Laurel Fertility Center, San Francisco, CA, United States.
Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, University of California Davis Medical School, Sacramento, CA, United States.
Front Endocrinol (Lausanne). 2023 Dec 12;14:1251102. doi: 10.3389/fendo.2023.1251102. eCollection 2023.
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.
随着生育年龄的增加，不孕、流产和染色体异常的情况越来越多，这主要是由于卵母细胞质量随年龄的增长而下降。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相老化中的作用。
从小鼠中取出卵母细胞（YB，8-14 周[w]）、退休繁殖者（RB，48-52w）和老年动物（OA，80-84w），在排卵触发后 13.5 和 17 小时进行评估。它们的卵透明带溶解时间（ZPDT）；卵浆微管动力学（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物。RB 的姐妹卵母细胞接受 NO 补充，并评估老化现象（AP）。所有卵丘-卵母细胞复合物均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 触发后 13.5 小时，与 YB 相比，RB 的卵母细胞具有明显更长的 ZPDT（37.8 ± 11.9 比 22.1 ± 4.1 秒[s]）、OMD（46.9 比 0%）、CG 丢失（39.4 比 0%）和正常 SM 减少（30.3 比 81.3%），表明 RB 卵母细胞的早期 AP 恶化，在 hCG 触发后 17 小时更为明显。当暴露于 SNAP 时，RB 的 AP 显著减少（ZPDT：35.1 ± 5.5 比 46.3 ± 8.9s，OMD：13.3 比 75.0%和 CG 丢失：50.0 比 93.3%），SM 得到改善（80.0 比 14.3%）。与 YB 相比，RB 的卵丘细胞（13.5 小时，46.7 ± 4.5 比 3.4 ± 0.7%；17 小时，82.2 ± 2.9 比 23.3 ± 3.6%）和卵母细胞（13.5 小时，57.1 比 0%；17 小时，100.0 比 55.5%）的 NT 阳性率显著升高。随着年龄的增长，从 YB 中取出的卵母细胞数量减少（29.8 ± 4.1 个/动物，RB 为 10.2 ± 2.1 个，OA 为 4.0 ± 1.6 个）。OA 的卵母细胞显示出 ZPDT 延长、CG 大量丢失、OMD 增加和纺锤体异常，以及原核形成，证实了自发减数分裂向间期的转变。
卵母细胞经历了透明带硬化、纺锤体和卵浆微管改变，以及皮质颗粒的过早释放，表明随着时间的推移，卵母细胞发生了自发的减数分裂-间期转变。这些变化也与 NO 不足和蛋白质硝化有关，并可通过补充 NO 供体得到缓解。
