Tmprss12 ^−/− mice were generated on the C57BL/6 background via Cas9/RNA-mediated gene targeting as described previously (Shen C. et al., 2013). All the mice were housed under specific pathogen free condition with unlimited access to food and water. The constant room was maintained at the temperature of 22–24°C with a light:dark cycle of 12:12. All the research involving animal experiments were approved by the ethics committees of Nanjing Medical University.

Total RNA was extracted from the sample by using Trizol reagent (Invitrogen, 15596-026) according to the manufacturer’s instructions. mRNA was reverse transcribed to cDNA using HiScript II Q Select RT SuperMix for qPCR (Vazyme, R232-01). PCR (polymerase chain reaction) was performed using 2 × Taq Plus Master Mix (Vazyme, P212-01), with primers specific for mouse Tmprss12. The PCR products were analyzed by agarose gel electrophoresis using mouse Actin as an internal control. Quantitative real-time PCR of cDNA was carried out using AceQ qPCR SYBR Green Master Mix (Vazyme, Q141-02) according to the manufacturer’s instructions with an ABI Q5 real-time PCR System (Applied Biosystems, Thermo Fisher Scientific). The primer sequences used for these experiments are listed in Supplementary Table 1.

Protein extracts from mouse tissues or sperms were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad, 1620177). Membranes were blocked with 5% nonfat milk and then incubated overnight with the primary antibodies: anti-TMPRSS12 (Santa Cruz Biotechnology, sc-249059; 1:500), anti-TEKTIN-T (Proteintech, 13518-1-AP; 1:500), anti-IQCG (Bioss, bs-9022R; 1:500), anti-CLIP170 (Proteintech, 23839-1-AP; 1:2,000), anti-MEIG1 (Bioss, bs-18778R; 1:500), anti-AGFG1 (ZENBIO, 513500; 1:1,000), anti-KLC3 (Santa Cruz Biotechnology, sc-398332E-7; 1:500), anti-COX2 (ZENBIO, 381136; 1:1,000), anti-COX3 (ZENBIO, 381270; 1:1,000), anti-MTATP (Proteintech, 55313-AP; 1:500), anti-MTCYB (Proteintech, 55090-1-AP; 1:500), anti-BAX (Proteintech, 50599-2-Ig; 1:5,000), anti-BCL2 (Proteintech, 12789-1-AP; 1: 2,000), anti-CASPASE3 (Proteintech, 19677-1-AP; 1:1,000), anti-HELLS (Abclonal, A5831; 1:1,000), anti-RAD51 (Abclonal, A6268; 1:500), anti-BCCIP (Abclonal, A8586; 1:500), anti-MGST1 (Abclonal, A16399; 1:1,000), anti-PGAM2 (Abclonal, A7917; 1:1,000), anti-β-ACTIN (Biogot, AP0060; 1:10,000), anti-α-TUBULIN (Biogot, BS1482M; 1:10,000). The washed membranes were incubated with the secondary antibodies according to the appropriate concentration. The specific protein bands were detected by an ECL kit (Thermo, 32109) and Bio-Rad gel imaging system.

Three adult Tmprss12 ^−/− mice and wild-type (WT) mice were used for fertility tests. Each male was matched with two females. Male and female mice were paired in this manner for 6 months and the number of live-born pups for each pairing was recorded.

The sperm count and motility was quantified by Computer Assisted Sperm Analysis detection with the IVOS II™ system (Hamilton Thorne, United States). Briefly, sperm from Tmprss12 ^−/− and WT mice were extracted from the cauda epididymis and incubated in human tubal fluid (HTF) culture medium (Easy Check, M1130) at 37°C. The sperm suspension was added to a counting chamber for analysis.

Testes from adult mice were fixed in modified Davidson’s fluid, embedded in paraffin and cut into 5-µm sections. Periodic acid-Schiff (PAS) staining was performed for testis sections according to the manufacturer’s protocol (Thermo, 87007) to determine the stage of spermatogenesis. Each stage with a distinct ordering of cell associations along the length of the seminiferous tubules was designated by Roman numerals, and approximately 200 tubules from each section were analyzed under microscopy (ZEISS, Germany). For sperm morphology analysis, sperm was washed three times in phosphate buffered saline (PBS) and transferred to slides. After immobilization with 4% PFA, sperm was stained with hematoxylin-eosin (HE) (Beyotime, C0105S). At least 200 sperm cells were analyzed per sample.

The prepared sections were blocked in 1% bovine serum album for 2 hours and then incubated with the goat polyclonal anti-TMPRSS12 (1:100, sc-249059; Santa Cruz Biotechnology) overnight at 4°C. The sections were incubated with fluorescently labeled secondary antibodies at room temperature for 2 h. After being counterstained with DAPI, the slides were viewed with a LSM700 confocal microscope (Zeiss, Germany).

Apoptosis detection of spermatogenic tubules and cells was conducted using a terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) BrightRed Apoptosis Detection Kit (Vazyme, A113-01), following the manufacturer’s directions. Briefly, the testis sections were equilibrated with TdT buffer for 20 min at room temperature. TdT buffer was removed and terminal transferase reaction mix was added. The reaction was performed for 1 h at 37°C. Sections were washed with PBS and then counterstained with DAPI. Slides were viewed under a LSM700 confocal microscope (Zeiss, Germany).

Chromosome spreads of prophase I spermatocytes were performed as previously described (Peters et al., 1997; Kolas et al., 2005). In brief, seminiferous tubules were separated, cut into pieces with scissors, and incubated in hypotonic buffer (0.45% NaCl) for 40 min. Cell suspensions were fixed with 1% paraformaldehyde containing 0.15% Triton X-100 and 0.5 M sodium borate and then air-dried on slides. Samples were blocked with 1x ADB (1% normal donkey serum, 0.03% BSA, and 0.05% Triton X-100) for 1 h and then incubated with primary antibodies at 37°C for 12–16 h. After being blocked with 1x ADB for 5–6 h at room temperature, the samples incubated in fluorescently labeled secondary antibodies at 37 °C for 1.5 h. The slides were viewed under a LSM700 confocal microscope (Zeiss, Germany). Primary antibodies were as follows: SYCP1 (1:100, NB300-229; Novus Biologicals), SYCP3 (1:500, ab97672; Abcam), γH2AX (1:500, ab2893; Abcam).

Sperm samples separated from cauda epididymis were fixed in 0.1 M phosphate buffer (pH7.3) containing 2.5% glutaraldehyde and 4% (vol/vol) glutaraldehyde with 2% (wt/vol) OsO4 and then embedded in Araldite. Transmission electron microscopy was performed by the Electron Microscopy Laboratory of Nanjing Medical University. Transmission electron microscopy imaging was conducted on a JEM-1010 transmission electron microscope (JEOL, United States, Inc.).

According to the manufacturer’s instructions (Beyotime, S0027), sperm samples were washed twice, resuspended in lysis buffer, vortexed and then placed on ice. ATP was measured by luminometric methods using commercially available luciferin/luciferase reagents in luminometer (TD-20/20, Turner Designs). An average of 5 × 10⁷ sperm were used for ATP analysis.

The JC-1 mitochondrial membrane potential detection kit (Beyotime, C2003S) was used to determinate the MMP. Briefly, sperm were incubated with JC-1 staining working fluid in the dark at 37°C for 20 min. After washed by PBS, the sperms were analyzed by an LSM700 confocal microscope (Zeiss, Germany) and flow cytometer. JC-1 monomers give green fluorescence while JC-1 aggregates produce red fluorescence. MMP was analyzed by relative ratio of red/green fluorescence.

Extracellular acidification and oxygen consumption were analysed by a glycolysis assay kit (Abcam, ab197244) and extracellular oxygen consumption assay kit (Abcam, ab197243), respectively, according to the manufacturer’s instructions. Briefly, sperm were plated at 50,000 cells per well in a 96-well plate, and then test reagent was added to the sample wells. For the oxygen consumption assay, two drops of prewarmed high-sensitivity mineral oil were added to each well before measurement. Fluorescence (Ex 360 nm Em 620 nm for the extracellular acidification assay; Ex 380 nm Em 645 nm for the oxygen consumption assay) was measured every 6 min for 1 h by using a microplate reader (BioTek Synergy Mx, United States).

Testes proteins were extracted from WT and Tmprss12 ^−/− mice in lysis buffer. The protein was loaded and separated by gel rehydration on 24-cm immobilized, PH3-10, nonlinear gradient strips for 2-D according to the previously described (Guo et al., 2010). After isoelectric focusing carried out in an IPGphor apparatus (GE Healthcare, San Francisco, CA, United States), the samples for second-dimension were separated in 12% polyacrylamide gels by an Ettan-Dalt 6 system (GE Healthcare). Subsequently, the gels were stained with silver staining. The sliver-stained gels were scanned and analyzed using 2D Elite Image Master software (GE Healthcare). The protein spots, which were shown >2-fold difference (p < 0.05, Student’s t-test) between WT and Tmprss12 ^−/− testes, were excised, dehydrated in ACN, and dried at room temperature. The protein spots were digested according to our previous described (Guo et al., 2010). The digests were immediately spotted onto 400-µm anchor chips (Bruker Daltonics). The protein spots were subsequently subjected to identification by MALDI-TOF/TOF analysis. All the samples were analyzed in a time-of-flight Biflex IV mass spectrometer (Bruker Daltonics). Tandem mass spectra were given by an Ultraflex II mass spectrometer (Bruker Daltonics) in LIFT mode and processed using FlexAnalysis software (version 2.4; Bruker Daltonics). Each acquired tandem mass spectrometry (MS/MS) queries were performed using the Mascot (version 2.1, Matrix Science, London, United Kingdom) search engine in the Uniport-House mouse database. All MS/MS identifications by Mascot were manually verified for spectral quality and y and b ion series matches.

All experiments were repeated at least three times. Significance was tested by using Two-way ANOVA or Student’s t test using Prism software (GraphPad Software, La Jolla, CA, United States). Values of p < 0.05 were considered to be statistically significant. All data are reported as means ± the standard deviation of the mean (SD).

The expression of Tmprss12 was assayed by RT-PCR performed with 11 mouse tissues, including heart, liver, spleen, lung, kidney, brain, muscle, testis, epididymis, uterus and ovary tissues. As shown in Figure 1A, Tmprss12 was exclusively expressed in testis tissue. By PCR and western blot detection, we found at the mRNA and protein levels that TMPRSS12 was first detected in the testes of 10-day-old mice, and its expression remained constant after 14 days of age (Figures 1B,C). We further detected the expression characteristics of TMPRSS12 in mouse testes by immunofluorescence analysis. The results demonstrated TMPRSS12 expression in germ cells within the seminiferous tubules and showed that its expression was initiated from spermatocytes and remained until step 12 elongated spermatids (Figure 1D). We also confirmed the expression of TMPRSS12 in mature sperm by PCR and western blot analysis (Figures 1E,F), and the immunofluorescence results showed positive signals of TMPRSS12 located in the acrosome (Figure 1G). The above results suggested the potential important role of TMPRSS12 in male reproductive function, which very likely influenced meiosis, spermiogenesis and acrosome-related sperm function.

To investigate whether TMPRSS12 is required for male fertility, Tmprss12 knockout mice were generated using the CRISPR/Cas9 system. The injection of a single guide RNA targeting Tmprss12 exon 2 produced knockout mice harbouring a 2-bp deletion. A representative image of the Sanger sequencing results for the verification of the Tmprss12 ^−/− mouse is shown in Figure 2A. We confirmed the lack of TMPRSS12 in the testes and sperm of Tmprss12 ^−/− mice by western blot and immunofluorescence assays (Figures 2B–D).

Mating tests were performed and continuous monitoring was conducted for 6 months to analyse the fertility of the mice. The female Tmprss12 ^−/− mice were fertile with litter sizes comparable to those of WT females. However, Tmprss12 ^−/− males were infertile and produced no litters (Figure 2E). These results suggest that TMPRSS12 is required for male reproduction.

To explore the possible reasons for infertility in Tmprss12 ^−/− mice, we first detected the sperm parameter by Computer Assisted Sperm Analysis (CASA). The results showed that the sperm count and motility were significantly decreased in Tmprss12 ^−/− mice compared to WT mice (Figure 3A).

The weight and size of the testis did not differ significantly, and the gross morphology and histology of the testis and sperm were similar between the WT and Tmprss12 ^−/− mice (Figures 3B–D). Moreover, we paid close attention to the spermatogenesis of Tmprss12 ^−/− testes by counting spermatogenic cells at all stages (Figure 3E). The counts of pachytene spermatocytes, diplotene spermatocytes, round spermatids and elongated spermatids all decreased significantly in Tmprss12 ^−/− mice compared with WT mice, which probably led to the decreased sperm count in Tmprss12 ^−/− mice.

To assess the reasons for the decrease in germ cells, we first evaluated the state of spermatogenic cells in the testes of Tmprss12 ^−/− and WT mice. Terminal deoxynucleotidyl transferase nick-end labelling (TUNEL) assays were performed to detect apoptotic cells in the testes of these two groups. We found that the numbers of apoptotic germ cells increased remarkably in Tmprss12 ^−/− mice compared with WT mice and the apoptotic cells were mainly spermatocytes (Figures 4A,B).

In spermatogenesis, meiosis is an important biological process that produces haploid germ cells. Abnormal meiosis interferes with the spermatocyte state and affects the progress of spermatogenesis. Based on our findings that spermatocyte apoptosis increased and the number of spermatogenic cells after the zygotene stage decreased, it is reasonable to speculate that TMPRSS12 may be involved in meiosis. Therefore, we examined the key biological events of meiosis in Tmprss12 ^−/− testes. We assessed chromosomal synapsis by immunofluorescence analysis using antibodies against SYCP1 and SYCP3, which are components of the synaptonemal complex (SC). Chromosome spreads from WT testes showed that the fluorescence signal of anti-SYCP3 in the pachytene stage completely overlapped with that of anti-SYCP1 around the SC, including 19 pairs of autosomes and partial synapsed sex chromosomes in the pseudoautosomal region (PAR). In the pachytene spermatocytes of Tmprss12 ^−/− testes, chromothripsis, no synapsed sex chromosomes, and abnormal SYCP1 signals in non-PAR were observed (Figure 4C). We quantified the spermatocytes of WT and Tmprss12 ^−/− testes and found that spermatocytes with abnormal synapses were significantly higher in Tmprss12 ^−/− mice than in WT mice (Figure 4D).

In addition, we monitored the meiotic recombination of homologues in spermatocytes. Recombination is initiated by DNA double-strand break (DSB) formation in leptotene spermatocytes. DSBs could be marked by the presence of phosphorylated H2AX (γH2AX). In normal meiosis, γH2AX is present on autosomal chromatin and distributed throughout the nucleus in leptotene spermatocytes, persists into the zygotene stage and is restricted to only the sex chromosomes at the pachytene stage. As shown in Figure 4E, γH2AX disappeared from autosomes following meiotic DSB repair and was restricted to the sex chromosomes in the pachytene stages of WT spermatocytes, but the γH2AX signal was distributed in Tmprss12 ^−/− pachytene spermatocytes and was not restricted to sex chromosomes, suggesting failure in the repair of meiotic DSBs. Moreover, the results of quantifying the abnormal spermatocytes in the two groups were significantly different (Figure 4F).

We further explored whether the apoptotic state of sperm was also involved in the decreased sperm count of Tmprss12 ^−/− mice. Mitochondrial membrane potential (MMP) reduction is an important indicator of early apoptosis. Therefore, we examined the MMP of sperm in Tmprss12 ^−/− mice. JC-1 dye was used to monitor the MMP, which was lower in Tmprss12 ^−/− mice than WT mice (Figures 5A–C). Furthermore, we detected key molecules in classic apoptosis pathways, including BAX, BCL-2 and CASPASE3 (Yang and Cortopassi, 1998; Antonsson et al., 2000; Barroso et al., 2006; Brunelle and Letai, 2009). The expression levels of BAX and CASPASE3 were increased while that of BCL-2 was decreased in Tmprss12 ^−/− mice, which indicated increased sperm apoptosis when TMPRSS12 was deficient (Figures 5D,E).

The CASA analysis revealed that the sperm motility of Tmprss12 ^−/− male mice was significantly lower than that of WT mice. To explore the reason for the decreased sperm motility, we further observed the ultrastructure of sperm in Tmprss12 ^−/− and WT mice under a transmission electron microscope. The results showed that the inner mitochondrial membrane cristae were blurred or absent in Tmprss12 ^−/− mice. According to the statistical analysis, the number of sperms with abnormal mitochondria in Tmprss12 ^−/− mice was significantly higher than that in WT mice (Figures 6A,B).

Mitochondria are the energy metabolism centres of cells and the main sites of oxidative phosphorylation for producing ATP. Thus, we detected the sperm ATP content, glycolysis, oxygen consumption and the key markers of the oxidative phosphorylation process in mitochondria. The decreased ATP content in the sperm of Tmprss12 ^−/− mice is shown in Figure 6C. Extracellular acidification revealed that the sperm of Tmprss12 ^−/− mice had glycolysis levels similar to those of WT mice (Figure 6D). However, the oxygen consumption assay showed that the oxygen consumption of sperm of Tmprss12 ^−/− mice was reduced compared with that of the sperm of WT mice (Figure 6E). COX2, COX3, MTATP and MTCYB are the key markers of the oxidative phosphorylation process in mitochondria (Feng et al., 2008; Heidari et al., 2016). The mRNA and protein levels of MTATP and MTCYB in the sperm of Tmprss12 ^−/− mice were reduced compared with those in WT mice (Figure 6F,G; Supplementary Figure 2). The above results suggested that an abnormal ultrastructure of mitochondria and impaired ATP synthesis in sperm are potential causes of decreased sperm motility.

To investigate whether the abnormal mitochondrial structure is caused by damaged spermiogenesis, we detected special molecules involved in spermiogenesis in the testes, including TEKTIN-T, IQCG, CLIP-170, MEIG1, AGFG1, and KLC3. TEKTIN-T and IQCG are essential for sperm flagellum formation (Tanaka et al., 2004; Li et al., 2014). CLIP-170 and MEIG1 are critical for manchette structures (Akhmanova et al., 2005; Zhang et al., 2009). AGFG1 is involved in acrosomal development (Kang-Decker et al., 2001). KLC3 plays an important role in mitochondrial migration and sperm tail midpiece formation (Zhang et al., 2012). Real-time PCR and western blot both showed that the expression of KLC3 decreased in the testes of Tmprss12 ^−/− mice (Figures 7A,B; Supplementary Figure 3).

As a serine protease, TMPRSS12 should theoretically regulate target proteins to play a specific role in spermatogenesis. To further investigate the mechanism of TMPRSS12 in male reproduction, we used 2-D electrophoresis combined with MALDI-TOF-TOF MS/MS to construct differential protein expression profiles of testes in Tmprss12 ^−/− and WT mice (Supplementary Figure 4). A total of 64 spots with 2-fold or more differential expression between two groups were identified using MALDI-TOF-TOF MS/MS. As a result, 54 unique proteins were identified successfully, including 39 proteins with decreased expression and 15 proteins with increased expression in Tmprss12 ^−/− mice (Supplementary Table 2). We analysed the molecular functions of these differentially expressed proteins by IPA (Ingenuity Pathway Analysis) and found that these proteins were mainly related to meiosis, apoptosis, mitochondrial function and cell adhesion (Figure 8A).

To certify the expression levels of differential proteins, some proteins related to key events were selected for an expression analysis by western blot. For example, lymphoid-specific helicase (HELLS) has been proven to be essential for meiotic progression, especially in chromosomal synapses (Zeng et al., 2011; Kollarovic et al., 2020). Recombinase RAD51 is necessary for DSB repair and homologous recombination (Hinch et al., 2020). BCCIP interacts with RAD51 to stimulate DNA pairing activity in homologous recombination (Kelso et al., 2017). PGAM2 activity could modulate mitochondrial function (Okuda et al., 2013). MGST1 is a glutathione transferase located in mitochondrial membranes that protects mitochondria against oxidative stress to ensure their normal biological function (Zhang et al., 2001; Johansson et al., 2010; Schaffert, 2011). The western blot results showed that the expression trend of these proteins was consistent with the differential expression profiles (Figures 8B,C).