Reproductive Medicine Center, Department of Obstetrics and Gynecology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
Institute of Reproductive & Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.
Front Endocrinol (Lausanne). 2023 May 24;14:1169378. doi: 10.3389/fendo.2023.1169378. eCollection 2023.
Human zona pellucida (ZP) plays an important role in reproductive process. Several rare mutations in the encoding genes (, , and ) have been demonstrated to cause women infertility. Mutations in have been reported to cause ZP defects or empty follicle syndrome. We aimed to identify pathogenic variants in an infertile woman with a thin zona pellucida (ZP) phenotype and investigated the effect of ZP defects on oocyte gene transcription.
We performed whole-exome sequencing and Sanger sequencing of genes were performed for infertilite patients characterized by fertilization failure in routine fertilization (IVF). Immunofluorescence (IF) and intracytoplasmic sperm injection (ICSI) were used in the mutant oocytes. Single-cell RNA sequencing was used to investigate transcriptomes of the gene-edited () rat model. Biological function enrichment analysis, quantitative real-time PCR (qRT-PCR), and IF were performed.
We identified a novel homozygous nonsense mutation of (c.1924C > T, p.Arg642X) in a patient with non-consanguineous married parents. All oocytes showed a thin or no ZP under a light microscope and were fertilized after ICSI. The patient successfully conceived by receiving the only two embryos that developed to the blastocyst stage. The immunofluorescence staining showed an apparently abnormal form of the stopped oocytes. We further demonstrated a total of 374 differentially expressed genes (DEGs) in the transcriptome profiles of rats oocytes and highlighted the signal communication between oocytes and granulosa cells. The pathway enrichment results of DEGs showed that they were enriched in multiple signaling pathways, especially the transforming growth factor-β (TGF-β) signaling pathway in oocyte development. qRT-PCR, IF, and phosphorylation analysis showed significantly downregulated expressions of Acvr2b, Smad2, p38MAPK, and Bcl2 and increased cleaved-caspase 3 protein expression.
Our findings expanded the known mutational spectrum of ZP2 associated with thin ZP and natural fertilization failure. Disruption of the integrity of the ZP impaired the TGF-β signaling pathway between oocytes and surrounding granulosa cells, leading to increased apoptosis and decreased developmental potential of oocytes.
人类透明带（ZP）在生殖过程中起着重要作用。编码基因（ZP2、ZP3 和 ZP4）中的一些罕见突变已被证明可导致女性不孕。ZP4 基因突变已被报道可导致透明带缺陷或空卵泡综合征。我们旨在鉴定一名透明带变薄（ZP）表型不孕女性的致病性变异，并研究透明带缺陷对卵母细胞基因转录的影响。
我们对表现为常规体外受精（IVF）受精失败的不孕患者进行了全外显子组测序和基因 Sanger 测序。对突变卵母细胞进行免疫荧光（IF）和胞浆内精子注射（ICSI）。对基因编辑（）大鼠模型的单细胞 RNA 测序用于研究转录组。进行了生物功能富集分析、实时定量 PCR（qRT-PCR）和 IF。
我们在一名非近亲结婚的患者中发现了一个新的纯合无义突变（c.1924C>T，p.Arg642X），位于 基因中。在光镜下，所有卵母细胞均显示透明带变薄或无透明带，经 ICSI 后受精。患者成功受孕，仅接受了两个发育至囊胚阶段的胚胎。免疫荧光染色显示卵母细胞的形态明显异常。我们进一步证明了大鼠卵母细胞转录组图谱中共有 374 个差异表达基因（DEGs），并强调了卵母细胞和颗粒细胞之间的信号通讯。DEGs 的通路富集结果表明，它们富集在多个信号通路中，特别是卵母细胞发育中的转化生长因子-β（TGF-β）信号通路。qRT-PCR、IF 和磷酸化分析显示 Acvr2b、Smad2、p38MAPK 和 Bcl2 的表达明显下调，cleaved-caspase 3 蛋白表达增加。
我们的发现扩展了与透明带变薄和自然受精失败相关的 ZP2 已知突变谱。透明带完整性的破坏干扰了卵母细胞和周围颗粒细胞之间的 TGF-β 信号通路，导致卵母细胞凋亡增加和发育潜能降低。
