RESUMO
Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for divergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of diverse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Sequência de Bases , Sítios de Ligação , Feminino , Células da Granulosa/metabolismo , Histonas/metabolismo , Humanos , Motivos de Nucleotídeos , Especificidade de Órgãos , Ovário/metabolismo , Ovulação/genética , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Elementos de RespostaRESUMO
Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre-granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development.
Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Animais não Endogâmicos , Linhagem Celular , Feminino , Camundongos , Técnicas de Cultura de Órgãos , Folículo Ovariano/citologia , Cultura Primária de Células , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras da Sinalização de Citocina/genéticaRESUMO
BACKGROUND: Polycystic ovary syndrome (PCOS) affects 5-10% of reproductive-aged women and is commonly associated with anovulatory infertility. Leukocytes, together with granulosa cells, may contribute to the pathogenesis of PCOS via their ability to secrete an array of cytokines implicated in follicle growth. The aim of this study was to examine leukocyte subtypes in follicular phase ovaries and to quantify cytokine and chemokine mRNA expression in follicular fluid cells obtained at the time of oocyte retrieval before IVF in women with and without PCOS. METHODS: Ovaries were immunostained for various leukocyte markers [CD3, CD4, CD14, CD15, CD45, CD45RA, CD45RO, CD57 and major histocompatibility complex (MHC) class II]. In addition, follicular fluid cells were subjected to quantitative RT-PCR to evaluate colony-stimulating factor-1 (CSF-1), granulocyte-macrophage (GM)-CSF, interleukins (IL-1beta, IL-6, IL-8 and IL-10), monocyte chemotactic protein (MCP-1) and tumour necrosis factor (TNFalpha) mRNA expression relative to beta-actin. RESULTS: CD45RO+ cells (activated/memory T lymphocytes) were reduced by 60% in the theca layer of follicles from PCOS women. The relative abundance of macrophages and neutrophils was unchanged. Cytokine and chemokine mRNA transcripts examined were not affected by PCOS status. There was an association between high BMI and high TNFalpha and low IL-6 mRNA expression in follicular cells. IL-6 expression was higher in women who subsequently achieved pregnancy. CONCLUSIONS: T lymphocytes potentially play a role in the local pathological mechanisms of PCOS. Further studies are required to identify their contribution to the aetiology of this common condition.
Assuntos
Antígenos CD/biossíntese , Quimiocinas/biossíntese , Citocinas/biossíntese , Líquido Folicular/citologia , Leucócitos/metabolismo , Ovário/citologia , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Adulto , Feminino , Fertilização in vitro , Líquido Folicular/metabolismo , Fase Folicular/metabolismo , Humanos , Gravidez , RNA Mensageiro/metabolismoRESUMO
Obesity is associated with a diverse set of metabolic disorders, and has reproductive consequences that are complex and not well understood. The adipose tissue-produced leptin has dominated the literature with regards to female fertility complications, but it is pertinent to explore the likely role of other adipokines--adiponectin and resistin--as our understanding of their biological functions emerge. Leptin influences the developing embryo, the functioning of the ovary and the endometrium, interacts with the release and activity of gonadotrophins and the hormones that control their synthesis. In this review such biological actions and potential roles of the adipokines leptin, adiponectin and resistin are explored in relation to female fertility and the complexity of the obese metabolic state.
Assuntos
Adiponectina/fisiologia , Tecido Adiposo/metabolismo , Infertilidade Feminina/metabolismo , Obesidade/metabolismo , Resistina/fisiologia , Animais , Glicemia/metabolismo , Feminino , Humanos , Insulina/metabolismo , Resistência à Insulina , Leptina/fisiologia , Camundongos , Camundongos Knockout , Modelos Animais , GravidezRESUMO
The luteinizing hormone (LH) surge initiates a cascade of proteolytic events that control ovulation. One of the genes induced by LH is the progesterone receptor (PR). Because mice with a mutant PR gene (PRKO) fail to ovulate and are infertile, we have used them as a model in which to determine PR target genes that might mediate the ovulatory process. The matrix metalloproteinases (MMPs: MMP2, MMP9, and MMP13) appear to be expressed in ovaries of PRKO mice in a manner similar to that in their wild-type littermates. However, the expression of two other types of proteases, cathepsin L (a member of the papain family) and ADAMTS-1 (A Disintegrin And Metalloproteinase with Thrombospondin-like motifs), are selectively induced in granulosa cells of preovulatory follicles by the LH surge. Maximal levels of these proteases are observed at 12-16 h after an LH surge, the time of ovulation. Furthermore, mRNAs encoding cathepsin L and ADAMTS-1 are reduced in the PRKO mice compared to their wild-type littermates. These novel observations indicate that these two proteases regulate some key step(s) controlling ovulation.
Assuntos
Endopeptidases , Ovulação/genética , Ovulação/fisiologia , Proteínas ADAM , Proteína ADAMTS1 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Catepsina L , Catepsinas/genética , Catepsinas/farmacologia , Ciclo-Oxigenase 2 , Cisteína Endopeptidases , Desintegrinas/genética , Desintegrinas/farmacologia , Feminino , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Isoenzimas/genética , Isoenzimas/farmacologia , Hormônio Luteinizante/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Metaloproteinases da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/farmacologia , Metaloendopeptidases/genética , Metaloendopeptidases/farmacologia , Camundongos , Camundongos Knockout , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Regiões Promotoras Genéticas , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , TransfecçãoRESUMO
Ovulation is a precisely timed process by which a mature oocyte is released from an ovarian follicle. This process is initiated by the pituitary surge of luteinizing hormone (LH), is temporally associated with transcriptional regulation of numerous genes, and is presumed to involve the synthesis and/or activation of specific proteases that degrade the follicle wall. The progesterone receptor (PR), a nuclear receptor transcription factor, is induced in granulosa cells of preovulatory follicles in response to the LH surge and has been shown to be essential for ovulation, because mice lacking PR fail to ovulate and are infertile. Using these mice as a model in which to elucidate PR-regulated genes in the ovulation process, we show that the matrix metalloproteinases MMP-2 and MMP-9 are not targets of PR during ovulation. In contrast, two other proteases, ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondin-like motifs) and cathepsin L (a lysosomal cysteine protease), are transcriptional targets of PR action. ADAMTS-1 is induced after LH stimulation in granulosa cells of preovulatory follicles and depends on PR. Cathepsin L is induced in granulosa cells of growing follicles by follicle-stimulating hormone, but the highest levels of cathepsin L mRNA occur in preovulatory follicles in response to LH in a PR-dependent manner. The identification of two regulated proteases in the ovary, together with their abnormal expression in anovulatory PR knockout mice, suggests that each plays a critical role in follicular rupture and represents a major advance in our understanding of the proteolytic events that control ovulation.
Assuntos
Catepsinas/genética , Desintegrinas/genética , Endopeptidases , Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases/genética , Ovário/fisiologia , Ovulação/genética , Progesterona/fisiologia , Receptores de Progesterona/fisiologia , Proteínas ADAM , Proteína ADAMTS1 , Animais , Catepsina L , Cisteína Endopeptidases , Feminino , Células da Granulosa/fisiologia , Hormônio Luteinizante/fisiologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Knockout , Fenótipo , Receptores de Progesterona/deficiência , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.
Assuntos
Monofosfato de Adenosina/metabolismo , Estradiol/farmacologia , Células da Granulosa/metabolismo , Receptores de Progesterona/genética , Sulfonamidas , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estradiol/análogos & derivados , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , Isoquinolinas/farmacologia , Hormônio Luteinizante/metabolismo , Hormônio Luteinizante/farmacologia , Dados de Sequência Molecular , Ovulação/efeitos dos fármacos , Ovulação/fisiologia , Alcamidas Poli-Insaturadas , Regiões Promotoras Genéticas , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
The proliferation and terminal differentiation of granulosa cells are critical for normal follicular growth, ovulation, and luteinization. Therefore, the in situ localization and hormonal regulation of cell cycle activators (cyclin D1, D2, and D3) and cell cycle inhibitors (p27Kip1 and p21Cip1) were analyzed in ovaries of mice and rats at defined stages of follicular growth and differentiation. Cyclin D2 mRNA was specifically localized to granulosa cells of growing follicles, while cyclin D1 and cyclin D3 were restricted to theca cells. In hypophysectomized (H) rats, cyclin D2 mRNA and protein were increased in granulosa cells by treatment with estradiol or FSH and were increased maximally by treatment with both hormones. In serum-free cultures of rat granulosa cells, cyclin D2 mRNA was rapidly elevated in response to FSH, forskolin, and estradiol, indicating that estradiol as well as cAMP can act directly and independently to increase cyclin D2 expression. The levels of p27Kip1 protein were not increased in response to estradiol or FSH. In contrast, when ovulatory doses of human CG (LH) were administered to hormonally primed H rats to stimulate luteinization, cyclin D2 mRNA and protein were rapidly decreased and undetectable within 4 h, specifically in granulosa cells of large follicles. Also in response to LH, the expression of the cell cycle inhibitor p27Kip1 was induced between 12 and 24 h (p21Cip1 was induced within 4 h) and remained elevated specifically in luteal tissue. A critical role for cyclin D2 in the hormone-dependent phase of follicular growth is illustrated by the ovarian follicles of cyclin D2-/- mice, which do not undergo rapid growth in response to hormones, but do express markers of FSH/LH action, cell cycle exit, and terminal differentiation. Collectively, these data indicate that FSH and estradiol regulate granulosa cell proliferation during the development of preovulatory follicles by increasing levels of cyclin D2 relative to p27Kip1 and that LH terminates follicular growth by down-regulating cyclin D2 concurrent with up-regulation of p27Kip1 and p21Cip1.
Assuntos
Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ciclinas/fisiologia , Células da Granulosa/citologia , Hormônios/farmacologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Supressoras de Tumor , Animais , Ciclina D2 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/genética , Inibidores Enzimáticos , Estradiol/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Folículo Ovariano/fisiologia , Ovário/química , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Aromatase (CYP19) mRNA is induced by follicle-stimulating hormone (FSH) in granulosa cells of preovulatory follicles and subsequently is rapidly diminished as a consequence of the luteinizing hormone (LH) surge. Primary cultures of rat granulosa cells were used to identify some of the cellular mechanisms by which FSH increases and LH decreases steady-state levels of aromatase mRNA. Induction of aromatase mRNA by FSH was increased by cycloheximide but was blocked by alpha-amanitin and the C-kinase activators gonadotropin-releasing hormone (GnRH) and phorbol 12-myristate 13-acetate (PMA). In contrast, the decrease in steady-state levels of aromatase mRNA by LH was mimicked by A-kinase (forskolin) and C-kinase (PMA or GnRH) activators. The decrease in aromatase mRNA was associated with decreased amounts of mRNA and protein for steroidogenic factor-1 (SF-1), a nuclear orphan receptor that binds and trans-activates the aromatase promoter, and with the A-kinase subunit type II (RII beta), which is required for mediating cAMP action in these cells. The down-regulation of aromatase, SF-1, and RII beta by each kinase activator and alpha-amanitin was prevented by cycloheximide when the drug was added in combination with the activator. If, however, cycloheximide was added 2 h after PMA (or LH), the drug did not prevent the rapid loss of mRNA. When granulosa cells were transfected with an aromatase CAT transgene, CAT activity was stimulated 10- to 20-fold by FSH and forskolin but not by PMA. Taken together, these results indicate that the A-kinase but not the C-kinase pathway can trans-activate the aromatase gene in immature granulosa cells, whereas the C-kinase, as well as A-kinase pathways, mimic the LH surge to decrease aromatase mRNA in preovulatory cells. By increasing degradation of aromatase mRNA and by inhibiting transcription, the LH surge rapidly terminates the granulosa cell pattern of gene expression while reprogramming the cells to express genes associated with ovulation and luteinization.
Assuntos
Aromatase/metabolismo , Regulação para Baixo , Hormônio Luteinizante/metabolismo , Ovário/enzimologia , Ovulação/metabolismo , Amanitinas/farmacologia , Animais , Aromatase/efeitos dos fármacos , Aromatase/genética , Gonadotropina Coriônica/farmacologia , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Hormônio Foliculoestimulante/farmacologia , Fatores de Transcrição Fushi Tarazu , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Proteínas de Homeodomínio , Hormônio Luteinizante/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator Esteroidogênico 1 , Testosterona/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.