Purpose

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.

Methods

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.

Results

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.

Conclusions

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.

中文翻译：

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鉴定和拯救导致第一次有丝分裂缺陷和不孕症的新型 TUBB8 突变。

目的

Tubulin beta 8 VIII 类 (TUBB8) 对人类的卵子发生、受精和植入前胚胎发育至关重要。尽管最近在不育女性的减数分裂受阻卵母细胞中发现了TUBB8突变，但对于这种基因突变导致的不孕症没有有效的治疗方法。本研究旨在进一步揭示患者家族中致不孕症的基因突变，探讨能否通过优化胚胎培养条件来挽救不孕症，最终达到使患者受孕的目的。

方法

对患者家属进行全外显子组序列分析和Sanger测序，筛选和鉴定候选突变基因。质粒的构建、体外转录、致病基因cRNA的显微注射和免疫荧光染色被用来概括观察到的患者不孕表型并了解致病原理。同时，在拯救实验中，在生发囊泡 (GV) 或中期 II (MII) 阶段的小鼠卵母细胞中过表达候选基因的突变型和野生型 cRNA。

结果

我们首先在编码区发现了一种新的可遗传TUBB8突变（c.1041C>A：p.N347K），该突变特别影响第一次有丝分裂并导致三代家庭中早期胚胎的发育停滞。我们进一步证明TUBB8突变可能导致异常的纺锤体组装。此外，在表达突变TUBB8的小鼠卵母细胞中额外表达野生型TUBB8 cRNA可以成功挽救所产生胚胎的发育缺陷并产生足月后代。

结论

我们的研究不仅定义了导致胚胎早期卵裂停滞的TUBB8新突变，而且为未来治疗此类女性不孕症提供了重要依据。