Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27
Filtrar
1.
Reprod Med Biol ; 23(1): e12571, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38510925

RESUMO

Purpose: LH induces the expression of EGF-like factors and their shedding enzyme (ADAM17) in granulosa cells (GCs), which is essential for ovulation via activation of the ErbB-ERK1/2 pathway in cumulus cells (CCs). Neurotensin (NTS) is reported as a novel regulator of ovulation, whereas the NTS-induced maturation mechanism in oocytes remains unclear. In this study, we focused on the role of NTS in the expression of EGF-like factors and ErbBs, and ADAM17 activity, during oocyte maturation and ovulation in mice. Methods: The expression and localization in GC and CC were examined. Next, hCG and NTS receptor 1 antagonist (SR) were injected into eCG-primed mice, and the effects of SR on ERK1/2 phosphorylation were investigated. Finally, we explored the effects of SR on the expression of EGF-like factors and ErbBs, and ADAM17 activity in GC and CC. Results: NTS was significantly upregulated in GC and CC following hCG injection. SR injection suppressed oocyte maturation and ERK1/2 phosphorylation. SR also downregulated part of the expression of EGF-like factors and their receptors, and ADAM17 activity. Conclusions: NTS induces oocyte maturation through the sustainable activation of the ERK1/2 signaling pathway by upregulating part of the EGF-like factor-induced pathway during oocyte maturation in mice.

2.
Reprod Med Biol ; 22(1): e12529, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37546178

RESUMO

Purpose: Since the developmental competence of oocytes cultured after in vitro maturation (IVM) is low, it is necessary to improve the IVM method for efficient offspring production. In this study, we revealed that transferrin (TF)-Fe3+ was accumulated in follicular fluid with increasing the follicular diameter, and that TF receptor (TFR1) was localized in granulosa cells of pig. Thus, we hypothesized that TF-Fe3+ would be a factor in the induction of developmental competence of porcine oocytes. Methods: To mimic the follicular development environment, cumulus-oocyte complexes (COCs) were cultured in pre-IVM medium (low dose of FSH) without or with Holo-TF (monoferric or diferric TF) or Apo-TF (non-iron bond TF). After pre-IVM without or with Holo-TF, COCs were cultured in IVM medium (high dose of FSH and EGF) without or with Holo-TF. Results: Cultivation with Holo-TF increased the expression of follicular development maker (Cyp19a1 and Ccnd2), E2 production, and proliferative activity of cumulus cells, whereas cultivation with Apo-TF did not show these positive effects. The treatment with Holo-TF during pre-IVM, but not during IVM, dramatically induced oocyte maturation with increasing the blastocyst rate. Conclusion: We succeeded in showing for the first time that the cultivation with Holo-TF in pre-IVM can produce embryos in pig with high efficiency.

3.
Mol Hum Reprod ; 27(7)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34057472

RESUMO

During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus-oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18ß-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Atresia Folicular/efeitos dos fármacos , Hidrocortisona/farmacologia , 11-beta-Hidroxiesteroide Desidrogenases/biossíntese , 11-beta-Hidroxiesteroide Desidrogenases/genética , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Indução Enzimática , Feminino , Hormônio Foliculoestimulante/fisiologia , Líquido Folicular/química , Regulação da Expressão Gênica , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Hidrocortisona/análise , Hidrocortisona/fisiologia , Metirapona/farmacologia , Modelos Biológicos , Progesterona/metabolismo , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/genética , Esteroide 11-beta-Hidroxilase/biossíntese , Esteroide 11-beta-Hidroxilase/genética , Esteroide 21-Hidroxilase/biossíntese , Esteroide 21-Hidroxilase/genética , Suínos
4.
J Reprod Dev ; 66(5): 475-483, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-32713881

RESUMO

Iron is important for many cellular functions, including ATP synthesis and cell proliferation. Insufficient of iron in the diet causes iron deficiency anemia (IDA), which often occurs in people living in the world. Since 50% of women with IDA show amenorrhea, the relationship of between iron deficiency and reproductive function was assessed using mice fed a low Fe diet (LFD). The estrous cycle in the LFD mice was blocked at diestrus, which impair follicle development, and fertility. Further, even LFD mice were injected with exogenous pregnant mare serum gonadotropin (PMSG), follicular development was ceased at the secondary follicle stage, and preovulatory follicles were not observed. Amount of ATP decreased in the ovary of the LFD mice, and expression of follicle development markers (Fshr, Cyp19a1, Ccnd2) and estradiol-17ß (E2) was low level compared to levels mice fed a normal diet. Feeding a normal diet with sufficient iron to the LFD mice for an additional 3 weeks completely reversed absence the effects of iron insufficient on the estrous cycle and infertility. Thus, iron restriction depresses ovary functions, especially follicular development from secondary follicle to antral follicles and infertility. These effects are fully reversible by supplementation of a normal diet containing iron.


Assuntos
Ferro/química , Folículo Ovariano/metabolismo , Trifosfato de Adenosina/metabolismo , Anemia Ferropriva , Animais , Aromatase/metabolismo , Peso Corporal , Ciclina D2/metabolismo , Estradiol/sangue , Estrogênios/metabolismo , Ciclo Estral/efeitos dos fármacos , Feminino , Hormônio Foliculoestimulante/metabolismo , Gonadotropinas Equinas/farmacologia , Ferro/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovulação/efeitos dos fármacos , Progesterona/sangue , RNA Mensageiro/metabolismo , Receptores do FSH/metabolismo
5.
Biol Reprod ; 95(4): 7, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27535959

RESUMO

The in vitro maturation (IVM) technique is beneficial for producing animal offspring, but the blastocyst rate is low after IVM. In this technique, cumulus-oocyte complexes (COCs) are collected from medium size follicles. The follicles are ultimately selected as large dominant follicles or atretic follicles; therefore it is possible that the COCs collected using IVM are contaminated by follicles that will develop into large follicles and induce atresia. In the dominant follicles, estradiol-17beta and progesterone induce the differentiation of follicular somatic cells which exhibit the ability to respond to ovulation during follicular development. Thus, we hypothesized that changes in the hormonal condition of healthy follicles are essential for oocyte maturation during IVM. In this study, we performed a comparative analysis of the steroid hormone concentrations in non-vascularized follicles (NVFs) and vascularized follicles (VFs). The estradiol-17beta concentration increased in medium VFs, whereas the level was low in NVFs of the same size. The progesterone concentration increased with large follicular size in VFs, but the level remained low in follicles of any size among NVFs. To improve the oocyte quality derived from NVFs, NVF COCs were cultured with FSH alone or FSH under theVF hormonal conditions. Cultivation under the VF hormonal conditions dramatically improved the proliferation and survival of cumulus cells, meiotic maturation of oocytes, cumulus expansion, and blastocyst rate following in vitro fertilization. Thus, the cultivation of NVF COCs under VF hormonal conditions improves the developmental potential to the blastocyst stage by NVF oocytes.

6.
Reprod Biol ; 23(1): 100710, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36470010

RESUMO

Postpartum endometritis is known to be associated with ovarian dysfunction in cows. Lipopolysaccharide (LPS) generated by Gram-negative bacteria is recognized by toll-like receptor 4 (TLR4), which leads to an inflammatory response by the generation of cytokines such as tumor necrosis factor-α (TNF-α) and interleukins. In this study, we investigated the effect of endometrial LPS on granulosa cell functions during early follicular development in cows. Uteri and follicles were obtained from a slaughterhouse and classified into either clinical endometritis (CE) or normal groups by vaginal mucus test. TLR4 mRNA and protein in normal cows were expressed in granulosa cells collected from follicles measuring 1-3 and 4-7 mm in a diameter, respectively. LPS content in endometrium and follicular fluid of CE cows was significantly higher than that in normal cows. Compared to normal cows, CE cows showed lower expression of follicular development markers (FSHR, CYP19A1, CCND2, and LHCGR) in granulosa cells, lower estradiol-17ß concentrations in follicular fluid, and lower granulosa cell proliferation. CE contraction significantly increased cytokine expressions (TNF, IL-1A, and IL-1B) in granulosa cells and suppressed apoptosis of granulosa cells compared to normal cows. LPS significantly suppressed the expression of follicular development markers and the production of estradiol-17ß in granulosa cells and reduced granulosa cells proliferation compared to cells cultured without LPS. LPS significantly increased cytokine expressions and suppressed granulosa cell apoptosis. Thus, the present results suggest that the existence of LPS in developing follicles is one of the causes of ovarian quiescence in cows.


Assuntos
Endometrite , Lipopolissacarídeos , Feminino , Humanos , Bovinos , Animais , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Citocinas/metabolismo , Endometrite/metabolismo , Células da Granulosa , Estradiol/metabolismo , Proliferação de Células
7.
J Reprod Dev ; 58(5): 510-4, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23124701

RESUMO

In mammalian preovulatory follicles, LH stimulation induces the ovulation process, including follicular wall rupture, granulosa cell luteinization, cumulus cell expansion and meiotic maturation of the oocyte. The receptor for LH (LHCGR) is expressed mostly in granulosa cells of preovulatory follicles, and is rarely expressed in cumulus cells or oocytes. The expression level in granulosa cells dramatically decreases after ovulation stimuli. Thus, a potent factor(s) secreted by granulosa cells is required to stimulate not only granulosa cells via an autocrine manner but also cumulus cells and/or oocytes via a paracrine pathway. Recent reports showed that granulosa cells and cumulus cells express EGF-like factors that activate the EGF receptor (EGFR)-mitogen-activated protein kinase3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase1/2 (ERK1/2)) pathway in both cell types. EGF-like factors are composed of a signal sequence, transmembrane domain and EGF domain, suggesting that release of the EGF domain by a specific enzyme is essential for interaction with the EGFR to induce the ovulation process. In our studies, TACE/ADAM17, which is known to be a proteolytic enzyme of EGF-like factors in many types of tissue, was found to be expressed in FSH/LH-stimulated granulosa cells and cumulus cells together with activation of the EGFR-MAPK3/1 pathway. When TACE/ADAM17 activity was decreased by a specific inhibitor or siRNA technique, granulosa cell luteinization, cumulus expansion and oocyte maturation were suppressed in an in vitro culture. Thus, TACE/ADAM17 is one of the key genes expressed in both granulosa cells and cumulus cells for induction of the ovulation process.


Assuntos
Células do Cúmulo/metabolismo , Fator de Crescimento Epidérmico/análogos & derivados , Receptores ErbB/agonistas , Células da Granulosa/metabolismo , Sistema de Sinalização das MAP Quinases , Ovulação/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteólise
8.
Biol Reprod ; 85(5): 1073-82, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21778143

RESUMO

During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.


Assuntos
Comunicação Celular/fisiologia , Células do Cúmulo/metabolismo , Dinoprostona/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oócitos/metabolismo , Animais , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Técnicas In Vitro , Modelos Animais , Nitrobenzenos/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores do FSH/metabolismo , Receptores de Prostaglandina E/metabolismo , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Suínos
9.
Endocrinology ; 162(11)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34431998

RESUMO

In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulate cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared with the control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum; however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.


Assuntos
Colesterol/biossíntese , Células da Granulosa/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Ovulação/efeitos dos fármacos , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Ovulação/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo
10.
Anim Sci J ; 91(1): e13493, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33314533

RESUMO

Although successful fertilization is completed by only 150 sperm in the pig oviduct, more than 50,000 sperms are required to achieve a fertilization rate of more than 70% by pig in vitro fertilization (IVF). In this study, to improve the efficiency of pig IVF, the effects of hypoxic conditions and treatment with creatine and methyl-beta cyclodextrin (MßCD) on the glycolytic pathway were investigated. Under low O2 conditions, zig-zag motility was strongly induced within 30 min; however, the induction disappeared at 60 min. Although caffeine suppressed zig-zag motility under low O2 conditions, creatine induced and sustained zig-zag motility until 120 min. Additionally, pretreatment with MßCD for 15 min greatly enhanced zig-zag motility via ATP production in sperm incubated with creatine under low O2 conditions. Sperm pretreated with MßCD were used for IVF in medium containing creatine under low O2 conditions. A fertilization rate of approximately 70% was achieved with only 1.0 x 104 sperms/mL, and there were few polyspermic embryos. Therefore, our novel method was beneficial for efficient production of pig embryos in vitro. Moreover, the zig-zag motility may be a novel movement which boar capacitated sperm exhibit in the culture medium.


Assuntos
Anaerobiose/fisiologia , Creatina/farmacologia , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Fertilização/efeitos dos fármacos , Motilidade dos Espermatozoides , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/fisiologia , Suínos/fisiologia , beta-Ciclodextrinas/farmacocinética , Animais , Sinergismo Farmacológico , Ejaculação/fisiologia , Feminino , Fertilização/fisiologia , Masculino , Interações Espermatozoide-Óvulo/fisiologia , beta-Ciclodextrinas/farmacologia
11.
Reproduction ; 136(1): 9-21, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18456902

RESUMO

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.


Assuntos
Células do Cúmulo/metabolismo , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Oogênese/genética , Suínos/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Western Blotting/métodos , Técnicas de Cultura de Células , Gonadotropina Coriônica/farmacologia , Células do Cúmulo/efeitos dos fármacos , Primers do DNA/genética , Relação Dose-Resposta a Droga , Estradiol/análise , Estradiol/farmacologia , Feminino , Líquido Folicular/química , Expressão Gênica/efeitos dos fármacos , Gonadotropinas Equinas/farmacologia , Células da Granulosa/metabolismo , Oogênese/efeitos dos fármacos , Progesterona/análise , Progesterona/farmacologia , RNA Mensageiro/análise , Receptores do FSH/genética , Receptores do LH/genética , Receptores de Progesterona , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superovulação , Testosterona/análise
12.
Mol Endocrinol ; 21(10): 2487-502, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17595323

RESUMO

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.


Assuntos
Quimiocinas/metabolismo , Exocitose/genética , Regulação da Expressão Gênica , Ovulação/genética , Proteína 25 Associada a Sinaptossoma/genética , Animais , Gonadotropina Coriônica/farmacologia , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Camundongos , Camundongos Mutantes , Ovulação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Proteína 25 Associada a Sinaptossoma/antagonistas & inibidores
13.
J Vet Med Sci ; 80(2): 368-374, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29269703

RESUMO

BNIP3 (BCL2/adenovirus E1B nineteen kilodalton interacting protein-3), a member of the BCL2 family, is activated under hypoxic conditions and induces apoptosis or mitochondrial autophagy for adapting cells to hypoxia. The physiological roles of BNIP3 in the mammalian ovary are still unclear. In order to understand the role of BNIP3 in the bovine ovary, we examined its mRNA and protein expressions of BNIP3 in follicular granulosa cells and corpus luteum (CL). BNIP3 mRNA and protein expressions in granulosa cells from large follicles (>10 mm) at the follicular stage were much higher than those in small follicles (2-8 mm). BNIP3 mRNA and protein expressions in the CL peaked at the early luteal stage. In bovine granulosa cells cultured for 6 hr under hypoxia (3% O2) and normoxia (20% O2), BNIP3 mRNA expression was higher under hypoxia. These results of the present study suggest that BNIP3 has some roles in luteal formation in the bovine ovary, and that the highly expressed BNIP3 in the granulosa cells from large follicles at the follicular stage is related to the roles of BNIP3 in the luteal formation.


Assuntos
Bovinos/metabolismo , Hipóxia Celular/fisiologia , Corpo Lúteo/metabolismo , Células da Granulosa/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Células Cultivadas , Corpo Lúteo/fisiologia , Ciclo Estral/fisiologia , Feminino , Expressão Gênica , Células da Granulosa/fisiologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise
14.
Endocrinology ; 148(12): 6164-75, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17901238

RESUMO

The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNFalpha-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNFalpha-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.


Assuntos
Proteínas ADAM/metabolismo , Células do Cúmulo/metabolismo , Receptores ErbB/metabolismo , Oócitos/metabolismo , Proteínas ADAM/genética , Proteína ADAM17 , Anfirregulina , Animais , Western Blotting , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Família de Proteínas EGF , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Epirregulina , Receptores ErbB/genética , Feminino , Flavonoides/farmacologia , Hormônio Foliculoestimulante/farmacologia , Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Hormônio Luteinizante/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Quinazolinas , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Transfecção , Tirfostinas/farmacologia
15.
Reprod Biol Endocrinol ; 4: 4, 2006 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-16457731

RESUMO

BACKGROUND: The objective of this study was to investigate whether the steroid hormone(s) secreted from cumulus-oocyte complexes (COCs) is a prerequisite for bovine oocyte maturation and cumulus expansion using aminoglutethimide (AGT), a P450 cholesterol side-chain cleavage inhibitor. METHODS: In experiment 1, COCs were cultured in maturation medium with various concentrations of AGT for 22 h to determine the effective concentration of AGT to inhibit steroid hormone secretion, meiotic maturation and cumulus expansion. In experiment 2, COCs were cultured in conditioned medium (CM) and TCM-199 medium with or without 10 mM AGT to check whether steroid hormones secreted from COCs were responsible for oocyte maturation and cumulus expansion. Experiments 3 and 4 were carried out to determine whether exogenous progesterone or estradiol-17beta was able to overcome the inhibitory effects of AGT on oocytes maturation and cumulus expansion. COCs cultured in 10 mM AGT-containing medium supplemented with various concentrations of progesterone or estradiol-17beta for 22 h were examined for oocyte maturation and cumulus expansion. RESULTS: Experiment 1 showed that a concentration of 10 mM AGT in medium was sufficient to block steroid hormone secretion, oocyte maturation and cumulus expansion, and that these inhibitory effects were fully reversible. In experiment 2, the addition of 10 mM AGT to CM did not significantly prevent oocyte maturation and cumulus expansion, implying that CM contains the steroid hormone(s) secreted from COCs, which are closely associated with oocyte maturation and cumulus expansion. The results in experiments 3 and 4 demonstrated that the addition of any concentration of progesterone or estradiol-17beta in the medium did not reduce the inhibitory effects of AGT on oocyte maturation and cumulus expansion. CONCLUSION: Our results indicate that bovine oocytes surrounded by cumulus cells are prevented from maturation and cumulus expansion through the inhibition of steroid secretion due to AGT, and that these inhibitory effects of AGT on oocyte maturation and cumulus expansions can not be overcome by the addition of either progesterone or estradiol-17beta in the medium. These observations suggest that some steroid hormone(s) other than P4 and E2 secreted from bovine COCs is essential for their meiotic maturation and cumulus expansion.


Assuntos
Hormônios Esteroides Gonadais/fisiologia , Oócitos/crescimento & desenvolvimento , Aminoglutetimida/administração & dosagem , Aminoglutetimida/farmacologia , Animais , Inibidores da Aromatase/administração & dosagem , Inibidores da Aromatase/farmacologia , Bovinos , Estradiol/farmacologia , Feminino , Hormônios Esteroides Gonadais/metabolismo , Oócitos/efeitos dos fármacos , Progesterona/farmacologia
16.
Endocrinology ; 146(1): 186-94, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15459117

RESUMO

Progesterone is produced from cholesterol in cumulus cells during meiotic resumption of porcine oocytes. In follicular cells, it has been shown that exogenous lipoprotein-bound cholesterol ester can be used for steroid hormone production. However, in serum-free medium, progesterone is also secreted by FSH- and LH-stimulated cumulus-oocyte complexes, suggesting that progesterone could be produced from de novo synthesized cholesterol in cumulus cells. In the present study, we investigated the expression of Delta14-reductase and Delta7-reductase, which are the members of the superfamily that converts acetyl-CoA to cholesterol in cumulus cells. The expression of both genes was analyzed by RT-PCR. Both Delta14-reductase mRNA and Delta7-reductase mRNA in cumulus cells, cultured until 4 h, were under the level of detection limit. In response to gonadotropins, both mRNA levels were dramatically up-regulated, reaching a maximum at 20 h. To clarify the role of induced enzymes in cumulus cells, cumulus-oocyte complexes were cultured with either Delta14-reductase inhibitor, AY9944-A-7, or Delta7-reductase inhibitor, BM15.766. The results indicated that these inhibitors significantly suppressed the progesterone production in cumulus cells and meiotic progression of oocytes. The inhibitory effects reached a maximum at 1 microM AY9944-A-7 or 20 microM BM15.766. The addition of 20 ng/ml progesterone overcame the inhibitory effects of both drugs on meiotic resumption of oocytes. These results imply that gonadotropin-induced expression and function of Delta14-reductase and Delta7-reductase in cumulus cells contribute to oocyte meiotic resumption via a progesterone-dependent pathway.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/farmacologia , Meiose/fisiologia , Oócitos/citologia , Folículo Ovariano/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Meios de Cultura/química , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Meiose/efeitos dos fármacos , Dados de Sequência Molecular , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Progesterona/biossíntese , Progesterona/farmacologia , Suínos , Fatores de Tempo , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/administração & dosagem , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/farmacologia
17.
Endocrinology ; 145(10): 4603-14, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15231699

RESUMO

ADAMTS-1, a member of the A disintegrin and metalloproteinase family of proteases, is expressed in rodent follicles via progesterone receptor (PR)-dependent pathways. However, the functional relationship between ADAMTS-1 expression and PR has not been studied extensively in other species. In the present study, we investigated the time-dependent changes in ADAMTS-1 expression in cumulus cells of porcine cumulus-oocyte complexes (COCs), and the roles of ADAMTS-1 in cumulus expansion during in vitro maturation of oocytes. ADAMTS-1 message was not detected in cumulus cells at the time of collection from the follicles. In response to gonadotropins, ADAMTS-1 mRNA was dramatically up-regulated and reached a maximum at 20 h. The level of mature ADAMTS-1 protein increased in a time-dependent manner with a maximum level at 40 h. The induction of ADAMTS-1 mRNA and protein was significantly decreased by the addition of PR antagonist RU486 to the cultures. However, RU486 did not affect the expression of ADAMTS-4 or factors that had been reported to be required for COC expansion (TSG-6, versican, HA synthase-2). COCs cultured with FSH and LH for 40 h exhibited prominent cumulus expansion. The expansion was reduced significantly by the addition of either RU486 or Galardin, a broad-spectrum matrix metalloproteinase inhibitor. These results suggest that the expression and induction of ADAMTS-1 through receptor-mediated action of progesterone in cumulus cells is one of the essential requirements for gonadotropin-regulated cumulus expansion of porcine COCs.


Assuntos
Desintegrinas/metabolismo , Regulação para Baixo , Antagonistas de Hormônios/farmacologia , Metaloendopeptidases/metabolismo , Mifepristona/farmacologia , Oócitos/citologia , Folículo Ovariano/citologia , Receptores de Progesterona/antagonistas & inibidores , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Dipeptídeos/farmacologia , Feminino , Metaloproteases/antagonistas & inibidores , Folículo Ovariano/metabolismo , Inibidores de Proteases/farmacologia , Receptores de Progesterona/metabolismo , Suínos , Fatores de Tempo
18.
J Mol Endocrinol ; 33(1): 209-25, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15291754

RESUMO

The present study aimed to investigate progesterone receptor (PR) gene expression in cumulus cells and their roles during meiotic resumption of porcine oocytes. The amount of PR-A or PR-B mRNA was analyzed by RT-PCR using primer sets for the PR-B region or the PR-A/B common region. The level of PR-B mRNA in cumulus cells was up-regulated by FSH and LH during the first 8 h of cultivation but the level significantly decreased at 12 h. However, a high level of total PR mRNA was maintained up to a cultivation period of 20 h. The level of PR-B protein in cumulus cells reached its maximum at 4 to 12 h, whereas PR-A predominated in cumulus cells of cumulus-oocyte complexes (COCs) at 20 h. Accompanying the shift in expression of PR isoforms, progesterone production in cumulus cells was significantly increased, and both the proliferative activity of cumulus cells during a 10- to 20-h cultivation period and the level of connexin-43, a major component of the gap junction, in cumulus cells significantly decreased. When COCs were cultured with FSH and LH for 10 h and then further cultured with additional RU486, there was a significant suppression in the shift in PR isoforms and in progesterone production, a loss of proliferative activity, and a decrease in connexin-43 mRNA in cumulus cells. Moreover, treatment with RU486 after 10-h cultivation of COCs inhibited the meiotic resumption of oocytes and cumulus cell expansion. These results suggest that the induction of PR isoforms in cumulus cells and their binding to progesterone appear to impact on proliferation and differentiation in a time-dependent manner, and the shift from PR-B to PR-A may help mediate certain events.


Assuntos
Meiose/fisiologia , Oócitos/citologia , Isoformas de Proteínas/fisiologia , Receptores de Progesterona/fisiologia , Animais , Sequência de Bases , DNA Complementar , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica , Hormônio Luteinizante/farmacologia , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Isoformas de Proteínas/genética , Receptores de Progesterona/genética , Homologia de Sequência do Ácido Nucleico , Suínos
19.
Mol Cell Endocrinol ; 209(1-2): 43-50, 2003 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-14604815

RESUMO

It has been reported that LH receptor (LHR) mRNA is not detected in cumulus cells of porcine cumulus-oocyte complexes (COCs) just after collection from small antral follicles. The present study showed that the formation of LHR in cumulus cells was up-regulated by the cultivation with 20 ng/ml FSH. When the newly synthesized receptors were stimulated by 1.0 microg/ml LH, significantly higher levels of cAMP and progesterone production in cumulus cells were observed as compared with those of COCs cultured with FSH. A loss of proliferative activity of cumulus cells was induced by the additional LH to FSH-containing medium; however, the inhibitory effect was overcome by progesterone receptor antagonist RU486. Furthermore, the addition of LH also accelerated ongoing GVBD in cumulus cells-enclosed oocytes. These results revealed that during in vitro meiotic maturation of porcine COCs, progesterone secreted by FSH- and LH-stimulated cumulus cells reduced proliferative activity of cumulus cells; the changes of cumulus cells might be involved in inducing meiotic resumption of porcine oocytes.


Assuntos
Hormônio Luteinizante/farmacologia , Oócitos/citologia , Folículo Ovariano/citologia , Animais , Diferenciação Celular , Divisão Celular , AMP Cíclico , Relação Dose-Resposta a Droga , Feminino , Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/genética , Luteolíticos/farmacologia , Meiose , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Progesterona/análise , Receptores do LH/biossíntese , Receptores do LH/genética , Suínos , Fatores de Tempo , Regulação para Cima
20.
Endocrinology ; 155(3): 1080-90, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24424050

RESUMO

During in vitro maturation of porcine cumulus cell-oocyte complexes and in vitro luteinization of porcine granulosa cells, FSH induces the expression of the protease TNFα-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) and the epidermal growth factor (EGF)-like factors, which activate the EGF receptor (EGFR)-MAPK3/1 pathway in both cumulus and granulosa cells. FSH is known to activate not only protein kinase A and p38MAPK pathways in both cell types but also activates protein kinase C (PKC). Because PKC-induced association of cellular-Sarcoma (c-Src) and TACE/ADAM17 is required for TACE/ADAM17 enzyme activation in some cancer cells, we hypothesized that PKC and c-Src impact TACE/ADAM17-mediated activation of EGFR signaling pathway in porcine granulosa and cumulus cells. When granulosa cells or cumulus cell-oocyte complexes were cultured with FSH, PKC activity and c-Src phosphorylation increased and were associated with increased TACE/ADAM17 enzyme activity. The PKC inhibitor calphostin C (CalC) and the c-Src inhibitor (4 amino 5 (4 chlorophenyl) 7 (t butyl)pyrazolo[3,4 d]pyrimidine [PP2]) suppressed TACE/ADAM17 enzyme activity, whereas these inhibitors did not affect Tace/Adam17 mRNA expression. Immunoprecipitation analysis showed that FSH mediated the association of c-Src with TACE/ADAM17 via a PKC-dependent mechanism. Either CalC or PP2 suppressed EGFR downstream signaling pathway (MAPK3/1) in these ovarian cell types and reduced cumulus expansion, meiotic maturation of oocytes, and progesterone production. The negative effects were overcome by the addition of amphiregulin. Collectively, these results indicate that activation of TACE/ADAM17 via a PKC-induced c-Src-dependent manner mediates proteolytic activation of the EGF-like factors that are involved in the induction of granulosa cell differentiation, cumulus expansion, and meiotic maturation of porcine oocytes in vitro.


Assuntos
Proteínas ADAM/metabolismo , Regulação Enzimológica da Expressão Gênica , Células da Granulosa/enzimologia , Oócitos/citologia , Ovário/enzimologia , Proteína Quinase C/metabolismo , Proteína ADAM17 , Animais , Diferenciação Celular , Células Cultivadas , Células do Cúmulo/citologia , Ativação Enzimática , Feminino , Hormônio Foliculoestimulante/metabolismo , Meiose , Naftalenos/química , Oócitos/enzimologia , Fosforilação , Progesterona/metabolismo , Ligação Proteica , Pirimidinas/química , Suínos , Fator de Necrose Tumoral alfa/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA