■多年来,平均生育年龄增加导致不孕症,流产,和染色体异常主要由年龄相关的卵母细胞质量下降引起。在这项研究中,我们研究了一氧化氮(NO)不足和蛋白质硝化在卵母细胞时间老化中的作用。
■从年轻的育种者中回收小鼠卵母细胞(YB,8-14周[w]),退休饲养员(RB,48-52w)和老年动物(OA,80-84w)在排卵触发后13.5和17小时。评估透明带溶解时间(ZPDT);卵质微管动力学(OMD);皮质颗粒(CG)状态和纺锤体形态(SM),作为卵母细胞质量的标志。将来自RB的同胞卵母细胞暴露于NO补充并评估衰老现象(AP)。对所有卵母细胞卵丘复合物进行荧光硝基酪氨酸(NT)免疫细胞化学和共聚焦显微镜检查,以评估形态和蛋白质硝化。
■在hCG触发13.5小时后,与YB相比,RB的卵母细胞ZPDT显着增加(37.8±11.9vs22.1±4.1秒[s]),OMD(46.9vs0%),CG损失(39.4vs0%),正常SM下降(30.3vs81.3%),表明在hCG触发后17小时,来自RB的卵母细胞中过早的AP恶化。当暴露于SNAP时,RBAP显着降低(ZPDT:35.1±5.5vs46.3±8.9s,OMD:13.3vs75.0%,CG损失:50.0vs93.3%)和SM改善(80.0vs14.3%)。卵丘细胞中NT阳性的发生率显着升高(13.5h,46.7±4.5vs3.4±0.7%;17小时,82.2±2.9vs23.3±3.6%)和卵母细胞(13.5小时,57.1vs0%;17小时,100.0vs55.5%)来自RB,与YB相比。回收的卵母细胞随着年龄的增长而减少(YB组中每只动物29.8±4.1,而RB中的10.2±2.1和OA中的4.0±1.6)。来自OA的卵母细胞显示增加的ZPDT,主要的CG损失,OMD和纺锤体异常增加,以及原核形成,确认自发减数分裂到间期。
■卵母细胞经历透明带硬化,改变纺锤体和卵质微管,皮质颗粒过早释放,指示自发减数分裂-间期转变,作为时间老化的函数。这些变化也与NO不足和蛋白质硝化有关,可以通过补充NO供体来缓解。
The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related decline in oocyte quality. In this study, we investigate the role of nitric oxide (NO) insufficiency and protein nitration in oocyte chronological aging.
Mouse oocytes were retrieved from young breeders (YB, 8-14 weeks [w]), retired breeders (RB, 48-52w) and old animals (OA, 80-84w) at 13.5 and 17 hours after ovulation trigger. They were assessed for zona pellucida dissolution time (ZPDT); ooplasmic microtubule dynamics (OMD); cortical granule (CG) status and spindle morphology (SM), as markers of oocyte quality. Sibling oocytes from RB were exposed to NO supplementation and assessed for aging phenomena (AP). All oocyte cumulus complexes were subjected to fluorescence nitrotyrosine (NT) immunocytochemistry and confocal microscopy to assess morphology and protein nitration.
At 13.5 h from hCG trigger, oocytes from RB compared to YB had significantly increased ZPDT (37.8 ± 11.9 vs 22.1 ± 4.1 seconds [s]), OMD (46.9 vs 0%), CG loss (39.4 vs 0%), and decreased normal SM (30.3 vs 81.3%), indicating premature AP that worsened among oocytes from RB at 17 hours post-hCG trigger. When exposed to SNAP, RB AP significantly decreased (ZPDT: 35.1 ± 5.5 vs 46.3 ± 8.9s, OMD: 13.3 vs 75.0% and CG loss: 50.0 vs 93.3%) and SM improved (80.0 vs 14.3%). The incidence of NT positivity was significantly higher in cumulus cells (13.5 h, 46.7 ± 4.5 vs 3.4 ± 0.7%; 17 h, 82.2 ± 2.9 vs 23.3 ± 3.6%) and oocytes (13.5 h, 57.1 vs 0%; 17 h, 100.0 vs 55.5%) from RB compared to YB. Oocytes retrieved decreased with advancing age (29.8 ± 4.1 per animal in the YB group compared to 10.2 ± 2.1 in RB and 4.0 ± 1.6 in OA). Oocytes from OA displayed increased ZPDT, major CG loss, increased OMD and spindle abnormalities, as well as pronuclear formation, confirming spontaneous meiosis to interphase transition.
Oocytes undergo zona pellucida hardening, altered spindle and ooplasmic microtubules, and premature cortical granule release, indicative of spontaneous meiosis-interphase transition, as a function of chronological aging. These changes are also associated with NO insufficiency and protein nitration and may be alleviated through supplementation with an NO-donor.