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1.
Reprod Sci ; 27(1): 278-289, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32046393

RESUMO

Ganglioside GT1b is well-known for its role in cytokine production and in activating epidermal growth factor receptor (EGFR)-mediated signaling pathways in cancer cells. However, there are no reports that clearly elucidate the role of GT1b in EGFR-mediated signaling pathways in porcine oocytes during the process of in vitro maturation (IVM). In this study, we investigated the role of GT1b in EGFR-mediated activation of the ERK1/2 pathway in porcine cumulus-oocyte complexes (COCs) at 44 h of IVM. Our data show that expression of the ST3GAL2 protein significantly increased in porcine COCs at 44 h irrespective of treatment with EGF. Meiotic maturation and mRNA levels of factors (HAS2, TNFAIP6, and PTX3) related to cumulus cell expansion significantly increased in COCs treated with 2 µM GT1b during IVM in the absence of EGF. They also increased in COCs treated with EGF/GT1b as compared to that in the other groups. Interestingly, protein levels of EGFR, phospho-EGFR, ERK1/2, and phospho-ERK1/2 dramatically increased in COCs treated with EGF/GT1b. Moreover, the rate of fertilization and the developmental competence of blastocyst were significantly higher in EGF/GT1b-treated COCs. Taken together, these results suggest that exogenous GT1b improves meiotic maturation and cumulus cell expansion in porcine COCs via activation of EGFR-mediated ERK1/2 signaling.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Receptores ErbB/metabolismo , Gangliosídeos/farmacologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Células do Cúmulo/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oócitos/metabolismo , Suínos
2.
Dev Reprod ; 21(4): 407-415, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29354786

RESUMO

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)-related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: 32.5±10.1% vs control: 77.8±5.3%). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, 200 µM) and melatonin (0.1 µM), in BIP/ GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, p-eIF2α, eIF2α, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

3.
J Reprod Dev ; 62(3): 249-55, 2016 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-26860251

RESUMO

Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs.


Assuntos
Blastocisto/citologia , Gangliosídeo G(M1)/análogos & derivados , Oócitos/citologia , Animais , Apoptose , Núcleo Celular/metabolismo , Células Cultivadas , Fase de Clivagem do Zigoto , Células do Cúmulo/citologia , Desenvolvimento Embrionário , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Feminino , Fertilização , Gangliosídeo G(M1)/fisiologia , Meiose , Metáfase , Ovário/metabolismo , Sialiltransferases/metabolismo , Suínos , beta-Galactosídeo alfa-2,3-Sialiltransferase
4.
J Vet Sci ; 17(3): 413-9, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26726020

RESUMO

Pelvic inflammatory disease (PID), which is one of the most problematic complications experienced by women with sexually transmitted diseases, frequently causes secondary infections after reproductive abnormalities in veterinary animals. Although the uterus is self-protective, it becomes fragile during periods or pregnancy. To investigate PID, bacteria or lipopolysaccharide (LPS) extracted from gram negative bacteria has been used to induce the disease in several animal models. However, when LPS is applied to the peritoneum, it often causes systemic sepsis leading to death and the PID was not consistently demonstrated. Hydrochloric acid (HCl) has been used to induce inflammation in the lungs and stomach but not tested for reproductive organs. In this study, we developed a PID model in mice by HCl and LPS sequential intracervical (i.c.) administration. The proinflammatory cytokines, interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α, were detected in the mouse uterus by western blot analysis and cytokine enzyme-linked immunosorbent assay after HCl (25 mg/kg) administration i.c. followed by four LPS (50 mg/kg) treatments. Moreover, mice exhibited increased infiltration of neutrophils in the endometrium and epithelial layer. These results suggest that ic co-administration of HCl and LPS induces PID in mice. This new model may provide a consistent and reproducible PID model for future research.


Assuntos
Citocinas/metabolismo , Modelos Animais de Doenças , Ácido Clorídrico , Inflamação/veterinária , Lipopolissacarídeos , Camundongos , Doença Inflamatória Pélvica/induzido quimicamente , Animais , Western Blotting/veterinária , Feminino , Humanos , Inflamação/induzido quimicamente , Camundongos Endogâmicos C57BL , Útero/imunologia , Útero/patologia
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