PRSS55 plays an important role in the structural differentiation and energy metabolism of sperm and is required for male fertility in mice

Abstract Orderly and stage‐specifically expressed proteins are essential for spermatogenesis, and proteases play a key role in protein activation and function. The present study aimed to investigate serine protease 55 (PRSS55), which was reported to play a role in sperm‐uterotubal junction (UTJ) migration and sperm‐zona pellucida (ZP) binding. We found that PRSS55 was specifically expressed in testicular spermatids and epididymal spermatozoa. By constructing knockout mice targeting all transcripts of Prss55, we demonstrated that deletion of Prss55 resulted in a serious decline of male fertility, with significantly increased sperm malformation and decreased sperm motility. In Prss55 −/− mice, increased structural abnormality, including deficient “9 + 2” microtubules, damaged peripheral dense fibre, and defective mitochondrial cristae, were found in sperm. In addition, sperm showed decreased expression of electron transfer chain molecules and lower ATP contents. These could be the potential causes of the astheno/teratozoospermia phenotype of the Prss55 −/− mice, and provided new evidence for the previously reported impaired sperm‐UTJ migration. Moreover, preliminary studies allowed us to speculate that PRSS55 might function by activating type II muscle myosin in the testis, which is involved in many processes requiring motivation and cytoskeleton translocation. Thus, PRSS55 is essential for the structural differentiation and energy metabolism of sperm, and might be a potential pathogenic factor in astheno/teratozoospermia. Our results provide an additional explanation for the male sterility of Prss55 −/− mice, and further reveal the role of PRSS55.


| INTRODUC TI ON
Infertility is commonly encountered in clinical practice, which not only affects marriages and families, but is also a worldwide reproductive health problem. Approximately 20% of couples of childbearing age worldwide suffer infertility, among which male factors account for about 50%. 1 Spermatozoa are the ultimate executor of the male reproductive process. The study of spermatogenesis has been a hot topic and focus in the field of reproductive biology, and understanding the pathogeneses of spermatogenesis disorders plays an important role in improving the diagnosis and treatment of male infertility.
Studies have shown that spermatogenesis is regulated by intricate gene and protein networks, and orderly and stage-specifically expressed genes and proteins play a key role. 2 Proteases are of interest, especially the largest serine protease family. They usually activate target substrate proteins by hydrolysing specific peptide segments, and then affect other downstream proteins, thus executing a cascade regulatory role. 3 Some members of this family play a cooperative role with other proteins. 4 Thus, proteases may participate in complex protein networks. A series of studies have shown the important roles of serine proteases in various physiological functions. 5 According to the Human Protein Atlas database analysis and literature review, male reproductive organs contain the most abundant serine proteases (Table S1), which suggests the importance of serine proteases in male reproduction. Some studies have focused on the potential roles of serine proteases in testicular spermatogenesis. For example, serine proteases PRSS41-43 were found to be involved in spermatocyte meiosis, and inhibition of PRSS41-43 induced meiosis arrest and germ cell apoptosis. 6,7 In addition, some PRSS members play an important role in sperm function. For example, deficiency of PRSS37 resulted in defective sperm migration into the oviduct and reduced the in vitro sperm-egg interaction, resulting in infertile male mice. 8,9 Mice lacking PRSS21 were subfertile; their spermatozoa exhibited decreased motility and an impaired ability to bind the zona pellucida (ZP) of oocytes. [10][11][12] However, some PRSS members, such as PRSS44, PRSS46, PRSS51 and PRSS54, were reported to be dispensable for male fertility; their deletion had no effect on mouse fertility and the routine testing indices of testis and sperm. 13,14 However, more members of the PRSS family need to be studied to reveal their role in male reproduction.
The present study focused on PRSS55, whose role in male reproduction is a matter of debate. Khan et al found that Prss55 KO mice were fertile and thus concluded that PRSS55 is dispensable for male fertility. 15 However, Shang et al and Kobayashi et al proposed that PRSS55 is essential for male reproduction; they found that deficiency of PRSS55 resulted in defective sperm migration from the uterus into the oviduct. 14,16 However, there were inconsistencies in their results. For example, Shang et al showed that Prss55 −/− sperm presented defective recognition and binding to zona-intact or zona-free oocytes, but the in vitro fertilization rate was not affected.
However, Kobayashi et al only found defective binding of Prss55 −/− sperm to zona-intact oocytes, and the in vitro fertilization rate was decreased significantly. In terms of knockout strategy, all the three research teams deleted two transcripts of Prss55 in the testes; however, the phenotypes were not exactly the same.
In the present study, we designed a single guide RNA (sgRNA) targeting exon 3 of Prss55, which could achieve knockout in mice by targeting all four transcripts of Prss55. We then conducted a series of experiments using this KO mouse model, aiming to determine the necessity of PRSS55 in male fertility and to explore whether there were more abnormal phenotypes, thus providing a more comprehensive analysis of PRSS55's functions.

| Animals
Two pairs of primers (Primer1, Primer2) were designed to verify all possible Prss55 transcripts in the testes. The primer sequences are listed in Table S2. Prss55 knockout (KO) mice were generated via Cas9/RNA-mediated gene targeting, as previously described, 17 and we designed an sgRNA for zygote injection that could target the knockout mice for all transcripts of Prss55. All mice were housed in a specific pathogen-free animal facility with a light:dark cycle of 12:12. All the animal experiments in this study were approved by the Institutional Animal Care and Use Committees of Nanjing Medical University, Nanjing, China.

| Assessment of fertility
To determine the fertility of the mice, a male was mated with two females. The female mice were separated from the male after the formation of a normal vaginal plug was detected. The number of offspring produced per female was recorded. The mean values for each genotype combination were then plotted.

| Sperm parameter analysis
Sperm were extracted and incubated in human tubal fluid (HTF) medium (Easycheck, Nanjing, China, M1120) supplemented with 10% foetal bovine serum (FBS) at 37°C. A 10-μL aliquot of the sperm samples was evenly distributed on a glass chamber slide and analysed using a Computer Assisted Sperm Analyzer (CASA) via the IVOS II system (Hamilton Thorne, Beverly, MA, USA).

| Measurement of sperm ATP in vitro
Sperm samples were washed twice, resuspended in lysis buffer, vortexed, and then placed on ice. ATP was measured using luminometric methods via commercially available luciferin/luciferase reagents according to the manufacturer's instructions (ATP Assay Kit; Beyotime Biotechnology, Shanghai China) in a luminometer (TD-20/20; Turner Designs, San Jose, CA, USA). An average of 3 × 10 7 sperm were used for ATP analysis.

| Histological analysis
The testes and epididymis tissues from adult wild-type (WT) and

| RT-PCR and qRT-PCR analyses
Total RNA was extracted from mouse tissues using Trizol (Invitrogen), following the manufacturer's directions, and mRNA was reverse transcribed to cDNA using a Prime Script™ RT Master kit (Takara).  Table S2.

| Western blotting analysis
Proteins were extracted using RIPA Lysis Buffer (Beyotime Biotechnology) and quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific). After denaturation at 100°C for 5 minutes, the samples were subjected to 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon PVDF membranes (Millipore, Billerica, MA, USA).
The membranes were subsequently blocked with 5% nonfat dry milk powder in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween20). Primary antibodies were applied overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature. Immunoreactive protein bands were detected using an ECL kit (Thermo Scientific, Waltham, MA, USA).

| Immunofluorescence analysis
Sections were obtained from samples that were fixed using

| Apoptosis detection analysis
Apoptosis detection of spermatogenic tubules and cells was achieved using a terminal deoxynulceotidyl transferase nick-endlabelling (TUNEL) BrightRed Apoptosis Detection Kit (Vazyme), following the manufacturer's directions. Slides were viewed under an LSM700 confocal microscope (Carl Zeiss AG).

| Immunoprecipitation from cell culture extracts
HEK293T cells were transfected with plasmids encoding a flagtagged PRSS55 fusion protein (for PRSS55 overexpression) using Lipofectomine 3000 (Thermo Fisher Scientific). Two days after transfection, cells were lysed using Pierce IP Lysis Buffer (Thermo Fisher Scientific) supplemented with 1% (vol/vol) protease inhibitor mixture (Selleck, Houston, TX, USA) for 45 minutes at 4°C, and then clarified using centrifugation at 12 000 g for 30 minutes. The lysates were precleared with 50 μL of Protein A/G magnetic beads (Selleck) for 1 hour at 4°C. The precleared lysates were incubated overnight with anti-Flag antibodies (EnoGene, E12-026-4; 2 μg) at 4°C. Lysates were then incubated with 100 μL of Protein A/G magnetic beads for 3 hours at 4°C. The beads were washed three times with immunoprecipitation (IP) Lysis Buffer and eluted with Pierce IgG Elution Buffer (Thermo Fisher Scientific). Samples were boiled in sodium dodecyl sulphate (SDS) loading buffer before being subjected to SDS-PAGE.

| Mass spectrometry analysis
For mass spectrometry analysis, elution was followed by in-solution digestion. Briefly, elution was first achieved using ultrafil-

| Statistical analysis
All assays were performed at least three times. Statistical analyses were evaluated using SPSS19.0 (IBM Corp., Armonk, NY, USA). The statistical significance of differences in the mean values was assessed using the t test. Values of P < .05 were considered statistically significant.

| Expression and location of PRSS55 in adult mice
The distribution of Prss55 mRNA in various tissues of mice was detected using RT-PCR. The cDNA samples were prepared from mouse tissues include heart, liver, spleen, lung, kidney, brain, testis, epididymis, ovary and uterus. As shown in Figure 1A, Prss55 mRNA was only expressed in male reproductive organs, with prominent expression in the testis, but weak expression in the epididymis. Testis maturation is age-dependent, and the first wave of spermatogenesis takes place within 35 postnatal days. To determine the temporal and spatial expression of Prss55 mRNA in testis, we examined the Prss55 mRNA abundance in testis of mice at 1, 2, 3, 4, 5, 6, 7 and 8 weeks old using RT-PCR. The results showed that Prss55 mRNA first appeared at the second week, and sharply increased at the third week, just before the initiation of spermatid elongation. Prss55 expression then increased gradually until the adult stage ( Figure 1B).
Immunofluorescence analysis was carried out to identify the expression and location of PRSS55 in adult mouse testis and epididymis. The results revealed that PRSS55 was restrictively expressed in the elongated spermatids at steps 12-16 of spermiogenesis in the testis ( Figure 1C). In the epididymis, PRSS55 was expressed in sperm rather than in epithelial cells ( Figure 1D). Such a specific expression pattern indicated the potential physiological role of PRSS55 during spermatogenesis and sperm function in mice.

| Generation of Prss55 knockout mice
By searching the NCBI database, we found that Prss55 not only had a known and confirmed transcript, but also had several predicted transcripts. The proteins translated from these different transcripts were inconsistent in sequence, but all had a trypsin-like serine protease domain. We confirmed that Prss55 encoded all these different transcripts in mouse testes (Figure 2A,B).
Gene editing using CRISPR/Cas9 technology combined with microinjection technology was used to construct Prss55 KO mice.
We targeted the 3rd exon, which is common to all predicted transcripts, to design the sgRNA for zygote injection, which could achieve knockout in mice by targeting all transcripts of Prss55 ( Figure 2C). After the extraction of DNA, PCR amplification was performed using specific primers ( Figure 2D). The results showed that a 53-bp deletion from Exon 3 of Prss55 could be detected. We then confirmed the absence of the PRSS55 protein in testis and epididymis of Prss55 −/− mice using immunofluorescence assays ( Figure 2E,F).

| PRSS55 deficiency impaired sperm motility, thus affected fertility
Mating tests were performed, and continuous monitoring for 6 months was used to analyse mouse fertility. We found that fe-  Figure 3D). In addition, apoptotic signals in the Prss55 −/− mouse testes were not increased significantly compared with those in the WT controls ( Figure 4A). BAX and BCL2 are the most important pair of proteins in apoptosis, which have opposite functions. BAX plays a pro-apoptotic role, whereas BCL2 plays an anti-apoptotic role. A series of regulations of the apoptotic pathway eventually activate caspase 3, which then promotes apoptosis. We found that the expression levels of these apoptosis-related proteins in the KO mice were not significantly different from those in the control mice ( Figure 4B). These results suggested that the absence of PRSS55 had no significant effect on apoptosis in the testis.
The morphology and histological structure of the epididymis also showed no obvious abnormalities in Prss55 −/− mice compared with those in WT mice ( Figure 5A,B). Further assessment of the indices of the epididymis sperm using CASA analysis showed that the sperm motility of Prss55 −/− mice was significantly lower than that of the controls ( Figure 5D) (P < .001), although there was no significant difference in sperm number between the two groups ( Figure 5C).

| Abnormal morphology, structure and ATP synthesis in Prss55 −/− mouse sperm
To determine the reason for the decreased sperm motility in the We then compared the ultrastructure of the sperm from the two groups using transmission electron microscopy. Compared with WT mice (Figure 6D

| Identification of potential target substrate of PRSS55
A PRSS55 overexpression plasmid expressing PRSS55 with a Flag label was transfected into HEK293T cells. After transfection of the PRSS55 expression plasmid, the cellular proteins were extracted, and anti-Flag antibodies were used to detect the transfection efficiency via Western blotting. The results showed that PRSS55 was successfully expressed in HEK293T cells ( Figure 8A). An immunoprecipitation experiment was carried out with proteins extracted from HEK293T cells transfected with the PRSS55-Flag plasmid.
The result showed that PRSS55 and proteins that might interact with PRSS55 were eluted ( Figure 8B). The eluate obtained by immunoprecipitation was subjected to mass spectrometry and several proteins were considered to be the potential substrates of PRSS55, which showed significantly decreased levels (>1.5 fold) or were absent in the experimental group, including several type II muscle myosin family members (MYL3, MYL6, MYH9 and MYH10) ( Figure 8C).
We further examined the expression of these four molecules in the  Khan et al found that the deletion of PRSS55 did not affect the fertility of male mice. 15 However, both Shang's team and Kobayashi's team believed that PRSS55 was essential for male reproduction, and they found that deficiency of PRSS55 affected specific sperm functions (including sperm-uterotubal junction (UTJ) migration and sperm-ZP binding), which impaired fertility, although their test results in some experimental indicators were different. 14,16 There are four transcripts of Prss55 in the testis, and failure to knock out all of them might lead to incomplete or unstable phenotypes. In this study, we constructed a gene knockout mouse model that targeted all the transcripts of Prss55, which theoretically, could reveal the role of PRSS55 more comprehensively. Through a series of experiments, we attempted to determine whether PRSS55 is necessary for male fertility and whether it is involved in other aspects of spermatogenesis or sperm function. The Prss55 −/− male mice exhibited a serious decrease in fertility. Analyses of the structure of reproductive organs, the number of germ cells, as well as the basic sperm indices in these mice, demonstrated that the decline in male fertility was not caused by a defect of sperm quantity, but by impaired sperm quality, manifesting as a significant decline in motility.

| D ISCUSS I ON
The motility of sperm depends on its differentiated normal tail structure (such as microtubules, peripheral dense fibres and mitochondria) and energy metabolism. The "9 + 2" microtubule complex constitutes the axoneme, which runs through the neck of sperm to the whole flagellum, and plays an important role in flagellum movement. The peripheral dense fibres contain abundant cysteine and disulphide bonds, which are very important for the stability and elastic retraction of sperm flagellum. The mitochondria wrap around the peripheral dense fibres in a spiral way to form a mitochondrial sheath, and ATP is produced for sperm movement by mitochondrial metabolism. 19 Thus, the sperm tail is an important structure to ensure normal sperm movement. In this study, we found that the mice lacking PRSS55 showed an abnormal extension and damaged structure of sperm tails. In these mice, sperm tails exhibited different degrees of folding and curling, and movement-related structures in the sperm tails all exhibited abnormalities, including deficiency of "9 + 2" microtubules, damage to peripheral dense fibres, and defects of mitochondrial cristae. We confirmed the above abnormal phenotypes from different aspects including morphological observation, structural assessment and molecular detection (such as the level of ODF1). Furthermore, abnormal energy metabolism in the Prss55 KO mice was also confirmed: their sperm ATP content was significantly lower than that of the WT mice. The energy for sperm motility The proteins in the immunoprecipitation eluent were identified using mass spectrometry. Five proteins were found to be absent and four proteins showed decreased levels (>1.5 fold) in the experimental group compared with those in the control group sperm itself is also an important reason for the defect in its UTJ migration. Studies have demonstrated that sperm movement ability plays an important role in its passage through the vagina, uterus, UTJ and oviduct. 22 Thus, the defect in sperm motility provides a further explanation for the subfertility phenotype of Prss55 −/− mice.
Our results indicated that PRSS55 might participate in the differentiation of spermatids and directly affect sperm function, which was consistent with the specific expression characteristics of PRSS55 in testicular spermatids and epididymal spermatozoa.
Kobayashi et al proposed that PRSS55 is expressed in testicular germ cells and retained in epididymis sperm, but might play a key role in epididymis sperm rather than testicular germ cells. 14  superfamily that binds to actin and generates power by hydrolysis of ATP. Myosin is divided into many categories, among which type II myosin includes myocyte myosin and non-muscle myosin. 23 Type II muscle myosin is expressed in all eukaryotic cells and is involved in many process requiring motivation and cytoskeleton translocation, such as cell migration, polarization, adhesion and maintenance of cell morphology, and signal transduction. [24][25][26] We confirmed the expression of these four potential substrates in the testis and sperm at the mRNA level (Myl3, Myl6, Myh9 and Myh10 were expressed in testis but only Myl6, Myh9 and Myh10 were expressed in sperm), indicating that PRSS55 may have different functional substrates in testis and sperm. As a serine protease, PRSS55 might function by activating these II muscle myosins in the testis, which then participate in sperm structural differentiation and further affect its function, a defect in which could lead to male infertility.
This study used a more comprehensive strategy to construct Prss55 KO mouse model to investigate the important role of PRSS55 in male fertility. Our results demonstrated that PRSS55 is essential for the structural differentiation and function maintenance of sperm. PRSS55 is likely to be a potential pathogenic factor in male astheno/teratozoospermia. More details of the mechanism of action of PRSS55 remain to be determined.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
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