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2.
Anim Reprod Sci ; 140(3-4): 180-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23886618

RESUMO

The role of either mTOR system/enzyme sirtuin1 (SIRT1) or transcription factor NF-κB in the direct control of ovarian function has not been estabished. The aim of our in vitro experiments was to examine the involvement of SIRT1 and the p65 and p50 subunits of NFκB in control of porcine ovarian granulosa cell functions and the interrelationships between SIRT1, NFκB (p65, p50) 30 and FSH in the ovary. Monolayers of primary granulosa cells were transfected with gene constructs encoding either SIRT1 or p65 and p50, and thereafter cultured with, or without, addition of FSH. The accumulation of markers of proliferation (cyclin B1 and cyclin-dependent protein kinase Cdc2/p34) and proteins p50, p65 and SIRT1 in the cells was detected by using SDS-PAGE/Western immunoblotting and immunocytochemistry. The secretion of progesterone (P4) and insulin-like growth factor I (IGF-I) was measured by using radioimmunoassay. It was observed that transfection of cells with a SIRT1 gene construct promoted accumulation of proliferation markers, Cdc2/p34, cyclin B1, decreased accumulation of p50 and p65 and stimulated release of P4 and IGF-I. Co-transfection of cells with cDNA p50 and cDNA p65 enhanced the accumulation of SIRT1 and the release of P4 but did not influence the release of IGF-I. Adding FSH to the culture medium stimulated accumulation of both subunits of NF-κB, as well as accumulation of Cdc2/p34, cyclin B1 and release of both P4 and IGF-I. The ability of FSH to promote NF-κB accumulation, the similarity of the main effects of FSH, SIRT1 and NF-κB, as well as the inability of NF-κB to substantially modify the the majority of FSH effects suggest that SIRT1/NF-κB system could be a mediator of FSH action on ovarian cell functions. On the other hand, SIRT1 was able to inhibit NF-κB and to change stimulatory the effect of FSH on NF-κB from stimulatory to inhibitory. This could suggest the existence of negative feedback control of FSH/NF-κB system by high amounts of SIRT1. Our observations (1) confirm the previous data on proliferation, P4 and IGF-I release in ovarian cells and their up-regulation by FSH, (2) demonstrate the presence of SIRT1, NF-κB/p50 and NF-κB/p65 in these cells, (3) show for the first time the involvement of SIRT1 and NF-κB in direct control of proliferation and secretory activity of ovarian cells, (4) represent the first data on interrelationships between FSH, SIRT1 and NF-κB within the ovary.


Assuntos
Hormônio Foliculoestimulante/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Ovário/metabolismo , Sirtuína 1/metabolismo , Suínos/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Western Blotting/veterinária , Proteína Quinase CDC2/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Imuno-Histoquímica/veterinária , Fator de Crescimento Insulin-Like I/metabolismo , Ovário/citologia , Progesterona/metabolismo , Estatísticas não Paramétricas , Transfecção/veterinária
3.
Artigo em Inglês | MEDLINE | ID: mdl-22877785

RESUMO

The purpose of the present study was to identify the role of age, nutritional state and some metabolic hormones in control of avian hypothalamic and ovarian ghrelin/ghrelin receptor system. We examined the effect of food restriction, administration of ghrelin 1-18, ghrelin antagonistic analogue (D-Lys-3)-GHRP-6, obestatin and combinations of them on the expression of ghrelin and ghrelin receptor (GHS-R1a) in hypothalamus and ovary of old (23months of age) and young (7months of age) chickens. Expression of mRNAs for ghrelin and GHS-R1a in both hypothalamus and largest ovarian follicle was measured by RT-PCR. It was observed that food restriction could promote the expression of ghrelin and GHS-R1a in hypothalamus and ovary of the old chickens, but in the young chickens it reduced expression of ghrelin and did not affect expression of GHS-R1a in the ovary. Administration of ghrelin 1-18 did not affect hypothalamic or ovarian ghrelin mRNA, but significantly increased the expression of GHS-R1a in hypothalamus, but not in ovary. (D-Lys-3)-GHRP-6, significantly stimulated accumulation of ghrelin, but not GHS-R1a mRNA in hypothalamus or ghrelin or GHS-R1a in the ovary. Ghrelin 1-18 and (D-Lys-3)-GHRP-6, when given together, were able either to prevent or to induce effect of these hormones. Obestatin administration increased expression of ghrelin gene in the hypothalamus, but not expression of hypothalamic GHS-R1a, ovarian ghrelin and GHS-R1a. Furthermore, obestatin was able to modify effect of both ghrelin and fasting on hypothalamic and ovarian mRNA for ghrelin GHS-R1a. Our results (1) confirm the existence of ghrelin and its functional receptors GHS-R1a in the chicken hypothalamus and ovary (2) confirm the age-dependent control of ovarian ghrelin by feeding, (3) demonstrate, that nutritional status can influence the expression of both ghrelin and GHS-R1a in hypothalamus and in the ovary (3) demonstrates for the first time, that ghrelin can promote generation of its functional receptor in the hypothalamus, but not in the ovary, (4) show that ghrelin1-18 and (D-Lys-3)-GHRP-6 could not only be antagonists in the action on chicken hypothalamus and ovaries, but also independent regulators and even agonists, and (5) provide first evidence for action of obestatin on hypothalamic ghrelin and on the response of hypothalamic and ovarian ghrelin/GHS-R1a system to food restriction. These data indicate the involvement of both hypothalamic and ovarian ghrelin/GHS-R1 systems in mediating the effects of nutritional status, ghrelin and obestatin on reproductive processes.


Assuntos
Galinhas/metabolismo , Privação de Alimentos , Grelina/antagonistas & inibidores , Hipotálamo/metabolismo , Ovário/metabolismo , Receptores de Grelina/metabolismo , Fatores Etários , Animais , Galinhas/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Grelina/metabolismo , Grelina/farmacologia , Humanos , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Oligopeptídeos/farmacologia , Ovário/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Grelina/genética , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Anim Reprod Sci ; 128(1-4): 73-84, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21996578

RESUMO

The aim of these in vitro studies was to examine the involvement of transcription factor NF-κB (p50/p50, p65/p65) and FSH in control of porcine ovarian granulosa cells functions and the possible role of dimers p50/p50, p65/p65 in mediating FSH actions on these cells. Monolayer of primary granulosa cells was transfected with plasmids encoding human p50 cDNA and p65 cDNA, and cultured with or without addition of FSH (0, 1, 10 or 100 ng/ml). The accumulation of proteins p50 and p65, as well as of proliferation markers (PCNA and MAPK/ERK1,2) and marker of apoptosis (Bax) in cells was detected by using SDS-PAGE-Western immunoblotting and immunocytochemistry. DNA fragmentation was evaluated by TUNEL assay. Release of hormones insulin-like growth factor I (IGF-I), progesterone (P(4)), oxytocin (OT), prostaglandins E(2) (PGE(2)) and F(2α) (PGF(2α)) was measured by using RIA. We observed, that p50/p50 promoted the accumulation of PCNA, MAPK/ERK1,2, the release of OT, PGF(2α); inhibited the occurrence of TdT-positive cells, the release of IGF-I and P(4), and did not influence the accumulation of Bax and the release of PGE(2). p65/p65 enhanced the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, PGE(2) and PGF(2α); decreased the percentage of cell containing TdT and did not affect the release of P(4). FSH stimulated the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, P(4), PGE(2); but reduced the proportion of TdT-positive cells and the release of PGF(2α). These observations suggest (1) the involvement of NF-κB (p50/p50) in stimulation of proliferation, inhibition of apoptosis and in either stimulation (OT, PGE(2)) or inhibition (IGF-I, P(4), but not PGF(2)) of hormones release by porcine ovarian granulosa cells; (2) the involvement of NF-κB (p65/p65) in stimulation of proliferation and mitochondrial/Bax-related apoptosis, inhibition of nuclear/TdT-related apoptosis and in stimulation of ovarian hormones (IGF-I, OT, PGE(2), PGF(2α), but not P(4)) release; (3) the role of FSH in up-regulation of both ovarian cell proliferation and mitochondrial/Bax-related apoptosis, in inhibition of nuclear/TdT-related apoptosis, in promotion of IGF-I, P(4), OT, PGE(2) and suppression of PGF(2α) release by porcine ovarian cells. The majority of results demonstrates the involvement of NF-κB (p50/p50 and p65/p65) and FSH in control of basic ovarian functions (proliferation, apoptosis, and secretory activity), but not the functional interrelationships between these regulators.


Assuntos
Células da Granulosa/fisiologia , Subunidade p50 de NF-kappa B/genética , Suínos/fisiologia , Fator de Transcrição RelA/genética , Animais , Apoptose , Proliferação de Células , DNA Complementar/genética , Relação Dose-Resposta a Droga , Feminino , Hormônio Foliculoestimulante/administração & dosagem , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica/fisiologia , Células da Granulosa/efeitos dos fármacos , Humanos , Subunidade p50 de NF-kappa B/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fator de Transcrição RelA/metabolismo
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