
==== Front
101585129
40574
Andrology
Andrology
Andrology
2047-2919
2047-2927

36428102
10.1111/andr.13350
nihpa1925039
Article
CRISPR/Cas9-mediated disruption of lipocalins, Ly6g5b, and Ly6g5c causes male subfertility in mice
Sakurai Nobuyuki 12
Fujihara Yoshitaka 127
Kobayashi Kiyonori 23
Ikawa Masahito 24567
1 Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, 6-1 Kishibeshinmachi, Suita, Osaka 564-8565, Japan
2 Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
3 Graduate School of Frontier Biosciences, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
4 Graduate School of Pharmaceutical Sciences, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
5 Graduate School of Medicine, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
6 The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Author’s contribution

Designed the research: N.S., Y.F., and M.I.; Performed the research: N.S., Y.F., and K.K.; Analyzed the data: N.S.; and Wrote the paper: N.S., Y.F., and M.I.

7 Correspondence: Yoshitaka Fujihara (fujihara@ncvc.go.jp) and Masahito Ikawa (ikawa@biken.osaka-u.ac.jp)
25 8 2023
25 11 2022
25 5 2024
10.1111/andr.13350https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
Background:

Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis-enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on Chromosomes 2 and 17 in the mouse, respectively.

Objective:

To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype.

Materials and methods:

We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c).

Results:

While the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3.

Conclusion:

These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability.

epididymis
knockout
Adam3
zona pellucida
sperm maturation
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pmcIntroduction

Spermatozoa acquire their ability to fertilize and capability of forward motility during epididymal transit. This event is called epididymal sperm maturation. The epididymis comprises a dense convoluted duct that measures just over one meter in mice (see review by Hinton et al.1) and is divided into the caput, corpus, and cauda. Caput-spermatozoa have very low fertilizing potential although rates vary between species. However, most spermatozoa in the distal part of the epididymis can fertilize oocytes (see review by Bedford2), suggesting that some key factors controlling sperm maturation in the epididymis exist in the caput and/or corpus segment. Our laboratory has been searching genes predominantly expressed in the epididymis using a transcriptome database3 and generating knockout (KO) mice to investigate their physiological functions.4–6 In the present study, we focused on epididymis-enriched lipocalin and Ly6 family genes.

The lipocalin family consists of many small, diverse secreted proteins and is defined by a highly conserved three-dimensional structure.7,8 Most lipocalin family genes are evolutionarily conserved in humans and mice, forming clusters on chromosome 2 in mice.9 LCN2 is secreted in the female reproductive tract and binds to spermatozoa. Although Lcn2 KO male mice are fully fertile, female mice show a significant reduction in pregnancy rate,10,11 suggesting that secreted LCN2 plays a key role in regulating sperm function in the female reproductive tract. Among the family, Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10 are predominantly expressed in the caput epididymis.9 KO mice for each of Lcn6, Lcn8, and Lcn9 showed that these genes were individually dispensable for male reproduction.12,13 However, these studies did not explore the possibility that other lipocalin family members complimented the roles of the disrupted lipocalin genes.

The Ly6 family genes belong to the superfamily of lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) proteins. Most Ly6 superfamily members are evolutionarily conserved in humans and mice. Although the Ly6/uPAR family proteins share a common structure, their expression patterns and functions vary.14 Our group reported that KO male mice for Ly6k, Lypd4, and Tex101, members of the Ly6/uPAR family localized in testes and on spermatozoa, were infertile, respectively.4,15,16 A subset of the Ly6/uPAR family, called PATE, consists of secretory proteins and is primarily in the epididymis. The PATE family forms a cluster on chromosome 9, and fertility is reduced in male mice when most of the family is deleted.4 In this study, we focused on Ly6g5b and Ly6g5c as a member of the Ly6/uPAR family. They are adjacently located in the MHC class III region of mouse chromosome 17 and encode secretory proteins.17 However, no research has analyzed the roles of Ly6g5b/c genes in vivo.

The CRISPR/Cas9-mediated KO mouse generation and phenotype screening are a cost-effective and labor-effective approach to identify essential genes in vivo quickly.18 Using the CRISPR/Cas9 system, we can quickly induce indel mutation by introducing a gRNA/Cas9 complex into zygotes, but there is a risk that unexpected transcription products remain or are newly produced. Therefore, we applied a new approach introducing two gRNA-Cas9 complexes that delete the entire or most of the target gene.5,19 This study generated mice lacking Lipocalin family genes on chromosome 2 and Ly6g5b/c genes on chromosome 17 and investigated their roles in epididymal sperm maturation.

Materials and Methods

Animals

All mice used in this study were purchased from Japan SLC. Mice were acclimated to a 12-hour-light/12-hour-dark cycle. All animal experiments were approved by the Animal Care and Use Committee of the National Cerebral and Cardiovascular Center and the Research Institute for Microbial Diseases, Osaka University. Mice were maintained on the B6D2F1 mouse strain. All KO mouse lines generated in this study are available through RIKEN BRC (https://mus.brc.riken.jp/en/) or CARD (http://cardb.cc.kumamoto-u.ac.jp/transgenic/) bioresource centers.

cDNA and RT-PCR

All tissues [brain, thymus, lung, heart, liver, spleen, kidney, testis, epididymis (caput, corpus, cauda), seminal vesicle, prostate, coagulation gland, ovary, and uterus] were collected from three C57BL/6NCr mice euthanized at 12 weeks old. These samples were homogenized in TRIzol (Ambion). Five μg of total RNA was reverse-transcribed to cDNA using SuperScript III First Standard Synthesis System, which contains 200 units of reverse transcriptase (Thermo Fisher Scientific), following the manufacturer’s instruction. Five ng of cDNA was used for PCR with primer sets (Table S1) and KOD DNA polymerase (KOD-FX Neo, TOYOBO). The amplification program was as follows: preincubation at 95°C for 5 min to activate the DNA polymerase, followed by 35 cycles of denaturation at 95°C for 30 s, annealing of the primers at 60°C for 30 s, and elongation at 72°C for 30 s.

Egg collection, electroporation, and embryo transfer

CARD HyperOva (0.1 ml/mouse, Kyudo) was injected into the abdominal cavity of B6D2F1 females, followed by human chorionic gonadotropin (5 units, ASKA pharmaceutical) and natural mating with B6D2F1 males 48 hours after HyperOva injection. After 20 hours, we collected fertilized eggs with 2 pronuclei for genome editing. The crRNA and tracrRNA (Merck) were diluted with nuclease-free water (non-DEPC treated, Thermo Fisher Scientific). The mixture was denatured at 95°C for 1 minute and allowed to anneal by cooling gradually to room temperature. Each gRNA was mixed with the Cas9 protein solution (Thermo Fisher Scientific), and then incubated at 37°C for 5 minutes to prepare the gRNA/Cas9 ribonucleoproteins (RNPs) [final concentration: 50 ng/μl Cas9 for 20 ng/μl of each gRNA] (Table S2). The gRNA/Cas9 RNP solution was placed between the electrodes, and then the electroporation was done with the following conditions [resistance value: 550~600 Ω, poring pulse: 225 V (voltage), 2 ms (pulse amplitude), 50 ms (pulse interval), 4 (pulse number), 10% (attenuation), + (polarity), transfer pulse: 20 V (voltage), 50 ms (pulse amplitude), 50 ms (pulse interval), 5 (pulse number), 40% (attenuation), +/− (polarity)]. Electroporated embryos were cultured overnight, and more than 90% survived. Twenty embryos were transplanted into the oviducts of each pseudo-pregnant ICR recipient. After 19 days, offspring were obtained by natural birth or Caesarean section.

Mating test

Eight-week-old KO male mice were caged with two B6D2F1 females per cage for 3 months. The number of delivered pups was counted, and they were removed immediately. Copulation was confirmed by checking for vaginal plugs every morning.

Histological analysis of epididymis and sperm morphology

Epididymides were fixed in Bouin’s fluid (Polysciences) at 4°C overnight. Increasing ethanol concentrations and xylene-dehydrated fixed epididymides were then embedded with paraffin. Paraffin sections (5 μm) were stained with Mayer hematoxylin solution for 3 minutes, counterstained with eosin Y solution [53% ethanol, 0.3% eosin, and 0.5% acetic acid] for 3 minutes, dehydrated in increasing ethanol concentrations, and finally mounted in Entellan new (Merck). The cauda epididymal spermatozoa were observed with phase contrast microscopy.

In vitro fertilization (IVF)

IVF using mouse spermatozoa was performed as described previously.20 Briefly, eggs collected from superovulated females 14 h after hCG injection were placed in the IVF medium (TYH or HTF). We used TYH for lipocalin quadruple KO and Ly6g5b/c KO, and switched to HTF for lipocalin quintuple KO because reagent supply became problematic during the COVID-19 situation. Epididymal sperm were collected from 3-month-old male mice and incubated in the IVF medium for 2 h for capacitation. Then, capacitated sperm were added to the medium containing eggs at a final concentration of 2 × 105 sperm/ml and cultured overnight.

Sperm-zona pellucida binding assay

Sperm-zona pellucida binding assay was performed with eggs from which the cumulus cells had been removed by treatment with bovine testicular hyaluronidase (175 U/ml, Merck) for 5 min. Cumulus-free eggs were placed in an IVF medium drop and inseminated. After 30 minutes of incubation, eggs were fixed with 0.25% glutaraldehyde. Spermatozoa bound to the zona pellucida were observed using an Olympus IX-73 microscope.

Immunoblot

Immunoblot analysis was performed as described previously.21 Briefly, to collect testicular germ cells, seminiferous tubules were minced in PBS, and the suspension was filtered to remove spermatozoa and pieces of tissue. Sperm samples were collected from the cauda epididymis. We collected these samples from three males. The samples were homogenized in lysis buffer containing 1% Triton X-100 and 1% protease inhibitor (Nacalai Tesque) and then were centrifuged (10,000 g for 20 min at 4°C), and the supernatants were collected. Protein lysates were applied 20 μg per lane, separated by SDS/PAGE under reducing conditions, and transferred to PVDF membranes (Merck). Blocking was performed by incubation in 0.5% Tween20 and 10% skim milk in PBS for 90 min. After blocking, blots were incubated with primary antibodies diluted in blocking solution (for ADAM3; 1:1000; Santa Cruz; cat# sc-365288, for BASIGIN; 1:1000; Santa Cruz; cat# sc-46700, for OVCH222; 1:500, for RNASE10; 1:100; LSBio; cat# LS-C296261, for GAPDH; 1:1000; Cell Signaling Technology; cat# 2118, for CMTM2A and CMTM2B23; 1:500, for IZUMO124; 1:1000, for ADAM1B25; 1:500, for ADAM2; 1:500; Merck; cat# MAB19292) overnight at 4°C, and then incubated with secondary antibodies conjugated with horseradish-peroxidase (1:5000; anti-mouse secondary antibody; Proteinsimple; cat# 042-205, anti-rat secondary antibody; Jackson ImmunoResearch Laboratories; cat# 112-035-167, anti-rabbit secondary antibody; Jackson ImmunoResearch Laboratories; cat# 111-035-144). The detection was performed using Chemi-Lumi One Super (Nacalai Tesque).

Statistical analysis

All values are shown as the mean ± SD of at least three independent experiments. Statistical analyses were performed using Student’s t-test inserted into Microsoft Excel after the data were tested for normality of distribution.

Results

Gene expression pattern

RT-PCR analyzed gene expression patterns of genes we targeted in this study with multiple mouse tissues (Figure 1A). All the genes are predominantly expressed in the caput epididymis, while Lcn5 is expressed in the epididymis and the uterus.

Fertility of mice lacking lipocalin family genes

Lipocalin family genes analyzed here are located on the mouse chromosome 2qA3 locus. While Lcn8, Lcn5, Lcn6, and Lcn10 form a cluster, Lcn9 is approximately 137 kb away from it (see the gene information at Ensemble, URL: http://asia.ensembl.org/Mus_musculus/Location/View?g=ENSMUSG00000026937;r=2:25547964-25551989). Because there are other genes in between, we first generated Lcn9 KO mice by introducing two gRNA/Cas9 protein complexes into pronuclear zygotes (Lcn9del/del, Supplementary Figure 1A) and analyzed male fertility. Lcn9del/del males had fertility comparable to wild-type (WT) males, and there were no apparent problems with sperm morphology and epididymal histology (Figure 1B, C, and D).

Next, we generated quadruple KO mice that deleted four lipocalin family genes, Lcn8, Lcn5, Lcn6, and Lcn10 (Figure 2A and Supplementary Figure 1B). Control males consistently fathered litters with 9 pups per plug, and they generated approximately 30 plugs during the course of the mating trial. However, the Lcn8-Lcn10 KO males fathered litters with less than 3 pups per plug, and the fertility was significantly lower than WT males (pups/plug ratio: 2.74 ± 1.37 in KO vs. 8.87 ± 0.38 in WT, P < 0.01, Figure 2B). There were no apparent defects in sperm morphology and epididymis histology of Lcn8-Lcn10 KO mice (Figures 2C and D). When we performed IVF, the Lcn8-Lcn10 KO spermatozoa efficiently fertilized cumulus-intact oocytes (Figure 2E) but barely bound to the zona pellucida when the spermatozoa were incubated with cumulus-free oocytes (Figures 2F and G).

Finally, we introduced two gRNA/Cas9 protein complexes to delete Lcn9 in Lcn8-10 KO zygotes and obtained quintuple KO mice. The Lcn8-10/9 KO male fertility was significantly lower than that of mice mated with WT males (pups/plug ratio: 0.36 ± 0.06 vs. 8.87 ± 0.38, P < 0.01, Figure 3A). There were no apparent defects in sperm morphology and epididymal histology of Lcn8-10/9 KO mice (Figures 3B and C). The quintuple KO sperm efficiently fertilized intact cumulus eggs in vitro (Figure 3D) but barely bound to the cumulus free zona pellucida (Figure 3E).

Fertility of mice lacking Ly6g5b and Ly6g5c genes

Ly6g5b and Ly6g5c are located on mouse chromosome 17qB1 locus (see the gene information at Ensemble, URL: https://asia.ensembl.org/Mus_musculus/Location/View?db=core;g=ENSMUSG00000043807;r=17:35332924-35334404). We generated Ly6g5b and Ly6g5c KO mice using the same method (Figure 4A and Supplementary Figure 1C). The Ly6g5b/c KO male fertility was significantly lower than that of WT males (pups/plug ratio: 3.07 ± 1.08 vs. 8.87 ± 0.38, P < 0.01, Figure 4B). There was no difference in sperm morphology and epididymal histology in Ly6g5b/c KO mice (Figure 4C and D). The Ly6g5b/c KO sperm efficiently fertilized intact cumulus eggs in vitro (Figure 4E). However, in the sperm binding assay, Ly6g5b/c KO spermatozoa barely bound to the zona pellucida when the spermatozoa were incubated with cumulus-free oocytes (Figure 4F).

Immunoblot analysis for ADAM3-related proteins

Adam3 KO spermatozoa can fertilize intact cumulus eggs but cannot bind to the zona pellucida,26,27 similar to the phenotype we found in our KO mice. Because of this similarity, we analyzed the ADAM3 protein levels by immunoblot analysis. ADAM3 protein exists on the sperm surface, and a 110 kDa precursor form of ADAM3 is processed into the 42 kDa mature protein during the transition from the testis to the epididymis.28 We used BASIGIN as a control because BASIGIN also localizes on the spermatozoa and is processed from 37 kDa to 26 kDa during the transfer from the testis to the epididymis.29 There was no apparent difference in ADAM3 levels in WT and KO testicular germ cell (TGC) samples (Figure 5). However, in cauda epididymal sperm samples derived from Lcn8-Lcn10 KO males, the ADAM3 signal was weaker than that of heterozygous mutant males (Figure 5A). In Lcn8-10/9 KO spermatozoa, the ADAM3 signal nearly disappeared (Figure 5B). In Ly6g5b/c mutants, the signal was also weak in homozygous mutant males (Figure 5C).

During epididymal sperm transition, Ovochymase 2 (OVCH2), and RNASE10 are secreted from the caput epididymis and are involved in ADAM3 processing.22,30 Therefore, we first examined them by immunoblot analysis, but we did not see any significant differences between WT and Lcn8-10/9 KO samples (Figure 5D). Finally, we examined sperm surface proteins, ADAM2 and CMTM2A/B, which are essential for ADAM3 maturation.23 There were no differences in ADAM2, but CMTM2A/B disappeared from Lcn8-10/9 KO spermatozoa. (Figure 5E).

Discussion

In mammals, immature spermatozoa generated in the testes must pass through the epididymis to gain their ability to fertilize.31 By knocking out genes in mice, we have searched for genes that are expressed in the epididymis and are involved in epididymal sperm maturation.4,5,22 Some epididymis-enriched genes are remarkably homologous to each other and are expected to play redundant roles; thus, the single KO approach is not a feasible way to study these genes. In this study, we took advantage of the CRISPR/Cas9 system by knocking out the epididymis-enriched Lcn8/5/6/10/9 and Ly6g5b/c gene clusters and found infertility phenotypes. It is important to remember that deletions of gene regions can potentially delete unidentified protein-coding regions, delete non-coding RNAs, or affect the expression of neighbor genes. Although the transgenic rescue approach is ideal for proving if the targeted gene is responsible for the phenotype, it is also difficult to choose which gene to express when we disrupt multiple genes. In this study, instead of adding a transgene, the additional defect observed with Lcn9 single KO underpins that Lcn9 codes protein that complements LCN protein functions.

As we did not see any overt morphological abnormalities and detected a normal amount of proteins involved in ADAM3 maturation, OVCH2 and RNASE10, in Lcn8-10/9 KO mice, lipocalins are not potentially critical for epididymal development per se. Instead, we found that sperm plasma membrane proteins CMTM2A/B disappeared from the Lcn8-10/9 KO spermatozoa suggesting that lipocalins affect ADAM3 localization through CMTM2A/B on the sperm heads. However, because the mechanisms of how CMTM2A/B regulates ADAM3 is unknown, further studies are needed to elucidate entire molecule mechanisms (Figure 5F). Furthermore, as secreted protein LCN6 binds to sperm heads and tails in humans,32 sperm affinity of lipocalins is also the key to understanding their molecular function.

Our study suggests that Ly6g5b/c genes are also involved in the ADAM3 maturation process in the epididymis. It should be noted that previous studies proved several Ly6 family proteins (e.g., LY6K, LYPD4, and TEX101) are individually required for sperm ADAM3 processing and male fertility.33 Therefore, it is not always true to consider family genes complementary. Intriguingly, Ly6 family proteins are expressed in various male reproductive organs (testis, spermatozoa, and epididymis). A transgenic approach with different promoters and/or swapping coding sequences by CRISPR/Cas9 mediated knockin might be helpful to assess individual functions in vivo. There still remain many proteins coded by gene clusters that seem to control epididymal sperm maturation.4,22 Our approach of deleting gene clusters will be helpful in deciphering the functions for epididymal sperm maturation and male fertility.

Supplementary Material

Sup1 Suppl. Fig. 1. Strategy for (A)Lcn9 (B) Lcn8-Lcn10, and (C) Ly6g5b/c KO mice generation. Gene information was obtained from Ensemble.

TableS1

TableS2

Acknowledgments

We thank Naoko Nagasawa, Eri Hosoyamada, and the Biotechnology Research and Development (nonprofit organization) for technical assistance, and Ferheen Abbasi and Julio M. Castaneda for critical reading of the manuscript.

Funding information

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants JP19J00865 to N.S. and JP20KK0155, JP21K19198, and JP21H02397 to Y.F. and JP19H05750 and JP21H05033 to M.I.; Japan Agency for Medical Research and Development Grant JP21gm5010001 to M.I.; Takeda Science Foundation Grants to Y.F. and M.I.; Mochida Memorial Foundation for Medical and Pharmaceutical Research Grant to Y.F.; Sumitomo Foundation Grant for Basic Science Research Project to Y.F.; Senri Life Science Foundation Grant to Y.F.; the Intramural Research Fund grants (21-2-6, 22-A-3, 30-2-5, and 31-6-3) for Cardiovascular Diseases of the National Cerebral and Cardiovascular Center to Y.F.; Eunice Kennedy Shriver National Institute of Child Health and Human Development Grants R01HD088412 and P01HD087157 to M.I.; and the Bill & Melinda Gates Foundation Grant INV-001902 to M.I.

Fig. 1. Multiple gene expression by RT-PCR and phenotype analysis of Lcn9 KO mice. (A) Gene expression of Lcn5, Lcn6, Lcn8, Lcn9, Lcn10, Ly6g5b, and Ly6g5c. Actin β (Actb) was used as the control. Epididymis (Epi). (B) Male fertility. There was no difference in the average pups/plug ratio of WT and KO males (P = 0.68). (C) Cauda epididymal spermatozoa from WT B6D2F1 and Lcn9del/del mice. Scale bars are 50 μm. (D) Histological analysis of the epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively.

Fig. 2. Phenotype analysis of Lcn8-Lcn10 KO mice. (A) Genomic structure of Lcn8, 5, 6, 10, and 9. Gene information was obtained from Ensemble (http://ensembl.org/index.html). (B) Number of pups per plug of WT female mice mated with KO male mice. WT B6D2F1 male mice were used as the control. * P < 0.01. (C) Cauda epididymal spermatozoa from (Lcn8-Lcn10)wt/del and (Lcn8-Lcn10)del/del mice. Scale bars are 50 μm. (D) Histological analysis of epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively. (E) In vitro fertilization ability analysis. (F and G) Sperm-zona pellucida binding assay. The average number of zona pellucida-binding spermatozoa obtained from heterozygote and homozygote mutant mice. Scale bars are 25 μm. * P < 0.01.

Fig. 3. Phenotype analysis of Lcn8-Lcn10 and Lcn9 KO mice. (A) Value of pups per plug of WT female mice mated with KO male mice. WT B6D2F1 male mice were used as the control. * P < 0.01. (B) Cauda epididymal spermatozoa from (Lcn8-10/9)wt/del and (Lcn8-10/9)del/del mice. Scale bars are 50 μm. (C) Histological analysis of the epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively. (D) In vitro fertilization ability analysis. (E) Sperm-zona pellucida binding assay. Average number of zona pellucida-binding spermatozoa obtained from heterozygote and homozygote mutant mice. * P < 0.01.

Fig. 4. Phenotype analysis of Ly6g5b/c KO mice. (A) Genomic structure of Ly6g5c and b. Gene information was obtained from Ensemble. (B) Value of pups per plug of WT female mice mated with KO male mice. WT B6D2F1 male mice were used as the control. * P < 0.01. (C) Cauda epididymal spermatozoa from (Ly6g5b/c)wt/del and (Ly6g5b/c)del/del mice. Scale bars are 50 μm. (D) Histological analysis of the epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively. (E) In vitro fertilization ability analysis. (F) Sperm-zona pellucida binding assay. The average number of zona pellucida-binding spermatozoa obtained from heterozygote and homozygote mutant mice. * P < 0.01.

Fig. 5. Immunoblot analysis. (A-C) Immunoblot analysis for ADAM3. The study was performed using testicular germ cells (TGC) and cauda epididymal spermatozoa obtained from (A) Lcn8-Lcn10, (B) Lcn8-10/9, and (C) Ly6g5b/c mutant mice. BASIGIN was used as the control. (D) Immunoblot analysis for OVCH2 and RNASE10 using protein lysates collected from WT and Lcn8-10/9 KO testis, caput, corpus, and cauda sperm. GAPDH was used as the control. (E) Immunoblot analysis for ADAM1B, ADAM2, CMTM2A, CMTM2B, and IZUMO1 using cauda sperm lysates collected from WT and Lcn8-10/9 KO mice. BASIGIN was used as the control. (F) Schematic view of this study.

Conflict of interest

The authors declare no conflict of interest.
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Reference

1. Hinton BT , Galdamez MM , Sutherland A , How do you get six meters of epididymis inside a human scrotum? Journal of andrology. Nov-Dec 2011;32 (6 ):558–64.21441421
2. Bedford JM . The bearing of epididymal function in strategies for in vitro fertilization and gamete intrafallopian transfer. Ann N Y Acad Sci. 1988;541 :284–91.3195911
3. Robertson MJ , Kent K , Tharp N , Large-scale discovery of male reproductive tract-specific genes through analysis of RNA-seq datasets. BMC Biol. Aug 19 2020;18 (1 ):103.32814578
4. Fujihara Y , Noda T , Kobayashi K , Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice. Proceedings of the National Academy of Sciences of the United States of America. Sep 10 2019;116 (37 ):18498–18506.31455729
5. Noda T , Sakurai N , Nozawa K , Nine genes abundantly expressed in the epididymis are not essential for male fecundity in mice. Andrology. Sep 2019;7 (5 ):644–653.30927342
6. Sun J , Lu Y , Nozawa K , CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproductiondagger. Biology of reproduction. Aug 4 2020;103 (2 ):183–194.32588039
7. Ganfornina MD , Gutierrez G , Bastiani M , Sanchez D . A phylogenetic analysis of the lipocalin protein family. Mol Biol Evol. Jan 2000;17 (1 ):114–26.10666711
8. Grzyb J , Latowski D , Strzalka K . Lipocalins - a family portrait. J Plant Physiol. Sep 2006;163 (9 ):895–915.16504339
9. Suzuki K , Lareyre JJ , Sanchez D , Molecular evolution of epididymal lipocalin genes localized on mouse chromosome 2. Gene. Sep 15 2004;339 :49–59.15363845
10. Berger T , Togawa A , Duncan GS , Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury. Proceedings of the National Academy of Sciences of the United States of America. Feb 7 2006;103 (6 ):1834–9.16446425
11. Watanabe H , Takeo T , Tojo H , Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation. Development. May 2014;141 (10 ):2157–64.24803661
12. Wen Z , Liu D , Zhu H , Deficiency for Lcn8 causes epididymal sperm maturation defects in mice. Biochemical and biophysical research communications. Apr 9 2021;548 :7–13.33631677
13. Yin Q , Shen J , Wan X , Liu Q , Zhou Y , Zhang Y . Impaired sperm maturation in conditional Lcn6 knockout mice. Biology of reproduction. Jan 1 2018;98 (1 ):28–41.29045572
14. Loughner CL , Bruford EA , McAndrews MS , Delp EE , Swamynathan S , Swamynathan SK . Organization, evolution and functions of the human and mouse Ly6/uPAR family genes. Hum Genomics. Apr 21 2016;10 :10.27098205
15. Fujihara Y , Okabe M , Ikawa M . GPI-anchored protein complex, LY6K/TEX101, is required for sperm migration into the oviduct and male fertility in mice. Biology of reproduction. Mar 2014;90 (3 ):60.24501175
16. Fujihara Y , Tokuhiro K , Muro Y , Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa. Proceedings of the National Academy of Sciences of the United States of America. May 14 2013;110 (20 ):8111–6.23633567
17. Mallya M , Campbell RD , Aguado B . Characterization of the five novel Ly-6 superfamily members encoded in the MHC, and detection of cells expressing their potential ligands. Protein Sci. Oct 2006;15 (10 ):2244–56.17008713
18. Mashiko D , Young SA , Muto M , Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Dev Growth Differ. Jan 2014;56 (1 ):122–9.24372541
19. Lu Y , Oura S , Matsumura T , CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in micedagger. Biology of reproduction. Aug 1 2019;101 (2 ):501–511.31201419
20. Tokuhiro K , Ikawa M , Benham AM , Okabe M . Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility [corrected]. Proceedings of the National Academy of Sciences of the United States of America. Mar 6 2012;109 (10 ):3850–5.22357757
21. Fujihara Y , Oji A , Larasati T , Kojima-Kita K , Ikawa M . Human Globozoospermia-Related Gene Spata16 Is Required for Sperm Formation Revealed by CRISPR/Cas9-Mediated Mouse Models. Int J Mol Sci. Oct 21 2017;18 (10 ).
22. Kiyozumi D , Noda T , Yamaguchi R , NELL2-mediated lumicrine signaling through OVCH2 is required for male fertility. Science. Jun 5 2020;368 (6495 ):1132–1135.32499443
23. Fujihara Y , Oji A , Kojima-Kita K , Larasati T , Ikawa M . Co-expression of sperm membrane proteins CMTM2A and CMTM2B is essential for ADAM3 localization and male fertility in mice. Journal of cell science. Oct 8 2018;131 (19 ).
24. Inoue N , Ikawa M , Isotani A , Okabe M . The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature. Mar 10 2005;434 (7030 ):234–8.15759005
25. Ikawa M , Tokuhiro K , Yamaguchi R , Calsperin is a testis-specific chaperone required for sperm fertility. The Journal of biological chemistry. Feb 18 2011;286 (7 ):5639–46.21131354
26. Shamsadin R , Adham IM , Nayernia K , Heinlein UA , Oberwinkler H , Engel W . Male mice deficient for germ-cell cyritestin are infertile. Biology of reproduction. Dec 1999;61 (6 ):1445–51.10569988
27. Yamaguchi R , Muro Y , Isotani A , Disruption of ADAM3 impairs the migration of sperm into oviduct in mouse. Biology of reproduction. Jul 2009;81 (1 ):142–6.19339711
28. Nishimura H , Kim E , Nakanishi T , Baba T . Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface. The Journal of biological chemistry. Aug 13 2004;279 (33 ):34957–62.15194697
29. Saxena DK , Oh-Oka T , Kadomatsu K , Muramatsu T , Toshimori K . Behaviour of a sperm surface transmembrane glycoprotein basigin during epididymal maturation and its role in fertilization in mice. Reproduction. Mar 2002;123 (3 ):435–44.11882021
30. Krutskikh A , Poliandri A , Cabrera-Sharp V , Dacheux JL , Poutanen M , Huhtaniemi I . Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility. FASEB J. Oct 2012;26 (10 ):4198–209.22750516
31. Amann RP , Hammerstedt RH , Veeramachaneni DN . The epididymis and sperm maturation: a perspective. Reproduction, fertility, and development. 1993;5 (4 ):361–81.8153387
32. Chen J , Zhao Y , Feng W . Selection, preparation and characterization of scFv against human lipocalin 6 by phage display technology. Protein Expr Purif. Jul 2020;171 :105627.32205279
33. Fujihara Y , Miyata H , Ikawa M . Factors controlling sperm migration through the oviduct revealed by gene-modified mouse models. Exp Anim. May 10 2018;67 (2 ):91–104.29353867
