times and locations during development, allowing an interplay that might regulate activin action on growth and differentiation of the gland.
Asunto(s)
Activinas/fisiología , Folistatina/biosíntesis , Subunidades beta de Inhibinas/fisiología , Glándulas Mamarias Animales/crecimiento & desarrollo , Receptores de Activinas/biosíntesis , Receptores de Activinas/genética , Activinas/genética , Activinas/farmacología , Animales , Caseínas/biosíntesis , Caseínas/genética , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Folistatina/genética , Folistatina/farmacología , Regulación de la Expresión Génica , Subunidades beta de Inhibinas/biosíntesis , Subunidades beta de Inhibinas/genética , Subunidades beta de Inhibinas/farmacología , Inhibinas/biosíntesis , Inhibinas/genética , Lactancia/fisiología , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/fisiología , Embarazo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23- to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor beta (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. (3)H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 micro g/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.
Asunto(s)
Apoptosis/efectos de los fármacos , Dietilestilbestrol/farmacología , Inhibinas/farmacología , Folículo Ovárico/citología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Estradiol/biosíntesis , Estradiol/farmacología , Femenino , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inhibinas/biosíntesis , Progesterona/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Inhibins, produced mainly in the gonads, suppress FSH synthesis. The bioactive dimeric forms of inhibin (A and B) have been proposed as peripheral markers of Sertoli and granulosa cell function. The determination of serum dimeric inhibins from birth through adulthood reflects a distinct pattern of both inhibins in males and females. Concomitantly with the gonadotrophin surge, an important production of inhibin B is observed during the first months of life. In males, inhibin B levels are higher than in females and persist elevated up to childhood, whereas in females they decrease up to prepubertal levels by 6 months of age. In girls, high serum levels of inhibin A are observed during the first two months of life; thereafter, they are undetectable until puberty. An active secretion of inhibin B persists in both males and females in the period of maximal LHRH pulse generator restraint; however, the possible gonadotrophin dependence of this production remains controversial. At puberty, a progressive rise in serum inhibin B occurs concomitantly with the increased production of sex steroids in both males and females. A similar secretion pattern of inhibin A is observed in girls. This increment is mainly exerted by gonadotrophins and modulated by multiple paracrine/autocrine mechanisms within the ovary and the testis that regulate the dimerization of the inhibin subunits throughout pubertal maturation. The differences observed in males and females between circulating dimeric inhibins in relation to gonadotrophins and sex steroid concentrations from birth through puberty has opened a new perspective for research in human reproduction. These new markers may contribute to a better knowledge of the regulation of the hypothalamic-pituitary-gonadal axis function and the physiopathology of the mechanisms involved in sexual differentiation and/or fertility disorders.
Asunto(s)
Inhibinas/fisiología , Pubertad/fisiología , Adolescente , Envejecimiento , Animales , Niño , Preescolar , Dimerización , Femenino , Feto/metabolismo , Hormona Folículo Estimulante/biosíntesis , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Lactante , Recién Nacido , Inhibinas/biosíntesis , Inhibinas/sangre , Masculino , Ovario/fisiología , Caracteres Sexuales , Testículo/fisiologíaRESUMEN
In the present study, we have examined the role of hormones and growth factors in regulating dimeric inhibin production in immature rat granulosa cells. Purified granulosa cells from estrogen-primed immature rats were cultured under defined conditions. Inhibins A and B in the culture media were measured using a two-site enzyme-linked immunosorbent assay specific for each dimer. Under basal conditions, granulosa cells produced 14-fold more inhibin A than inhibin B (inhibin A, 2.0; inhibin B, 0.14 ng/ml, measured against human standards; average A/B apparent ratio, 14). Addition of increasing doses of FSH elicited dose-dependent increases in both inhibins, the effects being more pronounced on inhibin A than on inhibin B (9.4- and 4.1-fold increases, respectively; average A/B ratio, 34). Estradiol, when added alone, stimulated inhibin A production 3- to 6-fold, whereas minor changes were observed in inhibin B production. Insulin-like growth factor-I produced a similar stimulation of both inhibins (3-fold stimulation over control). This growth factor, however, induced a marked dissociation in the sensitivity of inhibins A and B to FSH stimulation, with maximal stimulation of inhibin B observed at comparatively lower concentrations of the gonadotropin. Transforming growth factor-beta (TGF-beta, 5 ng/ml) had a more marked stimulatory effect on inhibin B than on inhibin A production (7- to 14-fold vs. 2- to 5-fold for inhibin B and A, respectively). A more pronounced differential stimulation of inhibin B was also exerted by another member of the TGF-beta superfamily, activin A (A/B ratio, 0.66). This preferential stimulation of inhibin B by TGF-beta and activin A was amplified in the presence of FSH. Coculture of rat granulosa cells with freshly isolated bovine oocytes was also associated with a marked stimulation of inhibin B production (100-fold increase) and a comparatively lower stimulation of inhibin A (10-fold increase; A/B ratio, 1). The discrepancy between the proportion of inhibin dimers in serum (A/B ratio, 0.13) and those produced by untreated granulosa cells may suggest that intraovarian factors, such as TGF-beta, activin A, or oocyte-derived factor(s), are responsible for the shift of the ratio toward the predominance of inhibin B.
Asunto(s)
Células de la Granulosa/metabolismo , Inhibinas/biosíntesis , Animales , Bovinos , Técnicas de Cocultivo , Dimerización , Estradiol/farmacología , Femenino , Hormona Folículo Estimulante/farmacología , Humanos , Inhibinas/sangre , Factor I del Crecimiento Similar a la Insulina/farmacología , Oocitos/fisiología , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Membranes derived from bovine pituitary glands free of the neural lobe were used to investigate the presence of binding sites for inhibin, a glycoprotein produced by the ovarian granulosa cells capable of selectively suppressing FSH secretion from the pituitary gland. Optimal concentration of membranes (400 micrograms prot) and 125I-bovine inhibin (2 nM) were incubated in a medium containing 50 mM Tris-HCl pH 7.4, 0.01 M MgCl2 and BSA 0.01% in a final assay volume of 200 microliters at 37 degrees C for different time intervals. Non-specific binding was estimated using unlabelled inhibin in excess. The time course of specific 125I-bovine inhibin (2 nM) binding to bovine pituitary membranes is slow with 50% binding at approximately 20 min of incubation and reaching equilibrium at 90 min of incubation. The kinetic analysis shows an apparent pseudo first order association rate constant (Kob) equivalent to 4 x 10(-2) min-1. Following equilibrium with the tracer, a large excess of unlabelled inhibin (1.27 microM) was able to displace 84% of the specific binding within 120 min of incubation and 50% of the binding at approximately 40 min. The analysis under displacing conditions showed an apparent dissociation rate constant (K2) equals to 1.5 x 10(-2) min-1 and an apparent association rate constant (K1) equals to 1.3 x 10(9) M min-1. Thus, the estimation of the apparent kinetic equilibrium dissociation constant (Kd = K2/K2) of the binding of inhibin to bovine pituitary membranes was 1.2 nM. These results show for the first time the existence of bovine inhibin specific binding sites in bovine pituitary, and also that such a binding can take place in the absence of either gonadal and/or hypothalamic influences. They also contribute to the better understanding of the role of non-steroidal hormones such as inhibin, in the regulation of gonadotrophin secretion.
Asunto(s)
Membrana Basal/fisiología , Sitios de Unión/fisiología , Inhibinas/biosíntesis , Inhibinas/farmacología , Hipófisis/fisiología , Animales , Bovinos , Femenino , Hormona Folículo Estimulante/metabolismo , Líquido Folicular/metabolismoRESUMEN
Membranes derived from bovine pituitary glands free of the neural lobe were used to investigate the presence of binding sites for inhibin, a glycoprotein produced by the ovarian granulosa cells capable of selectively suppressing FSH secretion from the pituitary gland. Optimal concentration of membranes (400 micrograms prot) and 125I-bovine inhibin (2 nM) were incubated in a medium containing 50 mM Tris-HCl pH 7.4, 0.01 M MgCl2 and BSA 0.01 percent in a final assay volume of 200 microliters at 37 degrees C for different time intervals. Non-specific binding was estimated using unlabelled inhibin in excess. The time course of specific 125I-bovine inhibin (2 nM) binding to bovine pituitary membranes is slow with 50 percent binding at approximately 20 min of incubation and reaching equilibrium at 90 min of incubation. The kinetic analysis shows an apparent pseudo first order association rate constant (Kob) equivalent to 4 x 10(-2) min-1. Following equilibrium with the tracer, a large excess of unlabelled inhibin (1.27 microM) was able to displace 84 percent of the specific binding within 120 min of incubation and 50 percent of the binding at approximately 40 min. The analysis under displacing conditions showed an apparent dissociation rate constant (K2) equals to 1.5 x 10(-2) min-1 and an apparent association rate constant (K1) equals to 1.3 x 10(9) M min-1. Thus, the estimation of the apparent kinetic equilibrium dissociation constant (Kd = K2/K2) of the binding of inhibin to bovine pituitary membranes was 1.2 nM. These results show for the first time the existence of bovine inhibin specific binding sites in bovine pituitary, and also that such a binding can take place in the absence of either gonadal and/or hypothalamic influences. They also contribute to the better understanding of the role of non-steroidal hormones such as inhibin, in the regulation of gonadotrophin secretion