本文選題：卵母細(xì)胞　切入點(diǎn)：體外成熟　出處：《吉林大學(xué)》2016年博士論文

【摘要】：卵母細(xì)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(biāo)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(xì)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(duì)卵母細(xì)胞成熟及合子基因組啟動(dòng)發(fā)揮非常重要的作用。豬卵母細(xì)胞在成熟過(guò)程中受到母源基因所表達(dá)蛋白的影響,本文以不同發(fā)育時(shí)期的豬大卵泡(5mm)和小卵泡(3mm)為研究對(duì)象,通過(guò)3′-d A抑制母源基因多聚腺苷酸化后,檢測(cè)大、小卵泡的體外成熟率,并觀(guān)察卵丘擴(kuò)展情況,使用實(shí)時(shí)熒光定量PCR方法檢測(cè)大、小卵泡卵母細(xì)胞在GV、GVBD、MⅠ、MⅡ不同時(shí)期母源基因Cyclin B1、Cdc2、Cmos、BMP15、GDF9的表達(dá)量;并比較大、小卵泡卵母細(xì)胞在不同時(shí)期及多聚腺苷酸化抑制后m RNA的poly-(A)尾長(zhǎng)度。使用western blot方法檢測(cè)與Cyclin B1、Cdc2、C-mos、BMP15、GDF9基因密切相關(guān)的p38-MAPK的表達(dá)量,通過(guò)免疫熒光方法檢測(cè)α-Tubulin蛋白,以檢測(cè)卵母細(xì)胞紡錘體形成的情況,并用PI復(fù)染檢測(cè)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(xì)胞的成熟率較對(duì)照組顯著降低(p0.01),小卵母細(xì)胞的成熟率在使用抑制劑(3′-d A)后與對(duì)照組相比差異不顯著(p0.05)。大卵泡卵母細(xì)胞處理組在添加3′-d A后卵丘擴(kuò)展的等級(jí)比對(duì)照組顯著降低,而小卵泡卵母細(xì)胞處理組在添加3′-d A后卵丘擴(kuò)展情況與對(duì)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(shí)期的小卵泡卵母細(xì)胞中的表達(dá)量代償性升高。其中Cyclin B1對(duì)大卵泡卵母細(xì)胞的成熟促進(jìn)作用比對(duì)小卵泡卵母細(xì)胞的作用大。而C-mos、GDF9、BMP15的表達(dá)量主要對(duì)小卵泡卵母細(xì)胞成熟起到促進(jìn)作用。對(duì)照組大小卵泡卵母細(xì)胞GV、GVBD、MⅠ和MⅡ時(shí)期紡錘體形成推進(jìn)和染色體排列均比較正常。小卵泡卵母細(xì)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(shí)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(xì)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(huì)導(dǎo)致部分卵母細(xì)胞不能排出第一極體。多聚腺苷酸化對(duì)大卵泡卵母細(xì)胞的成熟率具有促進(jìn)作用,而對(duì)小卵泡卵母細(xì)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(duì)大卵泡卵母細(xì)胞的卵丘擴(kuò)展具有促進(jìn)作用,而對(duì)小卵泡卵母細(xì)胞的卵丘擴(kuò)展作用比較小。多聚腺苷酸化對(duì)卵母細(xì)胞內(nèi)紡錘體形成、染色體排列具有促進(jìn)作用,而且多聚腺苷酸化在促進(jìn)小卵泡卵母細(xì)胞紡錘體形成和染色體排列過(guò)程中會(huì)產(chǎn)生紊亂,且促進(jìn)作用產(chǎn)生的紊亂影響程度大于大卵泡卵母細(xì)胞。本研究為更好的理解卵母細(xì)胞成熟的機(jī)制做出基礎(chǔ)性研究,對(duì)胚胎研究中提高小卵泡卵母細(xì)胞的利用率具有促進(jìn)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(hào)：1719048