Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, 200092, China.
Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China.
J Assist Reprod Genet. 2020 Nov;37(11):2713-2722. doi: 10.1007/s10815-020-01945-w. Epub 2020 Sep 18.
Tubulin beta eight class VIII (TUBB8) is essential for oogenesis, fertilization, and pre-implantation embryo development in human. Although TUBB8 mutations were recently discovered in meiosis-arrested oocytes of infertile females, there is no effective therapy for this gene mutation caused infertility. Our study aims to further reveal the infertility-causing gene mutations in the patient's family and to explore whether the infertility could be rescued by optimizing the conditions of embryo culture and finally achieve the purpose of making the patient pregnant.
Whole-exome sequence analysis and Sanger sequencing were performed on patients' family members to screen and identify candidate mutant genes. Construction of plasmids, in vitro transcription, microinjection of disease-causing gene cRNA, and immunofluorescence staining were used to recapitulate the infertility phenotype observed in patients and to understand the pathogenic principles. Simultaneously, overexpression of mutant and wild-type cRNA of the candidate gene in mouse oocytes at either germinal vesicle (GV) or metaphase II (MII) stage was performed in the rescue experiment.
We first identified a novel heritable TUBB8 mutation (c.1041C>A: p.N347K) in the coding region which specifically affects the first mitosis and causes the developmental arrest of early embryos in a three-generation family. We further demonstrated that TUBB8 mutation could lead to abnormal spindle assemble. And moreover, additional expression of wild-type TUBB8 cRNA in the mouse oocytes in which the mutant TUBB8 were expressed can successfully rescue the developmental defects of resulting embryo and produce full-term offspring.
Our study not only defines a novel mutation of TUBB8 causing the early cleavage arrest of embryos, but also provides an important basis for treating such female infertility in the future.
微管蛋白βVIII 类（TUBB8）对于人类卵母细胞发生、受精和着床前胚胎发育是必不可少的。尽管最近在不孕女性阻滞减数分裂的卵母细胞中发现了 TUBB8 突变，但对于这种由基因突变引起的不孕，目前尚无有效的治疗方法。我们的研究旨在进一步揭示患者家族中导致不孕的基因突变，并探讨是否可以通过优化胚胎培养条件来挽救不孕，最终达到使患者怀孕的目的。
对患者家庭成员进行全外显子组测序和 Sanger 测序，筛选并鉴定候选突变基因。构建质粒，体外转录，显微注射致病基因 cRNA，并进行免疫荧光染色，以重现患者中观察到的不孕表型，并了解致病原理。同时，在候选基因的野生型和突变型 cRNA 在小鼠卵母细胞的生发泡期（GV）或中期 II（MII）阶段的过表达的挽救实验中进行。
我们首先在一个三代家族中鉴定出一个新的遗传性 TUBB8 突变（c.1041C>A：p.N347K），该突变特异性影响第一次有丝分裂，导致早期胚胎发育停滞。我们进一步证明，TUBB8 突变可导致纺锤体异常组装。此外，在表达突变型 TUBB8 的小鼠卵母细胞中额外表达野生型 TUBB8 cRNA 可以成功挽救胚胎发育缺陷，并产生足月后代。
本研究不仅定义了一种导致胚胎早期卵裂阻滞的 TUBB8 新突变，也为未来治疗此类女性不孕提供了重要依据。
