American journal of obstetrics and gynecology · 2024
Homozygous missense variations of APC12 cause meiotic metaphase I arrest in oocytes and female infertility.
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Lin Yunying, Wei Zhe, Zhang Ling, Yao Yejie, Huang Yi, Yao Guangxin, Wang Wangsheng, Hu Shuanggang, Ding Ying, Lu Yao, Bian Xuejiao, Dong Xinyi, Guan Hengyu, Huang Yunfei, Sun Yun
Аннотация
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 has not been completely investigated.
OBJECTIVE: 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<.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<.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<.01), which could be rescued by injecting Apc12 cRNA (53.44±1.20%).
CONCLUSION: 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.
OBJECTIVE: 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<.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<.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<.01), which could be rescued by injecting Apc12 cRNA (53.44±1.20%).
CONCLUSION: 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.