Ma Jiajia, Carney Tom J
Lee Kong Chian School of Medicine, Yunnan Garden Campus, Nanyang Technological University, Singapore, Singapore.
PLoS Biol. 2025 Jun 17;23(6):e3003181. doi: 10.1371/journal.pbio.3003181. eCollection 2025 Jun.
Successful initiation of animal development requires activation of the egg immediately prior to fusion of gamete pronuclei. In all taxa, this is initiated by waves of calcium transients which transverse across the egg. Calcium waves also occur at cleavage furrows during later blastula cytokinesis. Calcium is released from the endoplasmic reticulum through activation of inositol-1,4,5-trisphosphate (IP3) receptors. Only a subset of the mechanisms employed to generate IP3 during vertebrate egg activation are defined, with strong evidence that other critical mechanisms exist. Serine proteases have been long implicated in egg activation and fertilization. Here, we report that treatment of zebrafish eggs with serine protease inhibitors leads to defective calcium wave propagation and failed egg activation. We further show that mutation of zebrafish Protease-activated receptor 2a (Par2a) also results in severe disruption of egg activation, leading to failed chorion elevation and ooplasmic segregation. Milder par2a mutants progress further, but then show abnormal blastomere cleavage. We observed that par2a mutants show decreased amplitude and duration of calcium transients. Restoring Ca++ or direct injection of IP3 ligand rescues egg activation aborted by either serine protease inhibitor treatment or by mutation of Par2a. We thus show that serine protease activity is a critical regulator of IP3 and subsequent calcium wave amplification during zebrafish egg activation, and link this to intracellular calcium release via the protease receptor, Par2a. This constitutes a novel signaling pathway critical for successful fertilization.
动物发育的成功起始需要在配子原核融合之前立即激活卵子。在所有生物分类群中，这是由横穿卵子的钙瞬变波启动的。在后期囊胚胞质分裂期间，卵裂沟处也会出现钙波。钙通过肌醇-1,4,5-三磷酸（IP3）受体的激活从内质网释放。在脊椎动物卵子激活过程中，用于产生IP3的机制中只有一部分已被明确，有强有力的证据表明还存在其他关键机制。丝氨酸蛋白酶长期以来一直被认为与卵子激活和受精有关。在此，我们报告用丝氨酸蛋白酶抑制剂处理斑马鱼卵子会导致钙波传播缺陷和卵子激活失败。我们进一步表明，斑马鱼蛋白酶激活受体2a（Par2a）的突变也会导致卵子激活严重受阻，导致绒毛膜抬高失败和卵质分离。Par2a突变较轻的个体发育进程会进一步推进，但随后会出现异常的卵裂球分裂。我们观察到Par2a突变体的钙瞬变幅度和持续时间降低。恢复Ca++或直接注射IP3配体可挽救因丝氨酸蛋白酶抑制剂处理或Par2a突变而中止的卵子激活。因此，我们表明丝氨酸蛋白酶活性是斑马鱼卵子激活过程中IP3及随后钙波放大的关键调节因子，并将其与通过蛋白酶受体Par2a的细胞内钙释放联系起来。这构成了一条对成功受精至关重要的新信号通路。
