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

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.


Introduction
Human infertility, due to a wide range of heterogeneous phenotypes, is defined as being unable to achieve a clinical pregnancy despite one year of regular and unprotected intercourse [1]. As a widespread health issue, infertility affects 10-15% of couples [2]. Causing about 45-50% infertility cases, male infertility is clinically diagnosed as azoospermia,

LRRC46 Is Specifically Expressed in Adult Testes
Analysis of RNA profiling data in the Mouse ENCODE project and NCBI (https://www. ncbi.nlm.nih.gov/gene/69297) (accessed on 1 May 2021), indicated that Lrrc46 mRNA is present at very high levels in adult testes, at low levels in adult ovaries and lungs, and at only baseline levels in other mouse organs. Corresponding to the expression analysis in the NCBI, the RT-PCR analysis of multiple organs show that Lrrc46 is expressed abundantly in adult testis, at a low level in the adult ovary, and is not apparently expressed in other organs ( Figure S1a).
At the protein level, immunoblotting of LRRC46 showed expression in the adult testis while no expression in other organs, including the heart, liver, spleen, lung, kidney, or ovary ( Figure 1a). A developmental time series analysis showed that that Lrrc46 was initially detected in the testis at postnatal day 21 (PD21), with the level increasing continuously from postnatal PD25 onward to a plateau at PD36, indicating that Lrrc46 might function in spermatogenesis (Figure 1b).

Generation of Lrrc46 Mutant Mice
With an ATG start codon in exon 1 and TGA stop codon in exon 8, the 6.81 kb testisspecific Lrrc46 gene is located on chromosome 11. To characterize the potential functions of Lrrc46 during spermatogenesis, Lrrc46 −/− mice were created using a CRISPR/Cas9mediated genome editing system from Cyagen Biosciences. Selecting exon 3 to exon 7 as the target sites (Figure 1c), 4075 bp were deleted. Via genomic DNA sequencing, the founder animals were genotyped and confirmed the genome edit. The total size of the Lrrc46 locus in Lrrc46 +/+ mice is 697 bp while the knockout mice was 398 bp (Figure 1d). Immunoblotting of testis extracts indicated that no LRRC46 protein was expressed in Lrrc46 −/− mice (Figure 1e). (e) Immunoblotting of LRRC46 in Lrrc46 +/+ and Lrrc46 −/− testes. Tubulin was used as the control. (f) The size of the testis was comparable between Lrrc46 +/+ and Lrrc46 −/− mice. (g) The body weights of Lrrc46 +/+ and Lrrc46 −/− male mice (n = 3 independent experiments); (h) The testis weights of Lrrc46 +/+ and Lrrc46 −/− male mice (n = 3 independent experiments); (i) The ratio of testis weight to body weight in the Lrrc46 +/+ and Lrrc46 −/− male mice (n = 3 independent experiments); (j) The average litter size of Lrrc46 +/+ and Lrrc46 −/− male mice in 3 months (n = 5 independent experiments). KO males were completely sterile. Data are presented as the mean ± SD. **** p < 0.0001, ns (no significant differences).

Lrrc46 KO Does Not Impact Testes Size but Causes Male Infertility
At the gross and then histological levels, examining the testis structure of adult male mice led to our identification of phenotypic changes in the Lrrc46 knockout male mice. Compared to the wild-type (WT) mice, the in the Lrrc46 knockout mice showed no significant differences in the testis size, testis weight, body weight, or the ratio of testes weight to mouse body weight (Figure 1f-i). We further examined the fertility of Lrrc46 male and female knockout mice. Exhibiting normal mounting behaviors, the Lrrc46 knockout male mice produced copulatory plugs after mating to WT adult female mice, but produced no pups (Figure 1j). In contrast, Lrrc46 knockout female mice generated offspring after mating with similarly aged wild-type males ( Figure S1e). HE-staining of ovary sections revealed no differences in the number or morphology of follicles between Lrrc46 +/+ and Lrrc46 −/− female mice ( Figure S1b). These results establish that Lrrc46 is required for male fertility but has no effect in female fertility.

Lrrc46 Is Required for Spermatogenesis
HE-staining of testes sections revealed obvious phenotypes in the sperm within the seminiferous tubules of Lrrc46 −/− mice. Briefly, the seminiferous tubules of the Lrrc46 +/+ mice had a tubular lumen with flagella appearing from the developing spermatids, while the flagella were absent in the seminiferous tubules of Lrrc46 −/− mice ( Figure 2a, red arrow). Subsequent IF-staining for α/β-tubulin-a specific flagellum marker-confirmed the defects in sperm flagellum biogenesis resulting from Lrrc46 −/− male mice (Figure 2b). These observations clearly suggest that Lrrc46 functions in flagellum formation.
To characterize the potential functions of Lrrc46 during sperm development, we further performed PAS experiments. PAS analysis showed that all of the seminiferous epithelium components and stages could be detected in both the Lrrc46 +/+ and Lrrc46 −/− male mice ( Figure 2c). The most prominent defects were observed in the spermatids at all stages of spermatogenesis: abnormally elongated and constricted sperm head shapes were identified in the Lrrc46 −/− male mice seminiferous tubules. Some head defected mature spermatozoa were found in the Stage IX in the Lrrc46 −/− male mice seminiferous tubule, which indicated that the absence of Lrrc46 influences the normal sperm release (Figure 2c).
We next analyzed the sperm head shaping between Lrrc46 +/+ and Lrrc46 −/− male mice, and no differences in Lrrc46 −/− materials from the Golgi apparatus period (step 1 to step 3) to the capping period (step 4 to step 8) were observed. During acrosomal migration (step 9 to step 13) and the chromatin condensation period (step 14 to step 16), we found major morphological differences in the sperm heads between the seminiferous tubules of Lrrc46 +/+ and Lrrc46 −/− male mice. Interestingly, head shaping was initiated at step 9 to step 10, and the morphology of the elongated Lrrc46 −/− spermatid heads was roughly normal compared with that of Lrrc46 +/+ mice. During step 11 to step 18 of the spermatids head shaping period, the sperm presented abnormal, club-shaped head morphology in Lrrc46 −/− male mice while Lrrc46 +/+ mice presented normal, hook-shaped heads (Figure 2d). This phenomenon became apparent between step 11 and step 16, which indicated that the absence of Lrrc46 defects the normal sperm head shaping [65]. Together, these results indicate that Lrrc46 is required for normal spermatogenesis. The PAS and HE-staining analysis of the testis seminiferous tubule cross-sections of Lrrc46 +/+ and Lrrc46 −/− male mice. The arrows highlight germ cells at various stages of spermatogenesis. Defects in the nuclear shape of several elongating spermatids were clearly evident in the Lrrc46 −/− male mice seminiferous tubule (red circle). P: pachytene spermatocyte, D: diploneme spermatocyte, Z: zygotene spermatocyte, M: meiotic spermatocyte, rST: round spermatid, eST: elongating spermatid, spz: spermatozoa. (d) The PAS and HE-staining analysis of spermatids at different steps from Lrrc46 +/+ and Lrrc46 −/− male mice. During step 1 to step 10 spermatids of acrosome development period, the sperm acrosome morphology was roughly normal in the Lrrc46 −/− male mice. During step 11 to step 18, te spermatids head shaping period, the sperm had an abnormal, club-shaped sperm head morphology in Lrrc46 −/− male mice while Lrrc46 +/+ mice had normal, hook shaped heads.

Knockout of Lrrc46 in Mice Leads to MMAF Phenotypes and Low Sperm Motility
HE-staining of caudal epididymides transverse sections revealed only a few spermatozoa in the epididymal lumen of Lrrc46 −/− mice. In addition, some dead cells could be detected in the Lrrc46 −/− seminiferous tubules ( Figure 3a). Then, we examined the spermatozoa that were released from the caudal epididymis and found the sperm count in the Lrrc46 −/− mice to be significantly decreased compared with the Lrrc46 +/+ mice (Figure 3b). To further observe the single sperm morphological characteristics by PS, we found that the Lrrc46 −/− sperm exhibit MMAF phenotypes, which are short, coiled, irregular, or absent flagella ( Figure 4a). The percentage of the short, coiled, irregular, or absent flagella in the Lrrc46 knockout mice were higher than in the wild-type mice (Figure 4f-i). The ratio of spermatozoa with abnormal heads and flagella in the Lrrc46 −/− mice is significantly higher than in the wild-type mice (Figure 4d,e). IF experiments of anti-α/β-tubulin (red) antibodies, lectin peanut agglutinin (PNA, green), and DAPI (blue) in single spermatozoa also indicated that Lrrc46 −/− sperm exhibit MMAF phenotypes (Figure 4b). Using SEM to observe ultrastructure of sperm flagellum revealed that the Lrrc46 −/− sperm had a variety of abnormal head shapes, as well as coiled, short, or absent flagella while the spermatozoa from wild-type mice had normal morphology ( Figure 4c). Notably, we observed the mitochondrial sheath was defected in the midpiece of sperm flagellum (Figure 4a,c). IF analysis with Mito Tracker, which is used to visualize mitochondria, showed that the mitochondrial sheath was malformed in the Lrrc46 −/− spermatozoa ( Figure S1c,d).  The data are presented as the mean ± SD. * or ** p < 0.01. Via CASA, the Lrrc46 knockout male mice presented a significantly decreased sperm motility rate compared to Lrrc46 +/+ mice. The percentage of motile spermatozoa of the Lrrc46 +/+ mice was 77.67% and the progressive spermatozoa of the Lrrc46 +/+ mice was 21.67% (p < 0.01), while no motile spermatozoa were found for the Lrrc46 −/− mice (Figure 3c,d). We observed significant differences in sperm motility Lrrc46 −/− mice VAP, VSL, and VCL compared to the WT mice (Figure 3e-g) (p < 0.01), as three important parameters to further assess spermatozoa motility. These results indicated that Lrrc46 is important for maintaining normal sperm motility.

Loss of Lrrc46 Causes Sperm Flagellum Ultrastructure Abnormalities
Using TEM analysis, we further observed the sperm flagellum defects in Lrrc46 knockout mice in ultrastructure. Cross sections of the Lrrc46 +/+ mice sperm samples revealed the components of the sperm head and flagellum, including the acrosome of the head, mitochondrial sheath, the outer dense fibers of the midpiece, and the fibrous sheath of the principal piece. A TEM of the Lrrc46 +/+ mice sperm samples showed sperm native architecture, which included nine obvious doublet microtubules and a central pair microtubule structure (9+2) [10,11]. Further observation of the ultrastructure of sperm flagellum revealed that Lrrc46 −/− mice show hypertrophy and hyperplasia of mitochondria, disarrangement and hyperplasia of outer dense fibers, and an absence of the fibrous sheath, compared to doublet microtubules and a central pair microtubule structure (9+2) in the proper position in Lrrc46 +/+ mice sperm flagellum (Figure 5a). In the Lrrc46 −/− flagella, the central pair microtubule structure was absent or disrupted, various peri-axonemal microtubule components were also distorted, and nine double microtubule doublets split with each other (Figure 5b). We observed detachment of the acrosome from the nuclear envelope from step 11 to step 16 in the Lrrc46 −/− flagella ( Figure S2a,b), which indicates that the absence of Lrrc46 led to acrosomal defects in sperm. These sperm flagellum morphology defects in ultrastructure confirmed that Lrrc46 may function for sperm flagellum formation or maintaining sperm flagellum stability.

Abnormal Sperm Manchettes Morphology in Lrrc46 −/− Male Mice
Via the PAS (Figure 2c,d) and TEM (Figure 6), abnormally elongated and constricted sperm head shapes were observed in the Lrrc46 −/− male mice seminiferous tubules. The presence of long and abnormal spermatid heads suggests defects in the function of the manchette, which is involved in sperm head shaping. In order to identify the prominent causes of abnormal head shaping in Lrrc46 −/− male mice, we use the antibody against acetylated tubulin, a flagellum-specific marker. IF experiments of anti-α/β-tubulin antibodies, lectin peanut agglutinin (PNA), and DAPI in single spermatozoa showed that manchette formation was normal in step 8 spermatids in Lrrc46 −/− mice, while step 9 to step 13 spermatids of Lrrc46 −/− mice had abnormally long and deformed manchettes compared with the WT controls ( Figure 7a). These data indicated that the ablation of Lrrc46 caused abnormal spermatozoa manchette formation from step 8 to step 13. The manchette of Lrrc46 −/− spermatozoa displayed abnormal elongation.

Lrrc46 Is Located at All Sperm Flagellum Essentially at the Midpiece
Ablation of Lrrc46 affects the normal male spermatozoa head and flagellum, but how the LRRC46 protein works during sperm development is still unknown. We performed the IF of anti-Lrrc46 (red) antibodies, α/β-tubulin (green), and DAPI (blue) in spermatozoa from the Lrrc46 +/+ and Lrrc46 −/− male mice. The IF analysis shows that Lrrc46 expressed very strongly at the midpiece of the flagellum, and lightly throughout the whole wild-type sperm flagellum, whereas α-tubulin marked the whole tail in the elongated spermatids around step 15 to step 16, suggesting Lrrc46 an axonemal pattern (Figure 7b). No Lrrc46 staining was detected in the Lrrc46 −/− mice flagellum (Figure 7b). In the last phase of spermiogenesis, Lrrc46 appears to be required in spermatid elongation, and development of the sperm flagellum.

Discussion
In the present study, our generation and characterization of Lrrc46 −/− mice with deletion of exons 3-7 identified the essential role of Lrrc46 in spermatogenesis and male fertility. Lrrc46 is expressed strongly in mouse testes, and we demonstrate that Lrrc46 knockout mice represent a male infertility model with characteristic MMAF manifestations, including short, coiled, irregular, and absent flagella; these knockout mice have reduced total sperm count and abnormalities in sperm motility. TEM analysis of testis cross sections revealed that the short flagella of Lrrc46 −/− male mice are disordered at their fibrous sheaths, outer dense fibers, manchette, and mitochondria. Observations of the spermatozoa from Lrrc46 −/− male mice clearly revealed the detachment of the acrosome from the nuclear envelope. IF experiments in Lrrc46 +/+ mice sperm indicated that Lrrc46 is located at the flagellum midpiece, where it apparently functions in flagellum formation. Our study provides evidence that the dysregulation of Lrrc46 can deleteriously affect sperm flagellum development and stability.
Previous studies have indicated Lrrc46 was found as a novel candidate susceptibility gene in familial prostate cancer (PCa) [66], high-grade serous ovarian cancer (HG-SOC) [67,68], Glioblastoma [69], and Nasopharyngeal carcinoma (NPC) [70]. In a silico data study, Lrrc46 was first described in the testes, which is involved not only primarily in structure elaboration or maintenance of germ cell process, but also in acid nucleic metabolism [71]. In a presently prospective cohort study, they use array hybridization, quantitative real-time polymerase chain reaction, and Counter technology to find the specifically expressed genes that are associated with ectopic pregnancy (ECT) and miscarriage in women. They found five cilia-associated genes, including C20orf85, Lrrc46, Rsph4a, and Wdr49 [72]. Most of the cilia-associated genes were predicted to regulate tubulin and microtubule assembly, or be a part of cilia complexes. The above studies indicated that Lrrc46 was a cilia-associated gene. However, these studies did not give any analysis about Lrrc46 function in severe tumors at all. Our results identified an essential role for Lrrc46 in spermatogenesis and male fertility and show that Lrrc46 deficiency leads to an atypical (relatively severe) MMAF phenotype, thus improving our understanding of the mechanisms underlying sperm flagellum development and head shape during spermiogenesis. These findings can facilitate the development of improved treatments for gene-affected male infertile individuals.

Generation of Lrrc46 Knockout Mice via CRISPR/Cas9 Gene Editing System
The mouse Lrrc46 gene (Transcript: ENSMUSG00000020878) is 6.81 kb and located on chromosome 11, containing 8 exons, with an ATG start codon in exon 1 and a TGA stop codon in exon 8. Selecting exon 3 to exon 7 as the target site, Lrrc46 knockout mice were created using a CRISPR/Cas9-mediated genome editing system from Cyagen Biosciences. The gRNA and Cas9 mRNA were co-injected into fertilized eggs of C57BL/6 mice, to generate a targeted line with a 4075 bp base deletion, CGTCAGGAACGTCCTAGAAGAGGdel-4075bp AATCTCACTGACACCGTCCTAGG. The founder animals were identified by DNA sequence analysis. Intercross heterozygous-targeted mice were created to generate normal offspring. Using PCR amplification of genomic DNA that was extracted from mouse tails, all the mice's genotypes were distinguished. For the Lrrc46 knockout mice, the specific primers were forward: 5-TGGTATAATCCTGGCCCTCAAG-3 and reverse: 5-GTTCCTGATGCTGATGCAGTTC-3, yielding a 697 bp fragment. Similarly, forward:5-GCAGGACTGTGTTCAAGGCGGTG-3 and reverse: 5-TGGTATAATCCTGGCCCTCAAG-3 were performed for the Lrrc46 wild-type mice, yielding a 398 bp fragment. The PCR annealing temperature was 60.0 • C. The mice and animal care protocols were approved by the Regional Ethics Committee of the School of Medicine, Shandong University.

Tissue Collection and Histological Analysis
Lrrc46 +/+ and Lrrc46 −/− male mice (n = 5) for each genotype were used in this experiment. After being killed by euthanasia, all the samples were dissected, fixed in 4% paraformaldehyde (P1110, Solarbio, Beijing, China) or Bouin's for 24 h at 4 • C. After being dehydrated in 70% ethanol, all the samples were embedded in paraffin, then sectioned at 5 µm and mounted on glass slides for staining. The slides were then stained with hematoxylin and eosin (HE) and PAS staining routinely.

RNA Extraction, cDNA Synthesis, and RT-PCR
For the RT-PCR experiments to analyze the expression of Lrrc46 mRNAs in various organs, all the RNA was isolated from Lrrc46 wild-type adult mice organs, including heart, liver, spleen, kidney, colon, muscle, fat, brain ovary, and testis. All the cDNA samples were synthesized using the Prime Script TM RT Reagent Kit with gDNA Eraser and TB Green Premix Ex Taq (Takara, RR037A). All RT-PCR reaction experiments were started with an initial denaturation at 95 • C for 10 min, followed by 30 cycles of denaturation at 95 • C for 30 s, then annealed at 65 • C for 30 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min using a T100 Thermal Cycler (Bio-Rad). The specific forward and reverse primers were as followed: Lrrc46 primer Forward: 5-TCACACTCTCGGTGAACTGG-3 and Reverse: 5-GCTCCTATCACCATCTTCCTGT-3. β-actin was amplified as a housekeeping gene. β-actin primer Forward: 5-CATTGCTGACAGGATGCAGAAGG-3 and Reverse: 5-TGCTGGAAGGTGGACAGTGAGG-3, yielding a 351 bp fragment.

Immunoblotting
To prepare the tissue proteins, the tissues were extracted using lysis buffer (P0013, Biyuntian, Shanghai, China) and 1% (v/v) protease inhibitor cocktail (4693116001, Roche, Shanghai, China). After tissue grinding and ultrasonication, the samples were incubated on ice for 25 min. Then, the protein lysates were centrifuged at 12,000 rpm for 25 min at 4 • C. The supernatant liquid of the extracts was used for immunoblotting. The proteins were separated by SDS-PAGE and electro-transferred onto a nitrocellulose membrane. Then, the membrane was blocked in 5% skim milk overnight and then incubated with corresponding primary and secondary antibodies.

Mouse Sperm Collection
The caudal epididymides were surgically and immediately dissected from the Lrrc46 +/+ and Lrrc46 −/− male mice. They were squeezed out from the caudal epididymis, the sper-matozoa were released in 1 mL phosphate buffered saline (PBS) for 30 min at 37 • C, under 5% CO 2 atmosphere. Then the samples were provided for sperm counting, morphological analysis, and IF experiments.

Single Sperm Morphological Analysis
The sperm morphological analysis was evaluated accorded to the World Health Organization (WHO) guidelines. From every Lrrc46 wild-type and knockout mouse, more than 250 spermatozoa were analyzed. A total of 10 µL sperm samples from each mouse was mounted on glass slides and dried overnight. Then these glass slides were performed PS. Diluted at a ratio of 1:100, via hemocytometer all the sperm samples were counted at least five times.

Sperm Motility Assessment Using CASA
Lrrc46 +/+ and Lrrc46 −/− male mice (n = 10) for each genotype were killed by euthanasia. The cauda epididymis was quickly cut into pieces in 1 mL human tubal fluid (HTF) (Millipore) or PBS, then incubated for 5 min at 37 • C. After incubating, 10 µL sperm liquid taken from each sample was put on the glass slide (Hamilton Thorne, 80 micron 2X-CEl), and taken captures via a CCD camera. Via a computer-assisted animal motility system (Version.12 CEROS, Hamilton Thorne, Beverly, MA, USA), more than 250 spermatozoa from every sample were performed by computer-associated semen analysis (CASA), using an Olympus B x51 microscope through a 20X phase objective (Olympus, Tokyo, Japan). Via computer-associated semen analysis (CASA), the sperm samples were observed to evaluate the parameters of total sperm motility, including the motile spermatozoa, progressive spermatozoa, average path velocity (VAP), progressive velocity (VSL), the track speed (VCL), and so on.

Scanning Electron Microscopy (SEM)
Lrrc46 +/+ and Lrrc46 −/− male mice (n = 3) for each genotype were killed by euthanasia. The cauda epididymis was quickly cut into pieces in 1 mL PBS. After shredding, 10 µL sperm liquid taken from each sample was put on the coverslips. Fixed with 2.5% glutaraldehyde, all coverslips were post-fixed in osmium tetroxide, then washed with water for at least for three times, dehydrated through 50%, 70%, 95%, and 100% ethanol. Via Tousimis Autosamdri-810 Critical Point Dryer, the coverslips were dried at 55 • C, a critical point. The samples then mounted onto specimen stubs, these coverslips were sputter-coated with palladium, and viewed with a FEI Quanta ESEM 200 SEM.

Transmission Electron Microscopy Analysis (TEM)
Lrrc46 +/+ and Lrrc46 −/− male mice (n = 5) for each genotype were killed by euthanasia. All the adult testis and epididymis samples were cut into pieces of approximately 1 mm 3 , and fixed immediately in 2.5% (v/v) glutaraldehyde overnight for 4 • C. All the samples were immersed in 1% OsO4 for 1 h at 4 • C, gradually dehydrated using a series of acetone, and then embedded in 100% resin. Then, cutting on an ultramicrotome, the samples ultrathin sections were stained with uranyl acetate and lead citrate. Lastly, the sections were imaged and analyzed via a transmission electron microscope (JEM-1400, JEOL, Tokyo, Japan).

IF in Testes
Lrrc46 +/+ and Lrrc46 −/− male mice (n = 6) for each genotype were used in this experiment. After being killed by euthanasia, all the samples were dissected, fixed in 4% paraformaldehyde (P1110, Solarbio, Beijing, China) or Bouin's for 24 h at 4 • C. After being dehydrated in 70% ethanol, all the samples were embedded in paraffin, then sectioned at 5 µm and mounted on glass slides. These sections were deparaffinized in 100% (v/v) dimethylbenzene for 30 min, rehydrated in 100%, 95%, 80%, 75% (v/v) ethanol dimethylbenzene for 25 min, antigen retrieval in 10 mM sodium citrate buffer (pH 7.2) for 25 min, washed several times in PBS (pH 7.4), followed by a decrease to room temperature. After being dropped to room temperature, these sections were blocked with 5%BSA for 1 h. After blocking with 5% BSA or 5% bovine serum albumin for 1 h, these sections were perforated with 0.5% (v/v) Triton-X 100. Then the primary antibodies were added to the glass slides and incubated at 4 • C for 14-16 h. These were washed several times in PBS (pH 7.4), the secondary antibodies were added for 1.5 h at 37 • C, the DAPI were added for 10 min 37 • C. All the IF samples were imaged by a confocal microscopy which contained the Leica Andor Dragonfly spinning disc confocal microscope driven by Fusion Software. The images were performed with ImageJ Software (NIH, version 1.6.0-65) or Bitplane Imaris (version 8.1) software.

IF in Single Spermatozoa
Lrrc46 +/+ and Lrrc46 −/− male mice (n = 6) for each genotype were killed by euthanasia. The cauda epididymis was quickly cut into pieces in 1 mL PBS. A total of 10 µL mice sperm liquid that was taken from each sample was put on the glass slides and air-dried, then stored at −80 • C. All the sections were fixed in 4% PFA for 15 min, blocked with 5%BSA for 1 h at room temperature. After being blocked with 5%BSA or 5% bovine serum albumin for 1 h, these sections were perforated with 0.5% (v/v) Triton-X 100. Then the primary antibodies were added to the glass slides and incubated at 4 • C for 14-16 h. After being washed several times in PBS (pH 7.4), the secondary antibodies were added for 1.5 h at 37 • C, the DAPI were added for 10 min 37 • C.

IF in Testicular Germ Cells
Lrrc46 +/+ and Lrrc46 −/− male mice (n = 6) for each genotype were killed by euthanasia. All the testes were immediately fixed with 2% paraformaldehyde in 0.05% PBST (PBS with 0.05% Triton X-100) or 5 min. About 20 µm fixed seminiferous segment tubules were placed on the glass slides that contained 100 µL fixation solution, covered with coverslips and were pressed down on. Then these glass slides were frozen in liquid nitrogen and stored at stored at −80 • C. After the coverslips were removed, these slides were fixed in 4% PFA for 15 min at room temperature and blocked with 5%BSA for 1 h. After being blocked with 5%BSA or 5% bovine serum albumin for 1 h, these sections were perforated with 0.5% (v/v) Triton-X 100. Then the primary antibodies were added to the glass slides and incubated at 4 • C for 14-16 h. After being washed several times in PBS (pH 7.4), the secondary antibodies were added for 1.5 h at 37 • C, the DAPI were added for 10 min 37 • C.

Statistical Analysis
In this paper, all of the experiments were repeated at least three times, and all of the presented error bars were denoted as the mean ± SEM. The statistical analyses were performed using GraphPad Prism (version 8.02) by the Student's t-test with a paired, twotailed distribution. The differences were considered significant while the p values < 0.05 (*), 0.01 (**), or 0.001 (***).