LRRC46 Accumulates at the Midpiece of Sperm Flagella and Is Essential for Spermiogenesis and Male Fertility in Mouse

Abstract

The sperm flagellum is essential for male fertility. Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. MMAF phenotypes are understood to result from pathogenic variants of genes from multiple families including AKAP, DANI, DNAH, RSPH, CCDC, CFAP, TTC, and LRRC, among others. The Leucine-rich repeat protein (LRRC) family includes two members reported to cause MMAF phenotypes: Lrrc6 and Lrrc50. Despite vigorous research towards understanding the pathogenesis of MMAF-related diseases, many genes remain unknown underlying the flagellum biogenesis. Here, we found that Leucine-rich repeat containing 46 (LRRC46) is specifically expressed in the testes of adult mice, and show that LRRC46 is essential for sperm flagellum biogenesis. Both scanning electron microscopy (SEM) and Papanicolaou staining (PS) presents that the knockout of Lrrc46 in mice resulted in typical MMAF phenotypes, including sperm with short, coiled, and irregular flagella. The male KO mice had reduced total sperm counts, impaired sperm motility, and were completely infertile. No reproductive phenotypes were detected in Lrrc46−/−female mice. Immunofluorescence (IF) assays showed that LRRC46 was present throughout the entire flagella of control sperm, albeit with evident concentration at the mid-piece. Transmission electron microscopy (TEM) demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. About the important part of the Materials and Methods, SEM and PS were used to observe the typical MMAF-related irregular flagella morphological phenotypes, TEM was used to further inspect the sperm flagellum defects in ultrastructure, and IF was chosen to confirm the location of protein. Our study suggests that LRRC46 is an essential protein for sperm flagellum biogenesis, and its mutations might be associated with MMAF that causes male infertility. Thus, our study provides insights for understanding developmental processes underlying sperm flagellum formation and contribute to further observe the pathogenic genes that cause male infertility.

The lack of Tex44 causes severe subfertility with flagellar abnormalities in male mice

“…Its location is restricted to the midpiece, like TEX44. However, the knockout of Lrrc46 showed typical MMAF phenotypes including coiled, short, and irregular flagella [ 24 ]. The Tex44 -KO doesn’t seem to affect deeply the annulus as SEPTIN4 is normally detected at the right position and, in electron microscopy images, the expected triangular dense structure of the annulus is visible between the midpiece and principal piece, in spite of surrounding abnormalities.…”

Deficiency of the Tmem232 Gene Causes Male Infertility with Morphological Abnormalities of the Sperm Flagellum in Mice

“…After cutting into ultra-thin sections using an ultramicrotome, the sections were double stained with lead citrate along with uranyl acetate. Images of the stained sections were acquired using a JEM-1400 transmission electron microscope [ 17 ], and subsequent analysis was performed.…”

“…After dehydration, the samples were dried with a Tousimis Autosamdri-810 Critical Point Dryer and mounted on a specimen base. Prior to imaging and analysis, the coverslips were sputtered on a co-rail clamp, which was conducted using a FEI Quanta ESEM 200 SEM [ 17 ].…”

Novel biallelic mutations in TTC29 cause asthenoteratospermia and male infertility

“…With the development of next‐generation sequencing, the investigation of MMAF‐associated genes has progressed. To date, 42 MMAF‐associated genes have been reported, including AK7 (MIM:615364), AKAP3 (MIM:604689), AKAP4 (MIM:300185), ARMC2 (MIM:618424), WDR19 (MIM:608151), WDR63 / DNAI3 (MIM:617968), CEP135 (MIM:611423), CFAP43 / WDR96 (MIM:617558), CFAP44 / WDR52 (MIM:617559), CFAP47 (MIM:301057), CFAP53 (MIM:614759), CFAP58 (MIM:619129), CFAP61 , CFAP65 (MIM:614270), CFAP69 (MIM:617949), CFAP70 (MIM:618661), CFAP74 , CFAP91 (MIM:609910), CFAP251 / WDR66 (MIM:618146), CFAP206 , CCDC34 (MIM:612324), CCDC38 , CCDC39 (MIM:613798), CCDC40 (MIM:613799), DNAH1 (MIM: 603332), DNAH2 (MIM:603333), DNAH6 (MIM: 603336), DNAH8 (MIM:603337), DNAH10 (MIM:605884), DNAH12 (MIM:603340), DNAH17 (MIM:610063), DZIP1 (MIM:608671), DRC1 (MIM:615288), FSIP2 (MIM:618153), ODF2 (MIM:602015), QRICH2 (MIM:618304), SPEF2 (MIM:610172), SPAG6 (MIM:605730), STK33 (MIM:607670), LRRC46 , TTC21A (MIM: 611430), and TTC29 (MIM: 618735) (Baccetti et al, 2005 ; Chen et al, 2021 ; Cong et al, 2022 ; He et al, 2020 ; Li et al, 2021 ; Li et al, 2022 ; Liu et al, 2020 ; Liu et al, 2021 ; Lu et al, 2021 ; Ma et al, 2021 ; Ma et al, 2022 ; Ni et al, 2020 ; Sha et al, 2020 ; Shen et al, 2021 ; Sironen et al, 2020 ; Tang et al, 2017 ; Visser et al, 2011 ; Wu et al, 2021 ; Xu et al, 2020 ; Xu, Tang, et al, 2022 ; Xu, Yang, et al, 2022 ; Yin et al, 2022 ; Zhang et al, 2021 ; Zhang et al, 2022 ; Zhu et al, 2022 ). However, there are still many individuals with MMAF that cannot be causally diagnosed, which indicates that there may be other pathogenic mutations not yet discovered.…”

“…With the development of next-generation sequencing, the investigation of MMAF-associated genes has progressed. To date, 42 MMAF-associated genes have been reported, including AK7 (MIM:615364), AKAP3 (MIM:604689), AKAP4 (MIM:300185), ARMC2 (MIM:602015), QRICH2 (MIM:618304), SPEF2 (MIM:610172), SPAG6 (MIM:605730), STK33 (MIM:607670), LRRC46, TTC21A (MIM: 611430), and TTC29 (MIM: 618735) (Baccetti et al, 2005;Chen et al, 2021;Cong et al, 2022;He et al, 2020;Li et al, 2022;Liu et al, 2020;Liu et al, 2021;Lu et al, 2021;Ma et al, 2021;Ma et al, 2022;Ni et al, 2020;Sha et al, 2020;Shen et al, 2021;Sironen et al, 2020;Tang et al, 2017;Visser et al, 2011;Wu et al, 2021;Xu et al, 2020;Xu, Tang, et al, 2022;Xu, Yang, et al, 2022;Yin et al, 2022;Zhang et al, 2021;Zhang et al, 2022;Zhu et al, 2022). However, there are still many individuals with MMAF that cannot be causally diagnosed, which indicates that there may be other pathogenic mutations not yet discovered.…”