Assuntos
Estágio Clínico/organização & administração , Currículo/normas , Violência Doméstica/prevenção & controle , Educação de Pós-Graduação em Medicina/organização & administração , Capacitação em Serviço/organização & administração , Internato e Residência , Programas de Rastreamento/métodos , Corpo Clínico Hospitalar/educação , Corpo Clínico Hospitalar/psicologia , Estudantes de Medicina/psicologia , Atitude do Pessoal de Saúde , Competência Clínica/normas , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Avaliação das Necessidades , Valor Preditivo dos Testes , Análise de Regressão , Fatores de Risco , Inquéritos e Questionários/normasRESUMO
Previous studies on the porcine renal epithelial LLC-PK1 cell line demonstrated that oxalate exposure produces concentration-dependent effects on renal cell growth and viability via process(es) involving free radicals. The present studies were conducted to determine whether these findings could be extended to a renal proximal tubular epithelial cell line derived from the human kidney. These studies examined oxalate-induced changes in membrane integrity after short-term exposure (4 h) and changes in cell survival after longer-term exposure (24 to 72 h). Oxalate-induced changes were also assessed in the expression of two genes: egr-1, a zinc-finger transcription factor, and osteopontin, a protein associated with tissue remodeling. The present studies also determined whether oxalate-induced changes in either cell viability or gene expression depended on free radicals. Oxalate at a concentration > or = 175 microM (free) produced membrane damage within 4 h. This effect was inhibited by Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), a superoxide dismutase mimetic, but not by N-acetyl cysteine, a glutathione precursor, or by deferoxamine, an iron chelator. Acute oxalate-induced injury was followed by cell loss within 24 h, an effect maintained at 48 and 72 h at high concentrations of oxalate. Oxalate also promoted DNA synthesis. This mitogenic effect offset cell loss at lower oxalate concentrations (88 microM) leading to a small but significant increase in cell number at 72 h. Treatment with oxalate also increased expression of egr-1 mRNA within 1 h, a response that was attenuated by MnTMPyP; oxalate treatment for 8 h also increased abundance of osteopontin mRNA. These studies suggest that oxalate exposure produces changes in human renal cell growth and viability via a process(es) dependent on reactive oxygen intermediates. Such changes may play a role in the development and/or progression of renal disease via generation of reactive oxygen intermediates.
Assuntos
Proteínas Imediatamente Precoces , Túbulos Renais Proximais/efeitos dos fármacos , Oxalatos/toxicidade , Divisão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Proteína 1 de Resposta de Crescimento Precoce , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/citologia , RNA Mensageiro/análise , Fatores de Transcrição/genéticaRESUMO
LH concentrations were measured in serum collected at 10-min intervals from chronically ovariectomized female Syrian hamsters that had been maintained for 9 wk in stimulatory (long) or inhibitory (short) photoperiods. Short days reduced the number of detectable LH pulses during both the morning and the afternoon. Most short-day hamsters experienced a gradual afternoon rise in serum LH concentrations; this rise was not composed of multiple pulses. In separate groups of similarly treated hamsters, pituitary LH-beta mRNA abundance was significantly reduced by short-day exposure at both times of day even though serum LH concentrations rose in the afternoon. Estradiol treatment induced an afternoon surge of serum LH in both photoperiods, and eliminated the effect of photoperiod on LH-beta mRNA abundance in the afternoon. Serum prolactin (PRL) concentrations were not consistently influenced by day length in castrated hamsters with or without estrogen treatment, but PRL mRNA abundance was significantly suppressed by short-day exposure in all groups. The results indicate that day length exerts profound steroid-independent effects upon hypophyseal gene expression, and that the regulation of LH-beta mRNA abundance may be due to photoperiodic control of the neural GnRH pulse generator.
Assuntos
Expressão Gênica/fisiologia , Luz , Hormônio Luteinizante/sangue , Periodicidade , Adeno-Hipófise/fisiologia , Animais , Cricetinae , Estradiol/farmacologia , Feminino , Expressão Gênica/genética , Hormônio Luteinizante/genética , Mesocricetus , Ovariectomia , Prolactina/sangue , Prolactina/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Radioimunoensaio , Fatores de TempoRESUMO
Hormonal regulation of adenohypophyseal messenger ribonucleic acids (mRNAs) encoding preprotachykinin (PPT), prolactin (PRL) and thyrotropin beta subunit (TSH beta) was examined in juvenile and pubertal female rats. Hypothyroidism, initiated on day 2 (d2) or 22 (d22) of life, increased PPT and TSH beta mRNAs but decreased PRL mRNA 17 days later. Exogenous estradiol given for 3 days reduced PPT mRNA in pubertal (d38) but not juvenile (d18) euthyroid females; conversely, estradiol increased PRL mRNA on d18 but not d38. In hypothyroid females however, estradiol decreased PPT and TSH beta mRNAs at both ages and increased PRL mRNA in pubertal but not juvenile females. Thus, regulation of adenohypophyseal mRNAs by estradiol varies with age and thyroid status. In previous studies, adenohypophyseal tachykinins increased in male, but not female rats at puberty. This sex difference was not reproduced here by neonatal androgenization of females, suggesting that it is not mediated by hypothalamic sexual differentiation. However, PRL mRNA increased in androgenized females; this increase was prevented by ovariectomy, suggesting its medication by estradiol.
Assuntos
Envelhecimento/fisiologia , Estradiol/farmacologia , Hipotireoidismo/metabolismo , Adeno-Hipófise/metabolismo , Precursores de Proteínas/biossíntese , RNA Mensageiro/biossíntese , Taquicininas/biossíntese , Testosterona/farmacologia , Tireotropina/biossíntese , Análise de Variância , Animais , Estradiol/sangue , Feminino , Hormônio Luteinizante/biossíntese , Tamanho do Órgão/efeitos dos fármacos , Adeno-Hipófise/anatomia & histologia , Adeno-Hipófise/efeitos dos fármacos , Prolactina/biossíntese , Radioimunoensaio , Ratos , Substância P/biossínteseRESUMO
Hyperprolactinaemia disrupts fertility in many species, perhaps by inhibiting ovarian follicular steroidogenesis. The present studies measured oestradiol and progesterone secretion from isolated follicles from rats rendered hyperprolactinaemic in one of two ways. Sustained hyperprolactinaemia was induced by transplantation of two donor pituitary grafts under the renal capsule of adult female rats; grafts remained in place for 3 months. Transient hyperprolactinaemia was induced by pseudopregnancy initiated by cervical stimulation. Small antral follicles were isolated from both groups of rats 8-10 days after the previous vaginal oestrous smear and also from a control group of dioestrous female rats. Follicles were incubated for 3 h in the presence or absence of human chorionic gonadotrophin (hCG) or testosterone. Basal and hCG-stimulated oestradiol production were each reduced in follicles from both hyperprolactinaemic groups, relative to follicles from dioestrous control rats. In contrast, in the presence of testosterone, all groups of follicles produced comparable amounts of oestradiol. hCG stimulated comparable progesterone production by follicles from all three treatment groups. Testosterone elicited smaller increases in progesterone accumulation by follicles from all in-vivo groups. Reduced basal and gonadotrophin-stimulated, but not androgen-stimulated, oestradiol accumulation suggests that androgen production rather than aromatase activity in small antral follicles may be impaired by long-term hyperprolactinaemia.
Assuntos
Estradiol/biossíntese , Hiperprolactinemia/metabolismo , Folículo Ovariano/metabolismo , Testosterona/fisiologia , Animais , Estradiol/sangue , Feminino , Progesterona/biossíntese , Progesterona/sangue , Pseudogravidez , Radioimunoensaio , Ratos , Ratos Endogâmicos , Fatores de TempoAssuntos
Estradiol/farmacologia , Regulação da Expressão Gênica , Neurocinina A/genética , Adeno-Hipófise/fisiologia , Precursores de Proteínas/genética , RNA Mensageiro/genética , Substância P/genética , Taquicininas/genética , Testosterona/farmacologia , Tireoidectomia , Envelhecimento , Animais , Feminino , Masculino , Neurocinina A/metabolismo , Orquiectomia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Ratos , Valores de Referência , Caracteres Sexuais , Substância P/metabolismoRESUMO
Exposure to short daylengths arrests the oestrous cycle, provokes daily gonadotrophin surges and reduces the ability of exogenous oestradiol to trigger behavioural receptivity in golden hamsters. In order to examine neuroendocrine effects of photoperiod which might underlie these responses, ovariectomized hamsters were maintained under long or short photoperiods for 54 days before treatment with cholesterol or various doses of oestradiol-17 beta. Short days reduced the ability of low doses of oestrogen to prime hamsters for the induction of oestrus by progesterone. Upon repetition of oestrogen priming 2 weeks later, photoperiod was without significant influence on the concentrations of nuclear oestrogen receptors or cytosolic progestin receptors in a block of tissue containing the hypothalamus and preoptic area. Oestradiol treatment provoked significant increases in serum concentrations of LH and prolactin in the afternoon, but photoperiod did not alter the positive-feedback efficacy of this gonadal steroid hormone. Adenohypophysial LH-beta subunit and prolactin mRNAs were suppressed by short days in ovariectomized hamsters not treated with oestradiol. Oestradiol decreased expression of the LH-beta subunit gene in both stimulatory and inhibitory photoperiods, but increased prolactin mRNA abundance in both long and short days. Photoperiod therefore exerts pronounced steroid-independent effects on phasic LH and prolactin secretion, but regulation of adenohypophysial abundance of LH-beta subunit and prolactin mRNAs by oestradiol is not markedly influenced by daylength. Photoperiodic regulation of the priming effects of oestradiol on behavioural receptivity may result from modulation of events occurring subsequent to steroid-receptor interactions, or involve changes in receptor populations not detectable by the present methods.
Assuntos
Expressão Gênica/fisiologia , Luz , Adeno-Hipófise/metabolismo , Hormônios Hipofisários/metabolismo , Receptores de Estrogênio/metabolismo , Comportamento Sexual Animal/fisiologia , Análise de Variância , Animais , Northern Blotting , Cricetinae , Estradiol/administração & dosagem , Estradiol/fisiologia , Feminino , Hormônio Luteinizante/sangue , Mesocricetus , Ovariectomia , Periodicidade , Hormônios Hipofisários/genética , Prolactina/sangue , Prolactina/genética , RNA Mensageiro/metabolismo , Radioimunoensaio , Receptores de Progesterona/metabolismoRESUMO
Abstract Daylength regulates neuroendocrine function in male golden hamsters. Exposure to short days triggers gonadal regression and decreases serum luteinizing hormone (LH), prolactin and testosterone concentrations. Inhibitory photoperiods also amplify the negative feedback actions of androgens upon gonadotropin secretion. To examine whether these changes arise from altered adenohypophyseal gene expression, we measured the abundance of the messenger ribonucleic acids (mRNAs) encoding beta-LH, prolactin and proopiomelanocortin in anterior pituitaries of male golden hamsters which were either left intact, castrated, castrated and implanted with testosterone, or pinealectomized and maintained in either long (14 h light/10 h dark) or short (5 h light/19 h dark) days. Short days caused testicular atrophy in intact male hamsters and reduced serum LH in intact and castrated, testosterone-replaced hamsters. The relative abundance of beta-LH mRNA was suppressed by exposure to short days only in castrated hamsters. Serum prolactin was decreased by short days regardless of circulating testosterone concentrations. Prolactin mRNA abundance was decreased by short days in all pineal-intact groups. Castration reduced proopiomelanocortin mRNA abundance in long days and testosterone replacement reversed this effect. In the presence of testosterone, photoperiod influenced serum LH concentrations without altering hypophyseal abundance of beta-LH mRNA. In contrast, photoperiodic influences on prolactin secretion were correlated with alterations in steady-state mRNA abundance.
RESUMO
The distribution of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) mRNA within the suprachiasmatic nucleus (SCN) of rats was evaluated by immunocytochemistry and in situ hybridization. The pattern of VIP and PHI immunoreactivity corresponded closely to the distribution of VIP/PHI mRNA within the ventrolateral SCN. Clear hybridization signal was observed within the SCN of rats killed 5 h after light onset and in rats killed 2 h after the onset of the dark phase of the light-dark cycle. Visual examination of the grain density within the autoradiographs suggested that VIP/PHI mRNA may occur in higher concentrations shortly after the onset of darkness than 5 h after the onset of the light phase.
Assuntos
Peptídeo PHI/genética , RNA Mensageiro/genética , Núcleo Supraquiasmático/metabolismo , Peptídeo Intestinal Vasoativo/genética , Animais , Autorradiografia , Imuno-Histoquímica , Masculino , Hibridização de Ácido Nucleico , Peptídeo PHI/análise , RNA Mensageiro/análise , Ratos , Radioisótopos de Enxofre , Núcleo Supraquiasmático/citologia , Transcrição GênicaRESUMO
The physiological stimulation and inhibition of release of several peptide and protein hormones appears to be associated with transcription of the respective peptide- and protein hormone-encoding genes. In the current studies we investigated whether this was also true for the rat pituitary LH system. Using an anterior pituitary primary tissue culture system, we have analyzed the effects of 10(-9) M GnRH on beta LH gene transcription using a transcription run-on assay, and on nuclear beta LH RNA levels using a highly sensitive solution hybridization-S1-nuclease protection assay. GnRH-stimulated release of LH does not appear to be coupled to a significant change in the rate of beta LH gene transcription, but is associated with both a 2- to 3-fold increase in levels of the beta LH primary transcript and processing intermediates and a rapid decrease in the levels of fully processed beta LH mRNA in the nucleus. No significant change in beta LH cytoplasmic mRNA levels, however, was associated with GnRH-stimulated release of LH, in vitro. Our findings suggest that unlike several other peptide and protein hormone systems, stimulated release of beta LH by GnRH in vitro is not associated with an increase in beta LH gene transcription or cytoplasmic mRNA levels.
Assuntos
Hormônio Luteinizante/metabolismo , Hormônios Liberadores de Hormônios Hipofisários/farmacologia , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Luteinizante/genética , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , RNA/metabolismo , Ratos , Transcrição GênicaRESUMO
Proopiomelanocortin (POMC) peptide secretion from rat anterior pituitary corticotrophs and intermediate pituitary melanotrophs is stimulated by corticotropin-releasing hormone (CRH). CRH-stimulated secretion in the corticotrophs is inhibited by glucocorticoids in a complex fashion, involving both a fast, direct blockade of POMC secretion (minutes to hours) and a longer inhibitory action (hours to days) that decreases the amount of POMC peptide available for release. The current studies tested the ability of CRH to stimulate beta-endorphin (a peptide derived from POMC) secretion and POMC gene transcription in cultured anterior and neurointermediate lobe pituitary cells, and examined interactions between CRH and glucocorticoids in regulating POMC gene expression using an in vitro nuclear transcription run-on assay. In both tissues, CRH elicited a time-dependent stimulation of POMC gene transcription that was maximal at 60 min and remained elevated for at least 18 hr. Glucocorticoids rapidly inhibited POMC gene transcription fourfold in the anterior lobe with maximal effects within 20 min. Glucocorticoids also blocked CRH-stimulated POMC gene transcription in anterior pituitary cultures in a temporal manner paralleling their inhibitory effects on CRH-stimulated beta-endorphin secretion. In neurointermediate lobe cultures, the effects of glucocorticoids and CRH on POMC gene transcription were qualitatively similar to, but of lesser magnitude than those observed in the anterior lobe. These studies indicate that the regulation of POMC gene transcription by glucocorticoids and CRH is complex and that the two modulators do not function independently.
Assuntos
Hormônio Liberador da Corticotropina/fisiologia , Regulação da Expressão Gênica , Glucocorticoides/fisiologia , Hipófise/fisiologia , Pró-Opiomelanocortina/genética , Transcrição Gênica , Animais , Células Cultivadas , Dexametasona/farmacologia , Endorfinas/metabolismo , Masculino , Adeno-Hipófise/fisiologia , RNA Mensageiro/genética , Ratos , Fatores de TempoRESUMO
Substance P (SP) and substance K (SK) are mammalian tachykinin peptides derived from a single preprotachykinin-A (PPT-A) gene and are widely but selectively distributed in neural and endocrine tissues. SP is present in the rat anterior pituitary, and its content there varies with the thyroid status of the animal. The present studies were undertaken to determine whether the PPT-A gene is expressed in the anterior pituitary and if so, whether PPT-A messenger RNA (mRNA) abundance is regulated by thyroid hormone status. Male rats were surgically or chemically thyroidectomized or made hyperthyroid by thyroid hormone (T3) injection. Total RNA was isolated from individual anterior pituitaries, and PPT-A mRNA abundance was determined by dot blot procedures. In parallel groups of rats, anterior pituitaries were extracted for measurement of SP and SK by specific RIAs. Surgical or chemical thyroidectomy increased PPT-A mRNA abundance 4 to 6-fold and increased both SP and SK content in the anterior pituitary. Administration of T3 to thyroidectomized rats reversed the increase in both PPT-A mRNA abundance and SP and SK content in the adenohypophysis. T3 administration to euthyroid rats also decreased PPT-A mRNA abundance and SP and SK content in the anterior pituitary. The coordinate presence of PPT-A mRNA with SP and SK in the anterior pituitary strongly suggests that these peptides are synthesized within this gland.
Assuntos
Regulação da Expressão Gênica , Neuropeptídeos/genética , Adeno-Hipófise/fisiologia , Precursores de Proteínas/genética , Taquicininas , Hormônios Tireóideos/fisiologia , Animais , Masculino , Neurocinina A , Neuropeptídeos/metabolismo , Oxirredução , RNA Mensageiro/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The effect of body condition on ovarian follicular development was investigated in Scottish Blackface ewes in high and low body condition. Follicles were dissected from ovaries on days 11 and 12 of the luteal phase and 24 h after prostaglandin-induced luteal regression. Ewes in low body condition had a lower ovulation rate (low: 0.9; high: 1.8 P less than 0.05) and lower mean plasma levels of FSH during both the luteal (low: 54; high: 72 micrograms/l) and follicular (low: 34; high: 43 micrograms/l) phases of the cycle. Low body condition was associated with a reduced number of large (greater than or equal to 4 mm) follicles in both the luteal and follicular phases, and in low condition a lower proportion of these follicles was oestrogenic and potentially ovulatory as assessed by follicular fluid levels of oestradiol. However, within the different oestrogenic classifications of these large follicles there were no significant differences in the steroidogenic capacity as assessed by the concentrations of either oestradiol or testosterone in follicular fluid, basal and hCG-stimulated testosterone production, thecal 125I hCG binding or basal and testosterone-stimulated oestradiol production by granulosa cells in relation to body condition. These results suggest that body condition influences ovulation rate by altering the concentration of FSH in blood, which in turn affects the number of potentially ovulatory follicles growing beyond 4 mm.
Assuntos
Folículo Ovariano/fisiologia , Ovulação , Ovinos/fisiologia , Animais , Constituição Corporal , Peso Corporal/efeitos dos fármacos , Gonadotropina Coriônica/farmacologia , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Ovulação/efeitos dos fármacos , Prostaglandinas/farmacologia , Radioimunoensaio , Receptores do LH/metabolismo , Testosterona/metabolismo , Testosterona/farmacologiaRESUMO
Active immunization of 6 Damline ewes against LHRH during seasonal anoestrus resulted in an inhibition of ovarian cyclicity throughout 2 subsequent breeding seasons. This was associated with a significant suppression of plasma LH and FSH concentrations but no significant effect on prolactin. The ovaries of LHRH-immunized ewes 30 months after primary immunization contained no follicles greater than 2.5 mm in diameter and a greater proportion of follicles between 1 and 2 mm were atretic than in control ewes (N = 8). In-vitro production of testosterone and androstenedione were similar in follicles 1-2 mm in both control and LHRH-immunized ewes (N = 6) and all had little or no ability to secrete oestradiol. However, basal and hCG-stimulated progesterone secretion was suppressed in the follicles from LHRH-immunized ewes. These results show that follicular development beyond 2.5 mm in the ewe is dependent on adequate stimulation by both LH and FSH.
Assuntos
Hormônio Liberador de Gonadotropina/imunologia , Hormônio Liberador de Gonadotropina/fisiologia , Folículo Ovariano/fisiologia , Anestro , Animais , Anticorpos Monoclonais/imunologia , Feminino , Hormônio Foliculoestimulante/sangue , Atresia Folicular , Imunidade Ativa , Hormônio Luteinizante/sangue , Folículo Ovariano/metabolismo , Ovulação , Progesterona/metabolismo , Prolactina/sangue , OvinosRESUMO
To examine the effects of prolactin (Prl) and human chorionic gonadotrophin (hCG) on progesterone production by murine ovarian explants, immature female mice were injected with 4 IU pregnant mare's serum gonadotrophin (PMSG) to induce follicular maturation. After 24 or 40 h mice were killed, ovaries removed, cut into fragments and maintained as explants for 24 h in the presence or absence of ovine or human Prl (25-2500 ng/ml). None of these doses of Prl affected basal progesterone accumulation into media over 24 h. To determine if Prl could modify the capacity of ovarian explants to respond to gonadotrophin, ovaries were incubated with 25 IU/ml hCG for 3 h after an initial 24 h incubation period with or without Prl. Prl had no effect on basal progesterone accumulation but significantly enhanced hCG-stimulated progesterone accumulation during the 3 h incubation period. We conclude that Prl does not inhibit but may enhance progesterone secretion by pre-ovulatory follicles in the mouse.
Assuntos
Gonadotropina Coriônica/farmacologia , Gonadotropinas Equinas/farmacologia , Ovário/metabolismo , Progesterona/metabolismo , Prolactina/farmacologia , Animais , Feminino , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos , Fatores de TempoRESUMO
The present study was performed to determine if adenylate cyclase in granulosa cells was affected by the pituitary hormone FSH and the ovarian hormone estradiol. Results demonstrate that granulosa cells of intact immature rats exhibit considerably more FSH- than hCG-stimulated adenylate cyclase activity. The FSH-responsive enzyme system was not altered by hypophysectomy or by treating hypophysectomized rats with FSH alone, but was increased slightly by treatment with estradiol alone. Sequential treatment of rats with estradiol and FSH markedly increased both FSH- and LH/hCG-stimulated adenylate cyclase activities. Thus, FSH-responsive adenylate cyclase appears to be a constitutive component of granulosa cells in prenatal follicles which exhibits a pronounced increase during the development of preovulatory follicles, a change dependent on the synergistic actions of estradiol and FSH. Desensitization of FSH-responsive adenylate cyclase in granulosa cells of preantral and antral follicles was assessed by administering 5 micrograms human FSH to estradiol or estradiol/FSH-treated rats, respectively. FSH failed to induce desensitization of adenylate cyclase in granulosa cells of preantral follicles at 2 h, but did desensitize the enzyme system in granulosa cells of antral follicles. Furthermore, the desensitization of adenylate cyclase in granulosa cells of antral follicles was heterologous; both FSH and hCG exerted this effect. The causes of the differences in the response of adenylate cyclase to high concentrations of FSH at different stages of follicular development remain unclear. The absence of desensitization in preantral follicles may be required to permit a continuous nondisruptive pattern of follicular growth when small follicles are repeatedly exposed to gonadotropin surges, whereas desensitization is required for the cessation of follicular growth and luteinization.
Assuntos
Adenilil Ciclases/metabolismo , Estradiol/farmacologia , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/citologia , Folículo Ovariano/enzimologia , Animais , Diferenciação Celular/efeitos dos fármacos , Gonadotropina Coriônica/farmacologia , Feminino , Hipofisectomia , Folículo Ovariano/efeitos dos fármacos , RatosRESUMO
Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.
Assuntos
Gonadotropina Coriônica/metabolismo , Células da Granulosa/metabolismo , Ovário/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Estradiol/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , Radioisótopos do Iodo , Rim/metabolismo , Cinética , Substâncias Macromoleculares , Folículo Ovariano/metabolismo , Pseudogravidez/metabolismo , Técnica de Diluição de Radioisótopos , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores do LHRESUMO
Physiological concentrations of progesterone (20-100 ng/ml), maintained by the insertion of implants into 30-day-old rats, delayed first ovulation, and withdrawal of progesterone on day 47 of age synchronized first ovulation in rats. Inhibition of ovulation involved negative feedback regulation of tonic LH and FSH secretion, blockage of gonadotropin surges, and suppression of preovulatory, but not antral, follicular growth. Removal of implants resulted in a rapid decline in serum progestrone from 100 to 5 ng/ml within 0-12 h. Between 0-36 h there were progressive increases in serum concentrations of LH and FSH, enhanced accumulation of estradiol by individual follicles incubated in vitro with or without exogenous substrate, and marked progressive increases in the content of LH (but not FSH) receptors in both thecal and granulosa cells. These events were followed by gonadotropin surges at 48 h (1800 h on day 49), ovulation, and morphological and biochemical signs of luteinization, including decreases in follicular gonadotropin receptor content and estradiol accumulation, evident by 60 h. With the exception of changes in basal LH, this sequence of events is remarkably similar in time and pattern to that after the decline of progesterone on diestrous day 2 and ovulation on proestrus of a 5-day cycle. Although a direct effect of progesterone on ovarian follicular cell function cannot be excluded, the data suggest that subtle but sustained increases in LH (and possibly FSH) are required for the enhanced follicular accumulation of estradiol and LH-binding activity occurring between diestrus and proestrus of the rat estrous cycle. Thus, perhaps some of the mystery surrounding the endocrine events between diestrus and proestrus can be ascribed to changes in serum LH that have been too small and/or variable for current nonserial sampling methods and RIAs to detect reliably.