Immature follicular origins and disrupted oocyte growth pathways contribute to decreased gamete quality during reproductive juvenescence in mice

Egg quality dictates fertility outcomes, and although there is a well-documented decline with advanced reproductive age, how it changes during puberty is less understood. Such knowledge is critical, since advances in Assisted Reproductive Technologies are enabling pre- and peri-pubertal patients to preserve fertility in the medical setting. Therefore, we investigated egg quality parameters in a mouse model of the pubertal transition or juvenescence (postnatal day; PND 11-40). Animal weight, vaginal opening, serum inhibin B levels, oocyte yield, oocyte diameter, and zona pellucida thickness increased with age. After PND 15, there was an age-associated ability of oocytes to resume meiosis and reach metaphase of meiosis II (MII) following in vitro maturation (IVM). However, eggs from the younger cohort (PND 16-20) had significantly more chromosome configuration abnormalities relative to the older cohorts and many were at telophase I instead of MII, indicative of a cell cycle delay. Oocytes from the youngest mouse cohorts originated from the smallest antral follicles with the fewest cumulus layers per oocyte, suggesting a more developmentally immature state. RNA Seq analysis of oocytes from mice at distinct ages revealed that the genes involved in cellular growth signaling pathways (PI3K, mTOR, and Hippo) were consistently repressed with meiotic competence, whereas genes involved in cellular communication were upregulated in oocytes with age. Taken together, these data demonstrate that gametes harvested during the pubertal transition have low meiotic maturation potential and derive from immature follicular origins. Overall design: Oocytes were collected from four cohorts of mice: PND 13, PND 16 small, PND 16 large, and PND 40 mice. For each cohort, pooled oocytes were snap frozen in three replicates. Each replicate had 18-45 oocytes. RNA was isolated using RNeasy Plus mini kit (Qiagen, Germany, 74134). cDNA synthesis was performed using NEB Next Ultra II directional RNA library prep kit (New England Biolabs, Ipswich, MA, E7760S) after rRNA depletion (NEB Next rRNA depletion kit, E6310S) for Illumina next generation sequencing (HiSeq-50bp).

卵子质量决定生育结局，尽管学界已充分证实高龄生殖年龄会导致卵子质量下降，但青春期阶段卵子质量的变化规律仍有待阐明。这一研究认知至关重要，因为辅助生殖技术（Assisted Reproductive Technologies, ART）的进步已使得青春期前及围青春期患者能够在临床场景中进行生育力保存。为此，本研究针对青春期过渡阶段（即青少年期，以出生后天数postnatal day, PND计：PND 11-40）的小鼠模型开展了卵子质量参数相关研究。随着年龄增长，小鼠体重、阴道开口状态、血清抑制素B（inhibin B）水平、卵母细胞获取量、卵母细胞直径及透明带（zona pellucida）厚度均呈上升趋势。PND 15之后，卵母细胞随年龄增长逐渐获得体外成熟（in vitro maturation, IVM）后恢复减数分裂并发育至第二次减数分裂中期（metaphase of meiosis II, MII）的能力。但相较于老年组，低龄组（PND 16-20）的卵子染色体构型异常比例显著更高，且多数卵子停滞于减数分裂I末期（telophase I）而非MII期，提示存在细胞周期阻滞。最低龄组小鼠的卵母细胞源自最小的窦状卵泡，且每个卵母细胞周围的卵丘细胞层数最少，提示其发育成熟度更低。对不同年龄段小鼠卵母细胞的转录组测序（RNA Seq）分析显示，随着卵母细胞减数分裂能力的提升，参与细胞生长信号通路（PI3K、mTOR及Hippo）的基因表达持续被抑制；而随着年龄增长，参与细胞通信的基因表达则呈上调趋势。综合上述结果，本研究数据表明，青春期过渡阶段获取的配子减数分裂成熟潜能较低，且源自发育未成熟的卵泡。实验设计：本研究从四组小鼠中收集卵母细胞：PND 13组、PND 16小卵泡组、PND 16大卵泡组及PND 40组。每一组的混合卵母细胞均设置3次生物学重复并快速冷冻保存，每个重复包含18~45个卵母细胞。总RNA提取采用RNeasy Plus Mini试剂盒（Qiagen，德国，货号74134）；在进行核糖体RNA（rRNA）去除（使用NEB Next rRNA去除试剂盒，货号E6310S）后，采用NEB Next Ultra II定向RNA建库试剂盒（New England Biolabs，美国马萨诸塞州伊普斯维奇，货号E7760S）完成cDNA合成，随后使用Illumina下一代测序平台（HiSeq，50bp读长）进行测序。