Department of Obstetrics/Gynecology, Joint Laboratory of Reproductive Medicine (SCU-CUHK), Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, China.
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, National Institute for Data Science in Health and Medicine, Xiamen University, Xiamen, Fujian, PR China.
Reproduction. 2024 Sep 16;168(4). doi: 10.1530/REP-23-0466. Print 2024 Oct 1.
PLCZ1 mutations are related to total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), characterised by abnormal oocyte oscillations. The novel PLCZ1 compound heterozygous mutations reported by this study were associated with TFF after ICSI, with one of the mutations indicating a gene dosage effect.
Oocyte activation failure is thought to be one of the main factors for total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), which could be induced by abnormal calcium oscillations. Phospholipase C zeta (PLCZ), a sperm factor, is associated with Ca2+ oscillations in mammalian oocytes. To date, some mutations in PLCZ1 (the gene that encodes PLCZ) have been linked to TFF, as demonstrated by the observed reduction in protein levels or activity to induce Ca2+ oscillations. In this study, normozoospermic males whose sperms exhibited TFF after ICSI and their families were recruited. First, mutations in the PLCZ1 sequence were identified by whole exome sequencing and validated using Sanger sequencing. Then, the locations of PLCZ1/PLCZ and the transcript and protein levels in the sperm of the patients were studied. Subsequently, in vitro function analysis and in silico analysis were performed to investigate the function-structure correlation of mutations identified in PLCZ1 using western blotting, immunofluorescence, RT-qPCR, and molecular simulation. Ca2+ oscillations were detected after cRNA microinjection into MII mouse oocytes to investigate calcium oscillations induced by abnormal PLCZ. Five variants with compound heterozygosity were identified, consisting of five new mutations and three previously reported mutations distributed across the main domains of PLCZ, except the EF hands domain. The transcript and protein levels decreased to varying degrees among all detected mutations in PLCZ1 when transfected in HEK293T cells. Among these, mutations in M138V and R391* of PLCZ were unable to trigger typical Ca2+ oscillations. In case 5, aberrant localisation of PLCZ in the sperm head and an increased expression of PLCZ in the sperm were observed. In conclusion, this study enhances the potential for genetic diagnosis of TFF in clinics and elucidates the possible relationship between the function and structure of PLCZ in novel mutations.
PLCZ1 突变与卵胞浆内单精子注射（ICSI）后的完全受精失败（TFF）有关，其特征是卵母细胞的异常摆动。本研究报道的新型 PLCZ1 复合杂合突变与 ICSI 后的 TFF 相关，其中一种突变表明存在基因剂量效应。
卵母细胞激活失败被认为是卵胞浆内单精子注射（ICSI）后完全受精失败（TFF）的主要因素之一，其可能由异常钙震荡引起。PLCZ，一种精子因子，与哺乳动物卵母细胞中的 Ca2+震荡有关。迄今为止，一些 PLCZ1（编码 PLCZ 的基因）突变与 TFF 有关，这是通过观察到的蛋白水平降低或诱导 Ca2+震荡的活性降低来证实的。在这项研究中，招募了 ICSI 后精子表现出 TFF 的正常精子男性及其家属。首先，通过全外显子组测序鉴定 PLCZ1 序列中的突变，并通过 Sanger 测序进行验证。然后，研究了患者精子中 PLCZ1/PLCZ 的位置以及转录本和蛋白水平。随后，通过 Western blot、免疫荧光、RT-qPCR 和分子模拟进行体外功能分析和计算分析，研究 PLCZ1 中鉴定出的突变的功能-结构相关性。通过 cRNA 微注射到 MII 期小鼠卵母细胞中检测 Ca2+震荡，以研究异常 PLCZ 诱导的钙震荡。鉴定出五个由主要结构域组成的复合杂合变异体，包括五个新突变和三个分布在 PLCZ 除 EF 手域外的主要结构域的先前报道的突变。当转染到 HEK293T 细胞中时，所有检测到的 PLCZ1 突变均不同程度地降低了转录本和蛋白水平。其中，PLCZ 的 M138V 和 R391*突变无法触发典型的 Ca2+震荡。在病例 5 中，观察到 PLCZ 在精子头部的异常定位和 PLCZ 在精子中的表达增加。总之，本研究增强了临床 TFF 遗传诊断的潜力，并阐明了 PLCZ 新突变中功能和结构之间的可能关系。
