■排卵后衰老是排卵卵母细胞的时间依赖性退化,是降低后代适应性的主要限制因素。该过程可能导致卵母细胞凋亡等细胞死亡途径的激活。
■我们评估了卵母细胞膜的完整性,卵发育能力,RT-qPCR检测凋亡相关基因的mRNA丰度。来自斑马鱼Daniorerio的卵母细胞在体内在28.5°C下保留24小时排卵后(HPO)。使用锥虫蓝(TB)染色评估活力。体内卵母细胞衰老对后代发育能力的影响由受精后24小时的胚胎存活决定,孵化,和幼虫畸形率。
■受精,卵母细胞活力,0HPO时的孵化率分别为91%、97%和65%,4HPO时分别降至62%、90%和22%,分别。在8HPO时,施肥能力降低到2%,而72%的卵母细胞仍有完整的质膜。在凋亡基因bcl-2(b细胞淋巴瘤2)中,bada(bcl2相关的细胞死亡激动剂a),组织蛋白酶D,组织蛋白酶Z,caspase6a,caspase7,caspase8,caspase9,apaf1,tp53(肿瘤蛋白p53),研究了cdk1(细胞周期蛋白依赖性激酶1),在24HPO时,抗凋亡bcl-2的mRNA丰度降低,促凋亡组织蛋白酶D增加。此外,与0HPO相比,tp53和cdk1mRNA转录本在24HPO降低。
■因此,TB染色未检测到衰老引起的卵母细胞能力丧失。TB染色,然而,可作为评价斑马鱼卵母细胞受精前质量的一种简便、快速的方法。一起来看,我们的结果表明斑马鱼卵母细胞衰老晚期细胞死亡途径的激活。
UNASSIGNED: Post-ovulatory aging is a time-dependent deterioration of ovulated oocytes and a major limiting factor reducing the fitness of offspring. This process may lead to the activation of cell death pathways like apoptosis in oocytes.
UNASSIGNED: We evaluated oocyte membrane integrity, egg developmental competency, and mRNA abundance of apoptosis-related genes by RT-qPCR. Oocytes from zebrafish Danio rerio were retained in vivo at 28.5°C for 24 h post-ovulation (HPO). Viability was assessed using trypan blue (TB) staining. The consequences of in vivo oocyte aging on the developmental competence of progeny were determined by the embryo survival at 24 h post fertilization, hatching, and larval malformation rates.
UNASSIGNED: The fertilization, oocyte viability, and hatching rates were 91, 97, and 65% at 0 HPO and dropped to 62, 90, and 22% at 4 HPO, respectively. The fertilizing ability was reduced to 2% at 8 HPO, while 72% of oocytes had still intact plasma membranes. Among the apoptotic genes bcl-2 (b-cell lymphoma 2), bada (bcl2-associated agonist of cell death a), cathepsin D, cathepsin Z, caspase 6a, caspase 7, caspase 8, caspase 9, apaf1, tp53 (tumor protein p53), cdk1 (cyclin-dependent kinase 1) studied, mRNA abundance of anti-apoptotic bcl-2 decreased and pro-apoptotic cathepsin D increased at 24 HPO. Furthermore, tp53 and cdk1 mRNA transcripts decreased at 24 HPO compared to 0 HPO.
UNASSIGNED: Thus, TB staining did not detect the loss of oocyte competency if caused by aging. TB staining, however, could be used as a simple and rapid method to evaluate the quality of zebrafish oocytes before fertilization. Taken together, our results indicate the activation of cell death pathways in the advanced stages of oocyte aging in zebrafish.