母源基因多聚腺苷酸化对猪卵母细胞体外成熟的影响

发布时间：2018-04-06 21:39

  本文选题：卵母细胞　切入点：体外成熟　出处：《吉林大学》2016年博士论文

【摘要】：卵母细胞的体外成熟包括核成熟和质成熟。核成熟以第一极体的排出为标志,质成熟的作用为积累稳定的m RNA和蛋白,用于维持受精和早期胚胎发育。细胞质中母源基因m RNA的多聚腺苷酸化,可以防止m RNA降解,对卵母细胞成熟及合子基因组启动发挥非常重要的作用。猪卵母细胞在成熟过程中受到母源基因所表达蛋白的影响,本文以不同发育时期的猪大卵泡(5mm)和小卵泡(3mm)为研究对象,通过3′-d A抑制母源基因多聚腺苷酸化后,检测大、小卵泡的体外成熟率,并观察卵丘扩展情况,使用实时荧光定量PCR方法检测大、小卵泡卵母细胞在GV、GVBD、MⅠ、MⅡ不同时期母源基因Cyclin B1、Cdc2、Cmos、BMP15、GDF9的表达量;并比较大、小卵泡卵母细胞在不同时期及多聚腺苷酸化抑制后m RNA的poly-(A)尾长度。使用western blot方法检测与Cyclin B1、Cdc2、C-mos、BMP15、GDF9基因密切相关的p38-MAPK的表达量,通过免疫荧光方法检测α-Tubulin蛋白,以检测卵母细胞纺锤体形成的情况,并用PI复染检测染色体排列情况。研究结果显示,由于3′-d A的腺苷酸化抑制作用,大卵泡卵母细胞的成熟率较对照组显著降低(p0.01),小卵母细胞的成熟率在使用抑制剂(3′-d A)后与对照组相比差异不显著(p0.05)。大卵泡卵母细胞处理组在添加3′-d A后卵丘扩展的等级比对照组显著降低,而小卵泡卵母细胞处理组在添加3′-d A后卵丘扩展情况与对照组相比并不明显。在加入3′-d A抑制剂后,C-mos、GDF9、BMP15在MⅡ时期的小卵泡卵母细胞中的表达量代偿性升高。其中Cyclin B1对大卵泡卵母细胞的成熟促进作用比对小卵泡卵母细胞的作用大。而C-mos、GDF9、BMP15的表达量主要对小卵泡卵母细胞成熟起到促进作用。对照组大小卵泡卵母细胞GV、GVBD、MⅠ和MⅡ时期纺锤体形成推进和染色体排列均比较正常。小卵泡卵母细胞在加入3′-d A抑制剂后纺锤体形成明显混乱,染色体排列也受到影响,MⅡ时期虽然能够正常排出第一极体,但是纺锤体受到很大影响。大卵泡卵母细胞在加入3′-d A抑制剂后纺锤体形成混乱,但不十分明显,然而会导致部分卵母细胞不能排出第一极体。多聚腺苷酸化对大卵泡卵母细胞的成熟率具有促进作用,而对小卵泡卵母细胞的成熟率具有较小的抑制作用。母源基因的多聚腺苷酸化对大卵泡卵母细胞的卵丘扩展具有促进作用,而对小卵泡卵母细胞的卵丘扩展作用比较小。多聚腺苷酸化对卵母细胞内纺锤体形成、染色体排列具有促进作用,而且多聚腺苷酸化在促进小卵泡卵母细胞纺锤体形成和染色体排列过程中会产生紊乱,且促进作用产生的紊乱影响程度大于大卵泡卵母细胞。本研究为更好的理解卵母细胞成熟的机制做出基础性研究,对胚胎研究中提高小卵泡卵母细胞的利用率具有促进意义。
[Abstract]:In vitro maturation of oocytes includes nuclear maturation and qualitative maturation.Nuclear maturation is marked by the excretion of the first polar body, and the role of qualitative maturation is to accumulate stable m RNA and proteins for maintaining fertilization and early embryonic development.The polyadenylation of mother gene m RNA in cytoplasm can prevent the degradation of m RNA and play a very important role in oocyte maturation and zygote genome initiation.Porcine oocytes were affected by proteins expressed by maternal genes during maturation. In this study, the porcine large follicles (5mm) and small follicles (3mm) at different developmental stages were studied.The tail length of m RNA in follicular oocytes at different stages and after polyadenylation inhibition.Western blot method was used to detect the expression of p38-MAPK, which was closely related to the gene of Cyclin B1c2Cdc2OBMP15GDF9. The 伪 -Tubulin protein was detected by immunofluorescence to detect the formation of oocyte spindle, and the chromosome arrangement was detected by Pi restaining.The grade of cumulus expansion in the large follicle oocyte treatment group was significantly lower than that in the control group, but that in the small follicular oocyte treatment group was not significantly higher than that in the control group.The expression of GDF9 BMP15 in follicular oocytes at M 鈪，

本文编号：1719048