本文選題：卵母細胞　切入點：體外成熟　出處：《吉林大學》2016年博士論文

【摘要】：卵母細胞的體外成熟包括核成熟和質(zhì)成熟。核成熟以第一極體的排出為標志,質(zhì)成熟的作用為積累穩(wěn)定的m RNA和蛋白,用于維持受精和早期胚胎發(fā)育。細胞質(zhì)中母源基因m RNA的多聚腺苷酸化,可以防止m RNA降解,對卵母細胞成熟及合子基因組啟動發(fā)揮非常重要的作用。豬卵母細胞在成熟過程中受到母源基因所表達蛋白的影響,本文以不同發(fā)育時期的豬大卵泡(5mm)和小卵泡(3mm)為研究對象,通過3′-d A抑制母源基因多聚腺苷酸化后,檢測大、小卵泡的體外成熟率,并觀察卵丘擴展情況,使用實時熒光定量PCR方法檢測大、小卵泡卵母細胞在GV、GVBD、MⅠ、MⅡ不同時期母源基因Cyclin B1、Cdc2、Cmos、BMP15、GDF9的表達量;并比較大、小卵泡卵母細胞在不同時期及多聚腺苷酸化抑制后m RNA的poly-(A)尾長度。使用western blot方法檢測與Cyclin B1、Cdc2、C-mos、BMP15、GDF9基因密切相關(guān)的p38-MAPK的表達量,通過免疫熒光方法檢測α-Tubulin蛋白,以檢測卵母細胞紡錘體形成的情況,并用PI復染檢測染色體排列情況。研究結(jié)果顯示,由于3′-d A的腺苷酸化抑制作用,大卵泡卵母細胞的成熟率較對照組顯著降低(p0.01),小卵母細胞的成熟率在使用抑制劑(3′-d A)后與對照組相比差異不顯著(p0.05)。大卵泡卵母細胞處理組在添加3′-d A后卵丘擴展的等級比對照組顯著降低,而小卵泡卵母細胞處理組在添加3′-d A后卵丘擴展情況與對照組相比并不明顯。在加入3′-d A抑制劑后,C-mos、GDF9、BMP15在MⅡ時期的小卵泡卵母細胞中的表達量代償性升高。其中Cyclin B1對大卵泡卵母細胞的成熟促進作用比對小卵泡卵母細胞的作用大。而C-mos、GDF9、BMP15的表達量主要對小卵泡卵母細胞成熟起到促進作用。對照組大小卵泡卵母細胞GV、GVBD、MⅠ和MⅡ時期紡錘體形成推進和染色體排列均比較正常。小卵泡卵母細胞在加入3′-d A抑制劑后紡錘體形成明顯混亂,染色體排列也受到影響,MⅡ時期雖然能夠正常排出第一極體,但是紡錘體受到很大影響。大卵泡卵母細胞在加入3′-d A抑制劑后紡錘體形成混亂,但不十分明顯,然而會導致部分卵母細胞不能排出第一極體。多聚腺苷酸化對大卵泡卵母細胞的成熟率具有促進作用,而對小卵泡卵母細胞的成熟率具有較小的抑制作用。母源基因的多聚腺苷酸化對大卵泡卵母細胞的卵丘擴展具有促進作用,而對小卵泡卵母細胞的卵丘擴展作用比較小。多聚腺苷酸化對卵母細胞內(nèi)紡錘體形成、染色體排列具有促進作用,而且多聚腺苷酸化在促進小卵泡卵母細胞紡錘體形成和染色體排列過程中會產(chǎn)生紊亂,且促進作用產(chǎn)生的紊亂影響程度大于大卵泡卵母細胞。本研究為更好的理解卵母細胞成熟的機制做出基礎(chǔ)性研究,對胚胎研究中提高小卵泡卵母細胞的利用率具有促進意義。
[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 偽 -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，

本文編號：1719048