Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella

Ying Shen,Jinyan Xu,Xiaoyu Yang,Weiyu Li,Xiaoliang Li,Feng Zhang,Ke Wang,Juan Cheng,Li Tang,Jianfeng Sun,Peng Xue ,Weiqi Kong,Kai Sheng,Lei Yan ,Fengyun Hu,Wenling Tu,Fuping Li,Shuying Li,Yihong Yang,Yuan Gao ,Mohan Liu,Haojuan Wu,Xudong Zhao,Huanxun Yue,Xiang Wang,Kailai Cai,Xiaohui Jiang,Xueguang Zhang,Chuan Jiang,Wei Xu

doi:10.1038/s41467-018-08182-x

Abstract

Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.

Highlights

morphological abnormalities of the flagella (MMAF) is not a rare autosomal-recessive inherited disorder in humans, the genetic etiology of a large proportion of MMAF cases remains elusive[4–6]
When we knocked down QRICH2 in NT2 cells which have been identified to express QRICH2, AKAP3, calcium binding tyrosine phosphorylation regulated (CABYR), ODF2, and testis specific serine kinase 4 (TSSK4), and the decreased protein amount of AKAP3, CABYR, ODF2, and TSSK4 was observed in these knocked-down cells compared to the control cells (Supplementary Fig. 5d), the decreased levels of the proteins which we focus on are caused by the reduced expression of QRICH2 directly but not the lack of sperm tail structures
To find out whether QRICH2 acted as a trans-acting factor to enhance the transcriptional activity of ODF2 and CABYR, chromatin immunoprecipitation (ChIP)-PCR was performed and the results showed that QRICH2 could bind to the promoter regions of CABYR and ODF2 (Fig. 6b)

Summary

Results

Loss-of-function mutations of QRICH2 in males with MMAF. Two infertile males from two consanguineous families were investigated in our study (Fig. 1a, b). To confirm the decreased expression of Akap[3], Tssk[4], Ropn[1], March[10], Cabyr, and Odf[2] was caused by the deficiency of Qrich[2] directly, but not by the sperm tail defects, we performed further research on heterozygous (Ht) QRICH2 mice which have the normal flagella development (Supplementary Fig. 5b). QRICH2 regulates the formation of outer dense fiber in sperm flagella by directly enhancing the transcription of ODF2 and stabilizing TSSK4 through inhibition of its ubiquitination degradation, which is a crucial factor for ODF2 phosphorylation Overall, these findings reveal that QRICH2 is required for maintaining the normal structure of sperm flagella by regulating key proteins in the related signaling pathways (Fig. 7a). Reduced sperm motility is the basic character for asthenospermia which is defined as a semen

Discussion

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Methods