Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulus-oocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; PNPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P幼年动物的体外胚胎生产成功率因卵母细胞固有品质低下而受损。常规体外成熟（in vitro maturation, IVM）会通过诱导自发减数分裂恢复，损害卵母细胞发育潜能。本研究开展一系列实验，以探究在常规IVM前设置成熟前培养阶段（pre-IVM）并维持减数分裂阻滞，是否能提升2月龄幼年山羊的卵丘-卵母细胞复合体（cumulus-oocyte complexes, COCs）的卵母细胞发育潜能。 实验1中，将COCs分别以0、50、100、200 nM的C型利钠肽（C-type natriuretic peptide, CNP）培养6小时与8小时，随后评估核相状态，结果显示生发泡（germinal vesicle, GV）破裂的发生率无显著差异。 实验2中，除设置与实验1相同浓度的CNP处理组外，额外加入10 nM雌二醇——已知其作为上游激动剂，可激活CNP的专属受体利钠肽受体2（natriuretic peptide receptor 2, NPR2）的表达。与对照组相比，200 nM CNP联合雌二醇处理组在培养6小时时的GV期卵母细胞占比显著升高（74.7% vs 28.3%；P）；经过6小时pre-IVM处理后，NPR2的表达被下调。 实验4中，对跨透明带突起的分析结果显示，pre-IVM可在卵母细胞回收后维持卵丘与卵母细胞间的通讯连接。 实验5与6中，对比了双相IVM方案（先以CNP与雌二醇进行6小时pre-IVM，再进行24小时常规IVM）与单纯24小时IVM对照组。结果显示，相较于对照组，双相IVM方案可提升卵母细胞内谷胱甘肽水平、降低活性氧（reactive oxygen species, ROS）含量，上调DNA甲基转移酶1（DNA methyltransferase 1, DNMT1）与五聚素3（pentraxin 3, PTX3）的表达，并提高囊胚发育率。