Obstetrics and Gynecology Hospital, NHC Key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), State Key Laboratory of Genetic Engineering at School of Life Sciences, Fudan University, Shanghai 200011, China; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai 200011, China; State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing 211116, China.
Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
Am J Hum Genet. 2020 Aug 6;107(2):330-341. doi: 10.1016/j.ajhg.2020.06.004. Epub 2020 Jul 2.
Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.
精子畸形是男性不育的直接因素。鞭毛形态多态异常（MMAF）是一种严重的弱精症，其特征是射出精液中的精子不动，鞭毛畸形和/或缺失。先前的研究表明 MMAF 存在遗传异质性。为了进一步确定 MMAF 的遗传因素，我们对 90 名中国 MMAF 受累男性进行了全外显子组测序。发现有 2 例（2.2%）携带双等位基因错义 DNAH8 变异，这些变异在对照人群中缺失或罕见，并且被多种生物信息学工具预测为有害。对来自法国、伊朗和北非的 167 名 MMAF 受累男性的第二个外显子组数据的重新分析，允许鉴定出另一名携带 DNAH8 纯合移码变异的男性。DNAH8 编码一种轴丝动力蛋白重链成分，在睾丸中优先表达。携带双等位基因 DNAH8 变异的男性的精子进行苏木精-伊红染色和电子显微镜分析，显示精子鞭毛的形态和超微结构高度异常。对携带双等位基因 DNAH8 变异的男性精子进行免疫荧光分析显示，DNAH8 及其相关蛋白 DNAH17 的染色缺失或明显减少。Dnah8 敲除雄性小鼠也表现出典型的 MMAF 表型和不育。有趣的是，使用 Dnah8 敲除雄性小鼠的精子进行胞浆内精子注射导致良好的妊娠结局。总之，我们从人类和小鼠的实验观察表明，DNAH8 对于精子鞭毛的形成是必不可少的，双等位基因有害的 DNAH8 变异导致男性不育和 MMAF。
