BACKGROUND Oocyte maturation arrest is a leading cause of female infertility. However, the genetic variables remain largely unknown. In oocytes, the activation of anaphase-promoting complex/cyclosome (APC/C) is a critical step in the transition from meiosis I to meiosis II. However, the causal relationship between variants in APC/C components and female infertility hasn't been completely investigated. OBJECTIVE(S) This study aims to find a novel gene and its pathogenic mutation as a cause for metaphase I arrest in oocytes, thus introducing a new APC/C component for screening causes of female infertility. STUDY DESIGN Whole-exome sequencing was performed on 30 infertile women with recurrent oocyte maturation arrest without known gene variants. A homozygous missense mutation in the APC12 gene (p.R8H) was identified as a candidate for oocyte metaphase I arrest in a consanguineous family member. The experiment was conducted in vitro with HEK293T cells and mouse oocytes. Methods such as oocyte microinjection, western blotting, co-immunoprecipitation, and immunofluorescence were used. A knockdown mouse model was generated to verify the function of Apc12 in causing oocyte metaphase I arrest. About 100 wild-type C57BL/6J mice and 50 gene-edited mice were used in this study. RESULTS APC12 p.R8H was identified in an infertile woman with oocyte metaphase I arrest. Microinjection of Apc12 mutant cRNA in mouse oocytes caused a considerably higher rate of metaphase I stage arrest (65.21±5.64% vs. 30.86±1.74%, p<0.01) with decreased APC12 protein expression and Securin accumulation compared to the control group, while oocytes injected with Apc12 cRNA showed comparable metaphase I arrest rate (31.51±3.05%). This fit the phenotype we identified in our case and suggested that Apc12 p.R8H was a loss-of-function mutation leading to oocyte metaphase I arrest. The in-vitro experiments in HEK293T cells suggested that the APC12 p.R8H mutation disrupted the interaction between APC12 and APC6, as well as impaired APC/C activity by disrupting Securin ubiquitination. Knocking down APC12 with siRNA in mouse oocytes led to metaphase I arrest (41.65±6.10% vs. 24.20±2.19%, p<0.01). Oocytes from Apc12+/- mice exhibited metaphase I arrest compared with oocytes from wild-type mice (72.29±0.51% vs. 23.33±5.82%, p<0.01), which could be rescued by injecting Apc12 cRNA (53.44±1.20%). CONCLUSION(S) We identified a pathogenic mutation in APC12 in a female patient and confirmed its relevance as a causative factor for metaphase I arrest in oocytes, implying its importance as an APC/C component in the pathophysiology of oocyte maturation arrest, which caused female infertility.

中文翻译：

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APC12 的纯合错义变异导致卵母细胞减数分裂中期 I 停滞和女性不育。


背景 卵母细胞成熟停滞是女性不孕的主要原因。然而，遗传变量在很大程度上仍然未知。在卵母细胞中，后期促进复合物/睫状体 （APC/C） 的激活是从减数分裂 I 过渡到减数分裂 II 的关键步骤。然而，APC/C 成分变异与女性不孕症之间的因果关系尚未得到完全研究。目的 本研究旨在寻找一种新的基因及其致病突变作为卵母细胞中期 I 期停滞的原因，从而引入一种新的 APC/C 成分来筛选女性不孕症的原因。研究设计 对 30 名复发性卵母细胞成熟停滞且无已知基因变异的不孕妇女进行了全外显子组测序。APC12 基因 （p.R8H） 中的纯合错义突变被确定为近亲家族成员卵母细胞中期 I 期停滞的候选者。该实验是在体外用 HEK293T 细胞和小鼠卵母细胞进行的。采用卵母细胞显微注射、western blotting、免疫共沉淀和免疫荧光等方法。生成敲低小鼠模型以验证 Apc12 在导致卵母细胞中期 I 期停滞中的功能。本研究使用了约 100 只野生型 C57BL/6J 小鼠和 50 只基因编辑小鼠。结果 在一名卵母细胞中期 I 期停滞的不孕妇女中发现 APC12 p.R8H。小鼠卵母细胞显微注射 Apc12 突变体 cRNA 导致中期 I 期停滞率相当高 （65.21±5.64% vs. 30.86±1.74%，p<0.01），与对照组相比，APC12 蛋白表达和 Securin 积累降低，而注射 Apc12 cRNA 的卵母细胞显示出相当的中期 I 期停滞率 （31.51±3.05%）。这符合我们在案例中鉴定的表型，并表明 Apc12 p.R8H 是一种功能丧失突变，导致卵母细胞中期 I 期停滞。HEK293T 细胞体外实验表明，APC12 p.R8H 突变破坏了 APC12 和 APC6 之间的相互作用，并通过破坏 Securin 泛素化损害了 APC/C 活性。在小鼠卵母细胞中用 siRNA 敲低 APC12 导致中期 I 期阻滞 （41.65±6.10% vs. 24.20±2.19%，p<0.01）。与野生型小鼠的卵母细胞相比，Apc12+/- 小鼠的卵母细胞表现出中期 I 期停滞 （72.29±0.51% vs. 23.33±5.82%，p<0.01），可以通过注射 Apc12 cRNA （53.44±1.20%） 来挽救。结论 我们在一名女性患者中发现了 APC12 的致病突变，并证实了其作为卵母细胞中期 I 期停滞的致病因素的相关性，这意味着它在导致女性不育的卵母细胞成熟停滞的病理生理学中作为 APC/C 成分的重要性。