Analysis of differential maternal mRNA expression in developmentally competent and incompetent bovine two-cell embryos
Analysis of differential maternal mRNA expression in developmentally competent and incompetent bovine two-cell embryos
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DOI：
10.1002/mrd.10385
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发表时间：
2004-02-01
影响因子：
2.5
通讯作者：
Lonergan, P
中科院分区：
文献类型：
--
作者：
Fair, T;Murphy, M;Lonergan, P
本研究的主要目的是鉴定与胚胎发育能力相关的mRNA转录本。在牛中，mRNA转录本、核糖体和生长期积累的蛋白质被用来维持成熟、受精和最初的细胞周期分裂，直到8- 16细胞阶段。早期卵裂的哺乳动物受精卵比其后期卵裂的对应物更可能发育到胚泡阶段，从而反映了它们起源的卵母细胞的内在质量。我们描述了结合这个成熟的模型，用于回顾性测定哺乳动物卵母细胞的发育能力与广泛筛选不同生物群体中差异基因表达的技术。未成熟的卵丘卵母细胞复合体从屠宰场卵巢上的表面可见卵泡中回收，洗涤，并进行常规体外成熟和受精。从授精后24至42小时（pi），以3小时的间隔从培养物中取出双细胞胚胎。鉴定了两个双细胞胚胎群体;早期分裂（27 hpi之前）的群体和晚期分裂（33 hpi之后）的群体。对两个群体的cDNA进行抑制性消减杂交，然后对差异表达的扩增子进行亚克隆和测序。使用BLAST算法将序列提交至NCB 1的非冗余和表达序列标签（EST）数据库。三个选定的候选基因的差异表达，被确定为pupillin在早期裂解合子上调，选择用于进一步研究;组蛋白H3，细胞周期蛋白B1，和GDF-9 B。使用定量真实的时间PCR，我们已经表明，组蛋白H3 A是显着更丰富的胚胎，最早切割。(C)2004 Wiley-Liss，Inc.
The main objective of this study was to identify mRNA transcripts associated with embryonic developmental competence. In cattle, mRNA transcripts, ribosomes, and proteins accumulated during the growth phase are drawn on to sustain maturation, fertilization, and the initial cell cycle divisions up to the 8- to 16-cell stage. Early cleaving mammalian zygotes are more likely to develop to the blastocyst stage than their later cleaving counterparts, thus reflecting the intrinsic quality of the oocytes from which they originated. We describe the combination of this well-established model for the retrospective determination of developmental competence in mammalian oocytes with a technique for wide screening of differential gene expression in different biological populations. Immature cumulus oocyte complexes were recovered from surface visible follicles on abattoir ovaries, washed, and submitted to routine in vitro maturation and fertilization. Two-cell embryos were removed from culture at 3-hr intervals from 24 to 42 hr post insemination (pi). Two populations of two-cell embryos were identified; those that cleaved early (before 27 hpi) and those that cleaved late (after 33 hpi). Suppressive subtractive hybridization was carried out on cDNA from the two populations, following which, differentially expressed amplicons were subcloned and sequenced. The sequences were submitted to the nonredunclant and expressed sequence tag (EST) databases at NCBl using the BLAST algorithm. The differential expression of three selected candidate genes that were identified as putatively upregulated in the early cleaving zygotes were chosen for further investigations; histone H3, cyclin B1, and GDF-9B. Using quantitative real time PCR we have shown that histone H3A is significantly more abundant in embryos that cleave earliest. (C) 2004 Wiley-Liss, Inc.