DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath

www.nature.com/cddis ARTICLE OPEN DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath Huan Wu 1,2,3,7, Yiyuan Liu1,7, Yuqian Li1,7, Kuokuo Li2,3, Chuan Xu1,4, Yang Gao1,4, Mingrong Lv1,4, Rui Guo2,5,6, Yuping Xu1,5,6, ✉ ✉ ✉ Ping Zhou1,5,6, Zhaolian Wei1,5,6, Rong Hua 2,3,4 , Xiaojin He 1,2,3 and Yunxia Cao 1,2,3 © The Author(s) 2023 1234567890();,: The axonemal dynein arms (outer (ODA) and inner dynein arms (IDAs)) are multiprotein structures organized by light, intermediate, light intermediate (LIC), and heavy chain proteins. They hydrolyze ATP to promote ciliary and flagellar movement. Till now, a variety of dynein protein deficiencies have been linked with asthenospermia (ASZ), highlighting the significance of these structures in human sperm motility. Herein, we detected bi-allelic DNALI1 mutations [c.663_666del (p.Glu221fs)], in an ASZ patient, which resulted in the complete loss of the DNALI1 in the patient’s sperm. We identified loss of sperm DNAH1 and DNAH7 rather than DNAH10 in both DNALI1663_666del patient and Dnali1−/− mice, demonstrating that mammalian DNALI1 is a LIC protein of a partial IDA subspecies. More importantly, we revealed that DNALI1 loss contributed to asymmetries in the most fibrous sheath (FS) of the sperm flagellum in both species. Immunoprecipitation revealed that DNALI1 might interact with the cytoplasmic dynein complex proteins in the testes. Furthermore, DNALI1 loss severely disrupted the transport and assembly of the FS proteins, especially AKAP3 and AKAP4, during flagellogenesis. Hence, DNALI1 may possess a non-classical molecular function, whereby it regulates the cytoplasmic dynein complex that assembles the flagella. We conclude that a DNALI deficiency-induced IDAs injury and an asymmetric FS-driven tail rigid structure alteration may simultaneously cause flagellum immotility. Finally, intracytoplasmic sperm injection (ICSI) can effectively resolve patient infertility. Collectively, we demonstrate that DNALI1 is a newly causative gene for AZS in both humans and mice, which possesses multiple crucial roles in modulating flagellar assembly and motility. Cell Death and Disease (2023)14:127 ; https://doi.org/10.1038/s41419-023-05653-y INTRODUCTION Sperm motility is critical for male fertility since the ejaculated sperm must self-propel its travel along the long female reproductive system to fertilize the egg and initiate life. Asthenozoospermia (ASZ) is a frequent form of primary male infertility, whereby over 40% of cases present some defects in sperm motility [1–3]. Sperm flagellum is uniquely designed for its key biological role of beating wave formation. This highly conserved and microtubular organelle is precisely organized by a core cytoskeletal structure known as the axoneme, as well as a variety of peri-axonemal elements [4]. The axoneme is permanently located in the center of the flagellum and consists of nine peripheral doublet microtubules (DMTs), which circumferentially surround a central microtubular pair (CP) to form the classical ‘9 + 2’ arrangements. Mechanical links formed by radial spokes, dynein arms, and nexin-dynein regulatory complex (N-DRC) maintain the integrity and coordinate the dynamics of these microtubules [5]. In addition, several accessory subunits, comprising the nine outer dense fibers (ODFs), mitochondrial sheath (MS), and fibrous sheath (FS), layer by layer encompass different segments of the central axoneme [6]. Based on these discrete subcellular structures along the longitudinal axis, the flagellum can be classified into three segments, namely, the middle, principal, and end pieces. ODFs are surrounded by the spiral MS in the middle region and by FS in the principal region. Distal to MS, FS is a tapered cylinder that consists of two longitudinal columns (LCs) with nearly symmetrical distribution, and several connecting circumferential ribs (CRs) distributed between the LCs [7]. The discovery of axonemal dyneins provides some insight into the mysterious mechanism of flagellar bending motion [8]. Axonemal dyneins are highly complicated molecular motors that 1 Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, 230022 Hefei, Anhui, China. NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract, Anhui Medical University, No. 81 Meishan Road, 230032 Hefei, Anhui, China. 3Key Laboratory of Population Health Across Life Cycle, Anhui Medical University, Ministry of Education of the People’s Republic of China, No. 81 Meishan Road, 230032 Hefei, Anhui, China. 4Anhui Province Key Laboratory of Reproductive Health and Genetics, Anhui Medical University, No. 81 Meishan Road, 230032 Hefei, Anhui, China. 5Anhui Provincial Engineering Research Center of Biopreservation and Artificial Organs, Anhui Medical University, No. 81 Meishan Road, 230032 Hefei, Anhui, China. 6Anhui Provincial Institute of Translational Medicine, Anhui Medical University, No. 81 Meishan Road, 230032 Hefei, Anhui, China. 7The authors contributed equally: Huan Wu, Yiyuan Liu, Yuqian Li. ✉email: ; ; Edited by Massimiliano Agostini 2 Received: 31 August 2022 Revised: 1 February 2023 Accepted: 3 February 2023 Official journal of CDDpress H. Wu et al. 2 contain a variable number of heavy (DHC), intermediate, light, and light intermediate chain (LIC) polypeptides that vary based on their molecular weight and cellular activity [9]. Given their distinct position within the axoneme, dynein motors can be further subdivided into two classes: outer dynein arms (ODAs) and inner dynein arms (IDAs). Defects within any axonemal dynein arms seriously impair ciliary motility in Chlamydomonas [10, 11]. In mammals, biallelic mutations in multiple genes involved in DHC biogenesis, such as DNAH1 (MIM: 603332) [12], DNAH2 (MIM: 603333) [13], DANH7 (MIM: 610061) [14], DANH8 (MIM: 610061) [15], DANH10 (MIM: 605884) [16], and DANH17 (MIM: 603340) [17], are reported to cause male sterility in humans and mice, which is characterized by severely diminished sperm movement and multiple morphological abnormalities of the flagella (MMAF). These findings not only emphasize the crucial role of DHC in flagellar assembly but also raises the question of how other chains function in unison during spermatogenesis. Dynein axonemal light intermediate chain 1 [DNALI1 (MIM: 602135)] encodes a LIC subunit of IDA in mammals [18, 19]. The orthologue DNALI1 gene, known as p28, was originally identified in Chlamydomonas and is known to interact with DHC2 during axonemal IDA assembly [20]. Notably, its orthologue genes in sea urchins and mice were subsequently identified as having a potential role in modulating sperm motility [19, 21]. This evidence suggested an interesting possibility that DNALI1 is indispensable in sperm flagellogenesis; however, the specific mechanism remains (...truncated)