【作者】 耿利英；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 本研究应用细胞培养、质粒提取、脂质体转染、RT-PCR(反转录PCR)、TUNEL(脱氧核糖核苷酸末端转移酶介导的缺口末端标记法)、RIA(放射免疫测定)和流式细胞分类等技术,利用本实验室构建的卵泡抑制素α亚基片段基因重组质粒即pEGISI,转染不同大小的牛卵泡颗粒细胞及卵母细胞,研究pEGISI在颗粒细胞、卵母细胞中的表达以及对颗粒细胞激素分泌、细胞增殖和细胞凋亡以及对卵母细胞成熟、体外受精和胚胎发育的影响,以探讨卵泡抑制素对卵泡发育的局部调节作用及作用机制。主要内容如下:1.pEGISI转染对卵泡颗粒细胞增殖、凋亡、激素分泌及共培养的卵母细胞和胚胎发育的影响为了了解抑制素基因转染卵泡的颗粒细胞后基因表达对颗粒细胞增殖、凋亡和激素分泌,以及转染的颗粒细胞同卵母细胞共培养对卵母细胞成熟、体外受精和胚胎发育的影响。本试验利用pEGISI质粒转染来源于牛中腔(直径,＞4mm-8mm)、小腔(直径,1mm-4mm)颗粒细胞,转染后48 h、96 h分别检测颗粒细胞的增殖、凋亡及雌激素和孕酮的分泌量,并检测共培养后的卵母细胞的发育情况。结果显示,脂质体介导的pEGISI转染颗粒细胞后,来自中腔卵泡的颗粒细胞增殖率(88.8±2.1%;平均值±标准差)显著低于对照组(100%)和转染pEGFP组(97.5±2.1%),对小腔卵泡颗粒细胞的增殖在各组之间无显著差异。pEGISI转染颗粒细胞后显著加剧了颗粒细胞的凋亡。转染颗粒细胞48 h后雌激素分泌量均增加,而孕酮分泌量下降。转染后的中腔卵泡颗粒细胞与卵母细胞共培养,降低了卵母细胞的成熟率(61.5±6.8%vs.71.2±5.7%,P＜0.05),但对体外受精和胚胎发育没有影响。这些结果表明,pEGISI在颗粒细胞中的超表达对颗粒细胞的增殖、凋亡和卵母细胞成熟有着重要的调节作用,其作用因颗粒细胞发育时期不同而异。2.pEGISI转染卵母细胞对其成熟及胚胎发育的影响为了了解抑制素基因对卵母细胞成熟及胚胎发育的影响,本试验利用pEGISI质粒直接转染卵母细胞,然后检测抑制素基因在卵母细胞中的表达,以及转染的卵母细胞成熟率和体外受精后胚胎发育情况,结果显示:(1)脂质体介导的抑制素片段转染能够成功地通过透明带进入卵母细胞,并且能够高效表达。(2)转染本身对卵母细胞成熟及体外受精能力没有伤害。(3)高效表达的抑制素片段对卵母细胞的成熟,体外受精以及胚胎发育没有影响。3.pEGISI转染卵母细胞复合体对其成熟及胚胎发育的影响为了进一步了解抑制素基因对卵母细胞复合体成熟及胚胎发育的影响。本试验用pEGISI质粒转染牛卵母细胞复合体,检测pEGISI在卵母细胞复合体中的超表达、卵丘细胞扩散、凋亡和激素分泌、以及卵母细胞复合体成熟、体外受精和胚胎发育的情况。结果显示:质粒pEGISI转染牛卵丘卵母细胞复合体,对中、小卵泡的卵丘细胞扩散有抑制作用,对中腔卵泡卵丘细胞的抑制作用尤为显著(69.8±2.7%,p＜0.05)。质粒pEGISI转染加剧了中、小卵泡卵丘细胞的凋亡,对中腔卵泡卵丘细胞的凋亡作用尤为显著(14.5±0.5%,p＜0.01)。此外,质粒pEGISI转染牛卵丘卵母细胞复合体后,促进了雌激素的分泌。转染后的牛中腔卵泡卵母细胞复合体成熟培养后,卵母细胞的成熟率降低(57.9±8.4%,p＜0.05),但对体外受精率和胚胎发育没有不良影响。更多还原

【Abstract】 Follicular inhibin is one of the important regulators of ovarian function.In monotocous mammalian,passive or active immunization against inhibin or pEGISI gene could be used to induce superovulation and twinning.Furthermore,inhibin is able to be used as one of best marker of ovarian cancer.However,little is known that inhibin gene plays a role on oocyte development in vitro.In this trial,effect of overexpression of inhibinα(1-32) fragment on bovine granulosa cell proliferation, apoptosis,steroidogenesis,oocyte maturation and embryo development were investigated respectively,by transfection with inhibin recombinant plasmid pEGISI. Techniques such as cell culture,plasmid extraction,RT-PCR,transfection,TUNEL, radioimmunoassay,and flow cytometry were used.The plasmid pEGISI was constructed previously in our laboratory.1.Effect of overexpression of inhibinα(1-32) fragment on bovine granulosa cell proliferation,apoptosis,steroidogenesis,and development of co-cultured oocytes.The objective of the present trial was to determine the effects of an inhibinα(1-32) fragment gene on proliferation,apoptosis,and steroidogenesis of bovine granulosa cells(GC) isolated from medium and small follicles(diameter＞4 to 8 mm and 1 to 4 mm,respectively),and the effect of GC,previously transfected with pEGISI,on oocyte maturation and in vitro embryo development.To enhance expression of the inhibinα(1-32) fragment,GC was transfected with pEGISI.Our results showed that transfection with pEGISI inhibited GC proliferation(88.8±2.1%; mean±SEM) compared to control and EGFP group(100%,97.5±2.1%) from medium follicles(P＜0.05),with no significant effect on GC from small follicles. Apoptosis was higher(P＜0.01) in transfected GC than in controls,transfection with pEGISI increased(P＜0.05) estradiol synthesis from both medium and small follicles(0.57±0.13 and 0.86±0.13 pg/mL vs.0.19±0.05 and 0.35±0.09 pg/mL in controls) after culturing for 48 h,with suppression(P＜0.05) in transfected GC after 96 h.Transfection reduced(P＜0.05) progesterone synthesis in GC from both medium and small follicles(24.5±3.4 and 75.4±4.6 ng/mL vs.45.42±5.33 and 117.32±11.99 ng/mL in the control) after culture for 48 h,with no significant difference after 96 h.Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control(61.5±6.8%vs.71.2±5.7%,P＜0.05),with no significant effect on embryo development.In conclusion, overexpression of inhibinα(1-32) fragment regulated GC development;effects on subsequent oocyte maturation were both time-and stage-dependent.2.Effect of overexpression of inhibinα(1-32) fragment on oocyte maturation and embryo development by transfectionIn this trial,denuded oocytes were transfected with pEGISI.The transfection efficiency,oocyte maturation,embryo development were investigated.The results showed that pEGISI could be transfected into oocyte through the zona pellucida with liposome and overexpressed in high efficiency(53%),which was observed under the fluorescence microscope.Transfection was not harmful to oocyte structure, maturation and fertilizability.The overexpression of pEGISI has no effect on oocyte maturation and embryo development.3.Effect of overexpression of inhibinα(1-32) fragment on bovine cumulus-oocyte complex maturation embryo development in vitroThe aim of this trial was to examine the effect of inhibin on the oocyte maturation and hormone secretion in vitro.For this purpose,we carried out the transfection of cumulus-oocyte complex(COC) with recombinant plasmid pEGISI,enhanced inhibin peptide fragment expression.The cumulus expansion,apoptosis,hormone secretion and oocyte maturation were investigated.The apoptosis of COC was assessed using TUNEL,and the release of progesterone,estradiol was evaluated using RIA technique.The transfection of pEGISI significantly decreased cumulus expansion and nuclear maturation in medium follicles(87.5%vs.69.8%,77.4%vs.57.9%),on the contrary,increased the cumulus expansion and nuclear maturation in the small follicles(55.9%vs.58.1%,50.3%vs.54.9%).The cumulus apoptosis was significantly increased in the pEGISI treatment group compared with the controls both in MFs and SFs.It was observed that transfection of COC with pEGISI increased the synthesis of progesterone,estradiol of COCs.The estradiol and progesterone synthesis in transfected cumulus cells of COCs were 6.9pg/mL and 0.47ng/mL, respectively,both of which were higher(P＜0.05) than that observed in the control group.These results suggested that inhibin is one of key regulator of COC development,effect of transfection with pEGISI on the COCs changed with different stage of follicles during in vitro maturation.更多还原