Abstract
There are approximately 20 000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.
Keywords: male infertility, phenotypic screening, gene knockout
Knockout model male mouse of 18 genes mainly expressed in the testis does not lose fertility.
Graphical Abstract
Graphical Abstract.
Introduction
Infertility is a major problem, especially in developed countries. One in seven couples in Europe and approximately one in six couples in Japan are unable to have children due to infertility. Approximately, 50% of infertility cases are thought to be caused by male reproductive factors [1, 2]. There is also a growing demand for male contraceptives in family planning. Historically, the development of contraceptives has relied primarily on hormones, which are generally effective when used by women. However, hormone-based contraceptives have serious side effects and poor tolerability. Outside of barrier protection, safe and effective contraceptive methods for men have not been established. Although the focus on contraceptive use has been on women, in recent years, there has been an increasing demand from couples for men to play a greater role in family planning [3]. Therefore, to develop male contraceptives, it is extremely necessary to elucidate the male reproductive control mechanism from the perspective of clinical application.
It has been suggested that 2375 genes in the mouse genome exhibit an expression pattern restricted to spermatogenic cells [4]. In the mouse RNA-seq dataset carefully selected by Robertson et al., out of 1262 testis-enriched genes, 549 genes had a phenotype, of which 242 genes had a phenotype of reduced fertility or male infertility [5]. In other words, not every gene has an important role. Furthermore, a group of genes that are not specifically expressed in the testis have also been shown to be significantly involved in the male reproductive system. The encoded proteins involved in the formation of cilia, which are present in multiple tissues such as in the lung and brain, also play an important role in the formation of flagella in sperm, and it has been reported that mutations in these genes cause ciliopathy and infertility [6–8].
Phenotypic analysis using the knockout (KO) model is useful for analyzing the functions of individual genes, and our laboratory has identified critical proteins such as SPATA33 [9] and TSKS [10] for spermiogenesis, NELL2 [11] and NICOL [12] for sperm maturation, IZUMO [13], SOF1, TMEM95, and SPACA6 [14] for fertilization. However, there are still many genes whose functions have not been identified. By using the genome editing system CRISPR/Cas9, it has become possible to construct KO models in a short period with simple experiments and analyze multiple strains in parallel.
In this study, we focused on 18 of these genes established KO mice, and screened their roles in male fecundity by conducting mating tests. The accumulated results demonstrated that the deficiency in any of them affected fertility.
Materials and methods
Animals
All animal experiments conducted in this study were conducted in compliance with the guidelines for animal experiments established and kept by Osaka University. The B6D2F1 strain for the background of KO mice and the ICR strain for the use of recipient mice were purchased from CLEA Japan Corporation (Tokyo, Japan) and SLC Corporation (Shizuoka, Japan).
mRNA expression pattern analysis from RNA-seq data sets
The mRNA expression patterns of target mouse genes and their orthologous human genes by tissue or by cell type were determined using MRGDv2 (https://orit.research.bcm.edu/MRGDv2) [15–18]. By searching this database, we selected a group of genes that are testis-specific or highly expressed for knockout targets.
Establishment of knockout mice strains
All KO lines were generated using the CRISPR/Cas9 system [19–21]. Gene sequence data were referred to the mouse GRCm39 version of Ensembl (https://www.ensembl.org/index.html). Two CRISPR RNAs (crRNAs) were designed for each gene allele, one near the ATG and the other near the stop codon, to delete the full-length region. Female mice were superovulated by inhibin anti-serum and eCG (IASe) (Kyudo Corp, Saga, Japan) administration followed by hCG. They were coupled with male B6D2F1 (BDF1) mice overnight and embryos at the two-pronuclear (2PN) stage were collected from the oviducts after cervical dislocation the next day. Then, each crRNA (Supplemental Figure 1) and trans-activating crRNA (tracrRNA) were annealed and mixed with the Cas9 protein. These ribonucleoproteins (RNPs) were introduced to zygotes by electroporation, and further cultured in vitro until they developed into the two-cell stage, which were then surgically transplanted into recipient pseudopregnant ICR mice. On day E19, the pups were delivered by cesarean section. Mice with the targeted allelic defect were identified by PCR screening from biopsied DNA samples and mated with the wild type. F1 heterozygotes were crossed to obtain F2 KO mice. The sequences of the missing alleles were analyzed by the Sanger method. To identify the genotype of mice, we designed primer pairs (Supplemental Figure 2) for WT and KO spanning the missing alleles, and then performed PCR.
For DNA sample preparation, a tissue sample was biopsied, placed into a tube containing lysis buffer and pronase K, and incubated at 57°C overnight. The PCR conditions were 40 cycles of denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Further annealing at 72°C was performed for 2 min.
Anatomical and histological observations
Testes collected from cervically dislocated mice of both WT and KO genotypes were imaged side by side under a BX50 (Olympus, Tokyo, Japan) microscope. To observe abnormal spermatogenesis within the seminiferous tubules, the testes were fixed in Bouin’s fixative (Polysciences, Inc., Warrington, PA) for 2 h at room temperature. After dehydration by immersing in ethanol at stepwise concentrations, the testes were embedded into paraffin. The paraffin block was sectioned into 5-μm thick sections by microtome (HM325, PMC holdings, Tokyo, Japan), mounted on MAS-coated slide glasses (Matsunami glass, Tokyo, Japan), and then PAS stained by established procedures. Briefly, the rehydrated sections were reacted with 1% (w/v) periodic acid (Nacalai Tesque, Kyoto, Japan) for 10 min, following Schiff’s reagent (Fujifilm Wako Chemicals, Tokyo, Japan) for 20 min. After the nuclei were counterstained with Mayer’s hematoxylin solution (Fujifilm Wako) for 5 min, the samples were dehydrated. A mounting medium was added, and the sample was covered with a cover glass. Spermatozoa from the cauda epididymis were released into TYH medium, then smeared on a glass slide and covered with a cover glass. Pictures were acquired using a microscope (BX53: Olympus, Tokyo, Japan) connected to a camera (DP74: Olympus, Tokyo, Japan).
Sperm motility analysis
Sperm from the cauda epididymis were suspended in TYH medium (100 μl per drop), covered with liquid paraffin, and cultured in an incubator at 37°C under a 5% CO2 atmosphere until motility was observed. After 10 min and 2 h of culturing, approximately 3 to 5 μl of sperm was collected from the upper layer of the TYH drop, and the sperm was added to another 100 μl of TYH drop. From there, 25 μl of the sperm suspension was added to a sperm motility measurement chamber (Leja®, Standard Count two Chamber Slide 100 micron), and various motility parameters (motility rates) were measured using a sperm motility analyzer (Hamilton Thorne, CEROS II). Motile %, average path velocity (VAP) μm/s, straight-line velocity (VSL) μm/s, and curvilinear velocity (VCL) μm/s were analyzed.
Motility parameters of sperm collected from the cauda epididymis were analyzed using CEROS II (Hamilton Thorne Biosciences, Beverly, MA, USA) equipped with a computer-assisted sperm parameter analysis (CASA) system. More than 200 spermatozoa were counted at each measurement time [21].
Mating test
One KO male mouse that had reached 8 weeks of age and three BDF1 females that were 8 weeks old were housed in the same cage and mated together for 8 weeks. A total of three male mice were examined. Thereafter, the male mouse was removed from the cages, and the females were kept for an additional three weeks. The total number of plugs, number of births, and number of pups born were recorded. Mating was performed in a similar manner using wild-type BDF1 male mice at the same time, and the results were compared with those of KO males.
Statistical data analysis
Statistical data analysis between WT and KO was performed by Welch t-test using Microsoft Office Excel (Microsoft Corporation, WA, USA). Differences were considered statistically significant if P < 0.05.
Results
We analyzed 18 candidate genes and compared the mRNA expression pattern of each to different tissues (Figure 1A). All genes other than Tmem235 & Dnajb3 had human orthologs and showed specific or highest expression in the testis. Focusing on individual cells or tissues, most of them showed strong expression from late meiosis to round spermatids, suggesting that they may function during the stages. Baz2a was found to be expressed throughout spermatogenesis from spermatogonia (Figure 1B).
Figure 1.
Different gene expression patterns of 18 genes in mouse and human tissues based on RNA-seq transcripts accumulated data (A). Expression patterns of target genes by cell type in each region of the testis and epididymis (B). The pattern of each tissue is displayed in a black-and-white gradation with a max TPM value of 30. Note that for EID3, TPM is shown with a maximum value of 100.
We successfully generated KO mice lines for all 18 genes using the CRISPR/Cas9 system and their phenotypes were analyzed. Information on the DNA deletion pattern in the target region, the number of litters obtained in the mating test, the testis size, and CASA-analyzed sperm motility parameters are listed in Tables 1 and 2, respectively. Frozen spermatozoa of each strain have been deposited and are available at the RIKEN Bioresource Center (Tsukuba, Japan) and Center of Animal Resources and Development (CARD: Kumamoto, Japan). Here, we show four representative analyses of genes that were not essential for male fertility: Dnajb3, Eid3, Ptpn20, and Spag8, whereas those of Antxrl, Art3, Art5, Baz2a, Ccdc70, Cfap107(1700012P22Rik), Cifipl (1700021F07Rik), Nupr2 (Nupr1l), Ppp1r36, Spmip3(1700016C15Rik), Tmem235, Tsacc, 1700011L22Rik, and 4930578I06Rik KO males are shown in Figures S1–S3 and Tables S1–S3.
Table 1.
Male fertility of the 18 mutant mouse lines
| Gene symbol | Genotype | Number of males | Number of plugs | Number of delivery | Number of pups | Average litter size (SD) |
|---|---|---|---|---|---|---|
| WT | WT/WT | 3 | 30 | 30 | 247 | 8.2 ± 2.7 |
| Antxrl | −245/−245 | 3 | 26 | 26 | 213 | 8.2 ± 2.4 |
| Art3 | −27,345/−27,345 | 3 | 21 | 20 | 173 | 8.7 ± 3.0 |
| Art5 | −1610/−1610 | 3 | 27 | 23 | 203 | 8.8 ± 2.2 |
| Baz2a | −18,246/−18,246 | 3 | 23 | 17 | 160 | 10.0 ± 4.2 |
| Ccdc70 | −790/−790 | 3 | 26 | 25 | 227 | 9.1 ± 1.4 |
| Cfap107 (1700012P22Rik) | −20,276/−20,276 | 3 | 24 | 23 | 208 | 9.0 ± 2.1 |
| Cimip1 (1700021F07Rik) | −5.692/−5692 | 3 | 33 | 25 | 239 | 9.6 ± 1.5 |
| Dnajb3 | −783/−783 | 3 | 28 | 27 | 224 | 8.3 ± 2.9 |
| Eid3 | 1096 + 1/−1096+ | 3 | 27 | 26 | 231 | 8.9 ± 1.6 |
| Nupr2(Nupr1l) | −338/−338 | 3 | 21 | 20 | 189 | 9.5 ± 1.8 |
| Ptpn20 | −25,443/−25,443 | 3 | 23 | 22 | 200 | 9.1 ± 2.4 |
| Ppp1r36 | −21,741/−21,741 | 3 | 24 | 23 | 192 | 8.3 ± 2.0 |
| Spag8 | −2336/−2336 | 3 | 24 | 21 | 202 | 9.6 ± 1.2 |
| Spmip3 (1700016C15Rik) | −20,122/−20,122 | 3 | 25 | 25 | 210 | 8.4 ± 2.5 |
| Tmem235 | −3832/−3832 | 3 | 28 | 28 | 229 | 8.2 ± 2.4 |
| Tsacc | −12,667/−12,667 | 3 | 21 | 20 | 247 | 10.5 ± 2.2 |
| 1700011L22Rik | −37,771/−37,771 | 3 | 29 | 26 | 222 | 8.5 ± 2.7 |
| 4930578I06Rik | −16,448/−16,448 | 3 | 19 | 18 | 132 | 7.3 ± 2.0 |
Mating test results. A total number of males, plugs, deliveries, and pups are shown. Average litter size was the average number of pups obtained at each birth.
Table 2.
Sperm motility analysis of the 18 mutant mouse
| Motility (%) | VAP(μ/s) | VCL(μ/s) | VSL(μ/s) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Gene symbol | Testis weight (mg) | 10 min | 120 min | 10 min | 120 min | 10 min | 120 min | 10 min | 120 min |
| WT | 106.2 ± 4.2 | 78.5 ± 0.3 | 69.7 ± 8.0 | 178.6 ± 20.0 | 150.6 ± 17.2 | 282.1 ± 51.7 | 249.1 ± 3.5 | 160.9 ± 22.0 | 132.2 ± 18.9 |
| Antxrl | 114.4 ± 11.9* | 70.3 ± 9.1 | 69.2 ± 7.3 | 149.5 ± 4.5 | 166.5 ± 42.8 | 239.8 ± 9.1* | 218.3 ± 6.2 | 133 ± 3.7 | 113 ± 2.6 |
| Art3 | 107.9 ± 10.7 | 79.3 ± 0.4 | 73.3 ± 8.8 | 143.7 ± 9.2 | 138.4 ± 3.7 | 256.1 ± 12.8 | 233.5 ± 14.6 | 113.2 ± 11.0 | 118.3 ± 10.5 |
| Art5 | 107.0 ± 9.8 | 80.4 ± 4.3 | 54.3 ± 7.6 | 150.9 ± 1.73 | 126.9 ± 10.1 | 234.8 ± 5.9* | 206.8 ± 13.4* | 136.9 ± 4.4 | 107.7 ± 18.8 |
| Baz2a | 98.7 ± 17.5 | 89.6 ± 2.0* | 73.9 ± 3.3 | 168.0 ± 12.9 | 164.5 ± 6 | 228.7 ± 19.2 | 252.8 ± 5.4 | 127.8 ± 16.5 | 145.7 ± 6.6 |
| Ccdc70 | 91.6 ± 6.2* | 84.4 ± 1.3* | 71.1 ± 2.6 | 148.8 ± 11.1 | 141.4 ± 4.1 | 228.7 ± 1.7 | 240.9 ± 2.1* | 127.8 ± 14.1 | 114.3 ± 11.0 |
| Cfap107 (1700012P22Rik) | 98.2 ± 9.9 | 79.7 ± 6.6 | 77.6 ± 12.7 | 140.4 ± 19.7 | 122.5 ± 9.2 | 228.7 ± 6.2 | 208.7 ± 3.0* | 128.7 ± 28.6 | 98.5 ± 14.9 |
| Cimip1 (1700021F07Rik) | 100.8 ± 9.8 | 78.5 ± 7.5 | 69.7 ± 13.2 | 178.6 ± 6.7 | 150.6 ± 1.8 | 282.1 ± 5.3 | 249.1 ± 5.7 | 160.9 ± 7.8 | 132.3 ± 2.3 |
| Dnajb3 | 77.4 ± 10.2* | 89.1 ± 3.2* | 85.5 ± 1.9 | 137.0 ± 5.2* | 138.1 ± 4.4 | 239.9 ± 5.6* | 227.0 ± 7.2* | 120.0 ± 5.1 | 119.8 ± 5.2 |
| Eid3 | 95.3 ± 7.0 | 90 ± 8.5 | 84.6 ± 11.0 | 154.7 ± 22.6 | 129.5 ± 9.0 | 264.0 ± 18.2 | 228.6 ± 4.9* | 135.1 ± 26.2 | 101.5 ± 10.9 |
| Nupr2 (Nupr1l) | 118.3 ± 5.7* | 74.7 ± 3.3 | 68.0 ± 7.2 | 140.6 ± 15.3 | 144.1 ± 12.9 | 228.7 ± 19.7 | 237.1 ± 31.0 | 127.8 ± 18.6 | 118.0 ± 10.7 |
| Ptpn20 | 102.3 ± 9.2 | 73.3 ± 4.0 | 67.6 ± 5.0 | 151.3 ± 20.6 | 146.6 ± 9.7 | 228.7 ± 17.8 | 250.6 ± 8.6 | 127.8 ± 25.9 | 120.1 ± 16.3 |
| Ppp1r36 | 108.716.2 | 80.84.9 | 73.0 ± 4.6 | 181.8 ± 8.8 | 182.2 ± 2.3 | 264.1 ± 20.6 | 249.4 ± 10.2 | 173.4 ± 7.7 | 170.8 ± 5.5 |
| Spag8 | 107.0 ± 9.8 | 92.1 ± 0.4 | 80.7 ± 6.0 | 143.3 ± 11.6 | 131.5 ± 11.0 | 246.9 ± 15.8 | 211.5 ± 14.0* | 125.9 ± 13.0 | 106.3 ± 15.1 |
| Spmip3 (1700016C15Rik) | 106.2 ± 10.0 | 79.1 ± 4.4 | 61.7 ± 13.7 | 150.8 ± 5.7 | 142.2 ± 10.5 | 228.7 ± 14.4 | 246.2 ± 6.5 | 127.8 ± 7.4 | 112.9 ± 13.2 |
| Tmem235 | 114.4 ± 16.2 | 88.4 ± 2.4* | 79.5 ± 3.3 | 158.9 ± 7.0 | 143.1 ± 2.4 | 269.3 ± 6.5 | 257.8 ± 5.5 | 140.7 ± 7.3 | 110.6 ± 2.8 |
| Tsacc | 114.4 ± 4.9 | 83.5 ± 5.3 | 73.4 ± 4.9 | 138.5 ± 9.0* | 140.4 ± 12.9 | 223.3 ± 3.9* | 216.5 ± 24.6 | 122.2 ± 6.1* | 123.0 ± 10.2 |
| 1700011L22Rik | 105.4 ± 7.8 | 68.6 ± 13.3 | 65.0 ± 6.1 | 150.6 ± 17.3 | 142.6 ± 10.6 | 255.8 ± 19.6 | 219.8 ± 12.5* | 132.3 ± 18.9 | 127 ± 9.8 |
| 4930578I06Rik | 115.6 ± 14.3 | 72.5 ± 6.8 | 72.8 ± 14.7 | 143.2 ± 20.4 | 143.1 ± 22.1 | 225.5 ± 33.1 | 233.8 ± 25.1 | 130.6 ± 20.4 | 108.9 ± 26.6 |
Analyses of testis weight and sperm motility parameters by CASA. Spermatozoa movement analysis was performed at 10 and 120 min of TYH culture. VAP: Average path velocity. VCL: Curvilinear Velocity. VSL: Straight Line Velocity. Asterisks indicate significant differences (P < 0.05) between WT data.
Dnajb3
DNAjb3 is encoded by a single exon, and by designing crRNA at two locations within this exon, a mutant with a deletion of 783 bp was obtained (Figure 2A and B). No overt abnormalities were observed in the KO mice. There were no abnormalities in testicular tissue morphology and histology (Figure 2C and D), as well as the spermatozoa (Figure 2E). The average litter size was 8.7 ± 2.9, which was not significantly different from the wild type (8.2 ± 2.7) (Table 1). Analysis of sperm motility patterns using CASA revealed that the motility of KO sperm was significantly higher after 10 min of incubation (Table 2). Furthermore, the VAP of KO sperm was lower after 10 min of incubation, and the VCL of the KO was lower than that of WT at both 10 and 120 min of incubation.
Figure 2.
Generation of Dnajb3 KO mice and analysis of male fertility phenotypes. Dnajb3 deletion strategy using CRISPR/Cas9 with crRNAs set at two locations in a single exon (A). PCR electrophoresis using primer pairs that distinguish between WT and KO. The designed positions of the primers are shown in (A). Sequence near the base deletion is part of the mutant DNA by Sanger sequencing. The broken line indicates the area where the defect occurred (B). Photographs of the testicular appearance of both genotypes. Bars = 2 mm (C). Photos of seminiferous tubule cross-section with PAS staining. Bars =200 μm (D). Morphological observation of sperm collected from the cauda epididymis. Bars = 100 μm.
Eid3
Eid3 is encoded by a single exon, and a 1096-bp deletion with 1-bp insertion mutant was obtained by designing crRNAs at two locations within the exon (Figure 3A and B). The average litter size was 8.6 ± 2.2, and no significant decrease was found (Table 1). No overt abnormalities were observed in the KO mice. No abnormalities were observed in the spermatogenic cycle (Figure 3C and D), or sperm morphology (Figure 3E). The motility parameters of KO sperm were significantly lower only in VCL at 120 min incubation (Table 2).
Figure 3.
Generation of Eid3 KO mice and analysis of male fertility phenotypes. Eid3 deletion strategy using CRISPR/Cas9 with crRNAs set at two locations in a single exon (A). PCR electrophoresis using primer pairs that distinguish between WT and KO. The designed positions of the primers are shown in (A). Sequence near the base deletion is part of the mutant DNA by Sanger sequencing. The broken line indicates the area where the defect occurred (B). Photographs of the testicular appearance of both genotypes. Bars = 2 mm (C). Photos of seminiferous tubule cross-section with PAS staining. Bars =200 μm (D). Morphological observation of sperm collected from the cauda epididymis. Bars = 50 μm.
Ptpn20
Ptpn20 is composed of 12 exons, and the ORF is located in exons 3–11. To select recognition sequences with high cleavage efficiency, we designed crRNAs in exons 4 and 10 (Figure 4A). As a result, a mutant lacking 25 443 bps in the target region was obtained (Figure 4B). As described in a previous paper, KO mice were found to develop hydrocephalus [22]. The average litter size was 9.1 ± 2.4 and appeared to have normal fecundity (Table 1). No abnormalities were observed in the spermatogenesis cycle (Figure 4C and D) or sperm morphology (Figure 4E).
Figure 4.
Generation of Ptpn20 KO mice and analysis of male fertility phenotypes. Ptpn20 deletion strategy using CRISPR/Cas9 with crRNAs set at two locations in a single exon (A). PCR electrophoresis using primer pairs that distinguish between WT and KO. The designed positions of the primers are shown in (A). Sequence near the base deletion is part of the mutant DNA by Sanger sequencing. The broken line indicates the area where the defect occurred (B). Photographs of the testicular appearance of both genotypes. Bars = 2 mm (C). Photos of seminiferous tubule cross-section with PAS staining. Bars =200 μm (D). Morphological observation of sperm collected from the cauda epididymis. Bars = 100 μm.
Spag8
The Spag8 allele was deleted by designing two crRNAs upstream of exon 1 and near the stop codon of exon 7 (Figure 5A). Mutants with a 2336 bp deletion were confirmed by PCR and sequencing (Figure 5B). Spag8 KO male mice were fertile, with a litter size of 9.6 ± 1.2 and there was statistically no significant difference to WT (Table 1). There were no overt abnormalities in the KO mice, the morphology of the external testis, or the prepared tissue specimens. (Figure 5C and D). T were no abnormalities in the external appearance of the constituent parts (Figure 5E) and overall motility of the spermatozoa but significantly lower values were detected in the motility rate at 120 min in the parameters measured by CASA (Table 2).
Figure 5.
Generation of Spag8 KO mice and analysis of male fertility phenotypes. Spag8 deletion strategy using CRISPR/Cas9 with crRNAs set at two locations in a single exon (A). PCR electrophoresis using primer pairs that distinguish between WT and KO. The designed positions of the primers are shown in (A). Sequence near the base deletion is part of the mutant DNA by Sanger sequencing. The broken line indicates the area where the defect occurred (B). Photographs of the testicular appearance of both genotypes. Bars = 2 mm (C). Photos of seminiferous tubule cross-section with PAS staining. Bars =200 μm (D). Morphological observation of sperm collected from the cauda epididymis. Bars = 25 μm.
Discussion
The generation of KO mice is effective in elucidating the functions of individual genes in vivo. However, generating KO mice by homologous recombination using ES cells requires enormous amounts of resources and time. The use of the CRISPR/Cas9 system has greatly improved efficiency, and it is now possible to generate KO mice for all genes. Our laboratory has previously conducted screenings targeting male fertility across several hundred gene knockout (KO) mouse lines, evaluating and reporting on genes whose functions were previously unknown [19–24]. The candidate genes we targeted in this article range from those for which there is little information other than high mRNA expression in the testis to those for which a certain degree of function or interacting factors have been predicted based on previous in vitro studies. Our results showed that mice created from knocking out these individual genes remained fertile and were not essential.
DNAJ family proteins have a J domain that binds to HSP70 and functions as co-chaperones [25]. Dnajb3, also called MSJ-1, was identified as a homolog expressed in spermatogenic cells [26]. Because the human DNAJB3 is classified as a pseudogene according to the Ensemble and NCBI database (as of 25 February 2024), the mRNA expression pattern was not shown here. However, a report has indicated its presence at the protein level in human PBMC [27], which requires further validation. Although the enriched expression pattern in the testes suggests that this gene is important for male reproductive function, no significant reduction was observed in the number of pups obtained from KO male mice. From these results, we concluded that it is not an essential gene.
Family proteins of EP300-interacting inhibitor of differentiation (EID) have been identified as factors that suppress skeletal muscle differentiation. So far EID1 [28], EID2 [29], and EID3 [30] have been reported. Like its human ortholog, Eid3 is highly expressed in the testis and was the target of our in vivo knockout screen. Verification of the function of EID3 in vitro predicted that it functions to suppress transcription in the nucleus [31]. However, our mating tests with Eid3 KO mice revealed that it does not affect the litter size. In contrast, sperm motility analysis showed a significant decrease in VCL after 120 min of incubation. This does not exclude the possibility that Eid3 deficiency affects the expression level or localization of proteins involved in sperm motility.
Based on the identified amino acid sequence information, tyrosine-protein phosphate non-receptor type 20 (Ptpn20) has been reported to be a tyrosine phosphatase localized in the spermatocytes cytoplasm [32]. A mouse model lacking Ptpn20 has already been established and reported to develop hydrocephalus [22], but no analysis has focused on a testicular phenotype. Here, our results clearly showed that the loss of Ptpn20 does not affect fertility, even though its mRNA expression level is a principle in the testis. It may not have a decisive role in spermatogenesis.
It has been experimentally reported using cell lines that sperm-associated antigen 8 (SPAG8) can promote the binding of ACT and CREM during post-meiotic transcriptional activation [33]. CREM was later reported to have decreased mRNA expression in NOA patients, suggesting a relationship with spermatogenesis [34]. Therefore, it was hypothesized that the KO of Spag8 would interfere with post-meiotic transcriptional activation resulting in a phenotypic appearance, but there was no significant difference in fertility between Spag8 KO and wild-type male mice.
In the present study, we newly investigated the in vivo functions of 18 genes that are abundantly expressed in the testis and analyzed individual knockout lines one by one. While some of the gene KO lines resulted in a decrease in sperm motility parameters, it was revealed that deficiency of each does not affect fertility. However, it should be noted that a few of the 18 target genes in this study have paralogs and a slight expression of paralogue is unlikely to complement the target gene function but we can not rule out their contribution. Therefore, we investigated the amino acid sequence homology [35] between the target and these paralogs and TPM values in the testis (Supplementary Table 3). While Antxr1 and Antxr2 are called paralogues, their TPM values in the testis were 2.0 and 8.4, which were markedly lower than those of Antxrl, 494.6. The TPM values in the testis for Baz2b are about five times lower than Baz2a (10.9 and 52.8, respectively). Interestingly, Tmem235 has many paralogues, Cacng2, Cacng3, Cacng4, Cacng5, Cacng7, Cacng8, and Tmem114, and their TPM values in the testis are comparable. To be conclusive, multiple gene KO studies would be needed.
Collectively, our results provide insight into the male contraceptive drug development. Because a single KO of our target genes did not result in male infertility, these encoded proteins are not suitable targets for drug development. Our continued approach will lead to the discovery of key genes for male infertility among the many genes of unknown function in the testis.
Supplementary Material
Acknowledgment
We thank the NPO, Biotechnology Research and Development for supporting the KO mice generation. We also thank Dr. Daisuke Mashiko for his helpful bioinformatic suggestions. We thank Ms. Ferheen Abbasi for her critical reading of the manuscript.
Footnotes
† Grant Support: This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI grants JP21H05033 (to MI), the Eunice Kennedy Shriver National Institute of Child Health and Human Development grant R01HD088412 (to MI).
Contributor Information
Kaito Yamamoto, Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Yuki Hiradate, Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Masahito Ikawa, Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Author contributions
K.Y. and M.I. designed the research. K.Y. and Y.H. performed the experiments. K.Y. and Y.H. analyzed the data and K.Y., Y.H., and M.I. wrote the manuscript.
Conflicts of interest: The authors have declared that no conflict of interest exists.
Data availability
The data underlying this article are available in the article and in its online supplementary material.
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