Table_2_Identification of zona pellucida defects revealed a novel loss-of-function mutation in ZP2 in humans and rats.docx

IntroductionHuman zona pellucida (ZP) plays an important role in reproductive process. Several rare mutations in the encoding genes (ZP1, ZP2, and ZP3) have been demonstrated to cause women infertility. Mutations in ZP2 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.
MethodsWe performed whole-exome sequencing and Sanger sequencing of genes were performed for infertilite patients characterized by fertilization failure in routine in vitro 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 (Zp2mut/mut) rat model. Biological function enrichment analysis, quantitative real-time PCR (qRT-PCR), and IF were performed.
ResultsWe identified a novel homozygous nonsense mutation of ZP2 (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 Zp2mut/mut 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.
DiscussionOur 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.

引言
人透明带（zona pellucida, ZP）在生殖过程中发挥重要作用。其编码基因（ZP1、ZP2和ZP3）的少数罕见突变已被证实可导致女性不孕。已有研究报道ZP2突变可引发透明带缺陷或空卵泡综合征。本研究旨在对1例表现为薄透明带表型的不孕女性进行致病变异鉴定，并探究透明带缺陷对卵母细胞基因转录的影响。

方法
我们对常规体外受精（in vitro fertilization, IVF）中受精失败的不孕患者开展了全外显子组测序与桑格测序。针对携带突变的卵母细胞，采用免疫荧光（immunofluorescence, IF）技术与卵胞浆内单精子注射（intracytoplasmic sperm injection, ICSI）进行相关检测。通过单细胞RNA测序分析基因编辑的Zp2mut/mut大鼠模型的卵母细胞转录组，并进行生物学功能富集分析、实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）及免疫荧光检测。

结果
我们在1对非近亲婚配夫妇的患者中，鉴定出1个全新的纯合无义突变ZP2（c.1924C>T, p.Arg642X）。光学显微镜下可见该患者的所有卵母细胞均呈现薄透明带或无透明带表型，且经ICSI后可成功受精。该患者移植仅有的2枚发育至囊胚期的胚胎后，成功实现妊娠。免疫荧光染色结果显示，停滞的卵母细胞形态存在明显异常。我们进一步在Zp2mut/mut大鼠的卵母细胞转录组中鉴定出共计374个差异表达基因（differentially expressed genes, DEGs），并重点解析了卵母细胞与颗粒细胞间的信号通讯。差异表达基因的通路富集分析结果表明，其显著富集于多条信号通路，尤其是卵母细胞发育过程中的转化生长因子-β（transforming growth factor-β, TGF-β）信号通路。qRT-PCR、免疫荧光及磷酸化分析结果显示，Acvr2b、Smad2、p38MAPK与Bcl2的表达水平显著下调，而cleaved-caspase 3的蛋白表达水平升高。

讨论
本研究的发现拓展了与薄透明带及自然受精失败相关的ZP2突变频谱。透明带完整性的破坏会损害卵母细胞与周围颗粒细胞间的TGF-β信号通路，进而引发细胞凋亡增加与卵母细胞发育潜能下降。