甲状腺激素受体α在猪有腔卵泡发育和孤雌激活胚胎发育过程中表达模式

Expression Pattern Analysis of Thyroid Hormone Receptor Alpha in Pig Antral Follicles and Parthenogenetic Early Embryos

本研究旨在探讨猪不同发育时期的卵泡甲状腺激素受体α(TRα)蛋白的表达规律,以及体外培养猪卵母细胞和早期胚胎中TRα的表达模式。采用化学发光免疫分析法(CLIA)测定了不同直径大小猪卵泡甲状腺激素(THs)的含量,运用免疫组化法对猪卵巢TRα进行定位染色,应用免疫荧光技术和QRT-PCR技术分别检测体外培养猪卵母细胞和孤雌激活早期胚胎TRα蛋白的表达定位和TRαmRNA水平表达变化规律。结果表明:猪卵泡中T3含量随着卵泡直径的增大呈现先下降后上升的趋势。免疫组化染色结果显示,阳性信号主要见于GC、TC及卵母细胞。随着卵泡的发育,TRα阳性细胞数量呈增长趋势,原始卵泡、初级卵泡、次级卵泡和成熟卵泡之间阳性细胞数量差异显著。QRT-PCR结果显示,猪TRα基因在不同成熟培养阶段的COCs均有表达,在孤雌激活早期胚胎1-细胞、2-细胞和4-细胞胚胎期显著高于8-细胞胚胎期、桑椹期和囊胚期(P【0.05)。免疫荧光观察到在猪体外成熟的COCs不同发育阶段颗粒细胞均表达TRα蛋白,在24~36h卵丘颗粒细胞表达阳性最强。TRα蛋白在猪孤雌激活早期胚胎不同阶段中均有表达,且8-细胞、桑椹胚和囊胚的阳性信号较强。结果提示,不同直径猪卵泡液中均存在THs,不同成熟阶段COCs和早期胚胎中TRα均呈现表达,推测THs可能通过与TRα结合而参与调控猪卵泡发生和早期胚胎发育。

Expression pattern of TR alpha in different developmental stages of porcine follicules,in vitro matured oocytes and early embryos were systemically investigated in the present study.The THs concentration of the different diameter sizes of pig follicular was determined by chemiluminescence immunoassay(CLIA)technique.And the localization of TR alpha in pig ovary were determined by immunohistochemical techniques.The expression pattern of TR alpha in pig oocytes and pre-implantation embryos were determined with immunofluorescence and QRT-PCR.The results showed that T3concentration in pig follicles was rised at first and then declined following the increase of follicle diameter.TR alpha-immunoreactive substance was mainly distributed over granular cells,theca cells and oocytes.The quantity of the TR alpha-immunoreactive andTR alpha-immunoreactive cells were increased following the development of follicles.The results of QRT-PCR showed that TR alpha was expressed through out the process of in vitro matured oocytes,and the relative expression level in 1-cell,2-cell and 4-cell stages were significant higher than that of 8-cell morula and blastula stage.Immunohistochemical analysis results showed that TRaclearly expressed following the increase of follicles,and reached peak at 24-36h.TR alpha protein expressed in different stages of early embryos,and its expression level was upregulated at8-cell,morula and blastocyst stage.THs existed in the pig follicle fluid,TR alpha expressed in different stages of COCs and early embryos,THs may be affected pig folliculogenesis and early embryonic development by banding with the TR alpha.