Testis‐specific serine kinase 3 is required for sperm morphogenesis and male fertility

The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis‐specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known.


INTRODUCTION
Oligoteratozoospermia, a cause of idiopathic infertility in men, is a failure of spermatogenesis leading to both a decrease in sperm counts (oligozoospermia) and the presence of abnormal sperm forms (teratozoospermia). 1 Genetic factors are considered to be one of the causes of nonobstructive oligozoospermia. 2 Multiple genes responsible for azoospermia have been reported to date, 3 and the utilization of this information for the development of infertility diagnosis, 4,5 or the development of novel contraceptives targeting crucial reproductive tract-specific proteins, 6,7 holds promise. Spermatogenesis, which takes place in the seminiferous tubule in the testis, demands a wellorganized process in which spermatogonia undergo mitosis, meiosis, and spermiogenesis. 8 During spermiogenesis, round spermatids are dramatically restructured into a suitable morphology for proper fertilization. Spermiogenesis is further divided into the three main phases: Golgi, acrosome cap/elongation, and maturation phases. 9 Phosphorylation is a posttranslational modification of proteins that is required for many cellular processes, including cell cycle progression, motility, metabolism, cell growth, and differentiation. 10 The occurrence of phosphorylation is critical for proper sperm function as it is for other somatic cells. Protein phosphorylation in mammalian sperm motility and metabolic systems has been documented. [11][12][13][14] Several studies support the importance of phosphorylation by kinases in spermatogenesis, but little is known about the mechanisms. [15][16][17] The testis-specific serine kinase (TSSK) protein family members were cloned as kinase-encoding genes with restricted expression to mouse testis. Each family member shares a similar, serine/threonine protein kinase catalytic domain. There are six members in the TSSK family: TSSK1, TSSK2, TSSK3, TSSK4, and TSSK6 are conserved in mouse and human, while TSSK5 is protein coding in the mouse and is a pseudogene in human. 16 Some of the gene knockout (KO) mouse models have been created and reported to affect male fertility. Tssk1/Tssk2 double KO and Tssk6 single KO mice display infertility, while Tssk4 single KO mice display subfertility. [18][19][20] However, detailed functions and substrates of these kinases remain unknown.
Investigation of potential substrates of kinases is important to understand the function and significance of the target kinases, and for future applications. Identification of the substrates of TSSKs has been approached in vitro, 16 but the numbers of those substrates are limited. Several strategies have been proposed and conducted to globally identify kinase substrates, such as phosphoproteomics analysis after artificial trigger or reaction using antikinase motif antibody. 21,22 One convincing strategy is global quantitative phosphoproteomics analysis using wild-type (WT)/heterozygous mutant (HET) and KO cells/tissues. 23 During bioinformatics analysis, this strategy can aid in the reduction of background signals and noise.
While Nayyab et al. 24 generated Tssk3 KO mice with local gene indels (−19 bp, −6+19 bp, and 47 bp) and reported similar phenotype results as our TSSK3 KO, the functional pathways and substrates of TSSK3 remain unknown. In the present report, we created Tssk3 KO mice with a deletion of 1551 bp, including most of the open reading frame, using the CRISPR/Cas9 system and revealed that TSSK3 plays an essential role in formation of sperm and male fertility. We additionally identified putative TSSK3 phosphorylation substrates using phosphoproteomics and our null mouse model. In particular, we discovered an alteration in the phosphorylation status of fertility-critical proteins in our studies.

Ethics statement
Mice were maintained in accordance with NIH guidelines, and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine.

Reverse transcription-polymerase chain reaction (RT-PCR)
mRNA was collected from tissues of C57BL6J/129SvEv hybrid mice and reverse transcribed into cDNA. Human multiple tissue cDNA was purchased from BD Biosciences. RT-PCR was performed with mouse or human cDNA as described previously 25

RNA-seq analysis
Quantitative heatmaps depicting the average transcripts per million (TPM) value per tissue per gene in a graphical output resembling conventional semiquantitative polymerase chain reaction (PCR) was performed as previously described. 26 This information includes RNAseq data from six mouse testis datasets, 27 nine mouse epididymis datasets, 26 10 purified mouse germ cell datasets, 28,29 and three purified mouse Sertoli cell datasets. 30 The raw values for these and other mouse and human tissues and cells can be found in Additional Files 3 and 4 (Tables S3 and S4) 31 The chemiluminescent signal was developed using a ChemiDoc Imaging System (BioRad, USA).

Male fertility assessment
Sexually mature male mice were housed with two WT of C57BL6J/129SvEv hybrid females for 4 months. During the mating period, the number of pups born per litter per male was counted.
Average litter sizes are presented as the average number of pups per litter from all of the males.

Sperm count
Sperm was extracted by cutting cauda epididymis 30 times and incubating in Toyoda, Yokoyama, Hoshi (TYH) medium at 37 • C and 5% CO 2 .
After 15 min incubation, sperm samples were applied to a chamber of a 100-µm deep counting slide (CellVision), and the sperm number was measured using the Hamilton Thorne CEROS II system.

Phosphoproteomics
The seminiferous tubules of testes from the adult mice were unrav- Dynamic exclusion was set to 20 s, and the isolation width was set to 0.7 m/z.

Phosphoproteomics analysis and data processing
Raw files were converted to mzML using MSConvert. 34 Precursor ion intensities were derived from the area under each elution curve, and reporter ions intensities were extracted, using MASIC. 35 We used the reference mouse database (2020-03-24) provided by RefSeq. 36 Philosopher 37 was used to add decoy reversed sequences and common contaminants. 37,38 Raw spectra were searched with MSFragger (v3.2) with mass calibration enabled. 39,40 For proteomic profiling, search settings included precursor ions with charge 2-6, precursor mass mode set to CORRECTED, and isotope error set to −1/0/1/2. Remove precursor peak was set to true and remove precursor range was set to +/− 1.5 Da. TopN was set at 150. Fully tryptic in silico digestion settings included amino acids from 7 to 50 amino acids in length with a mass range from 350-10,000 Dalton generated from cleavage after KR (including if followed by P) with a maximum of two miscuts, and with clip N-term methionine enabled. Allow multiple variable modifications were set to false. Precursor true tolerance was set at 10 ppm, and fragment mass tolerance was set at 0.02 Dalton. For phosphoprofiling, the following deviations in the search settings were made: peptide N-term acetylation was removed, and a maximum of three phosphorylations per peptide on STY (79.966331) was added to the dynamic modifications list. Isotope error was set to 0/1/2, and topN peaks was set to 300. Allowed modifications per peptide was set to true. Peptide validation was performed using semisupervised learning procedure in Percolator 41 as implemented in MokaPot. 42 The gene product inference and iBAQ-based quantification were carried out using the gpGrouper algorithm 43

Statistical analysis
Statistical significance was evaluated using the two-tailed unpaired Student t-test assuming unequal variances except as otherwise noted.
Data are represented as means ± SEM.

Generation of Tssk3 KO mice
We identified Tssk3 as a conserved testis-specific gene by a bioinformatic screen as previously described. 26, 45 We performed RT-PCR on reproductive and nonreproductive human and mouse tissues to confirm that both human TSSK3 and mouse Tssk3 are testis-specific ( Figure 1A,B). To determine the temporal expression pattern of mouse Tssk3 expression, all with less than 6 TPM, or ∼8-9-fold less than round spermatids (log2FC) ( Figure 1D).
To generate KO mice using the CRISPR/Cas9 system, we designed two single guide RNAs (sgRNA) for mouse Tssk3 to produce a large deletion of the coding region as shown ( Figure 1E)  Figure 1E). After the deletion was confirmed by Sanger sequencing, specific primers for the WT or KO allele were designed and used for genotyping ( Figure 1F). The KO mice did not show any obvious developmental abnormalities or differences in sexual behavior. The absence of TSSK3 protein was confirmed by Western blot analysis using anti-TSSK3 antibodies. Interestingly, when mature WT sperm was separated into head and tail fractions, Western blot analysis revealed that TSSK3 is localized predominantly, if not exclusively, to the tail of mature sperm ( Figure 1G). Sperm also displays a smaller form of TSSK3 of unknown significance.  Figure 1E amplify specific amplicons for the wild-type (WT) or KO alleles. The sizes of several DNA ladder bands are shown for comparison. (G) Western blot analysis using testis, sperm, and spermatozoa fractionated into sperm heads and sperm tails. TSSK3 protein is detected in the testes from WT and heterozygous (HET), and whole sperm and sperm tails from WT mice.

Tssk3 KO males are sterile
KO sperm demonstrating amorphous head morphology and thin sperm midpieces ( Figure 2D).  (Figure 3). In agreement with the report by Nayyab et al., 24 we found that elongated spermatids did not line the and VIII-since these two stages were nearly impossible to ascertain in Tssk3 KO mice through standard staging criteria-of all circular tubule cross-sections in the KO that contained vacuolization, 92% ± 3% were at stages IV-VI and 8% ± 3% were at stage IX ( Figure S1). We also noted a higher distribution of stages I-VI in the KO than in controls presumably due to the obvious block or inability of elongated spermatids to proceed normally along the spermiation pathway.

Microscopic analysis of control and
Analysis of epididymis histology revealed that only a few sperm were present in the lumen of KO mice, while, as expected, the lumen of WT mice was filled with sperm ( Figure 4A), which correlates with our sperm count results ( Figure 2C). We further investigated the morphology of sperm in the testes by transmission electron microscopy (TEM).
Similar to our light microscopy observations, we found abnormalities present through TEM. Although acrosomal shape and the manchette in KO elongated spermatids at step 9 appeared normal, at steps 14 and 15, they showed unstructured morphology and degradation, while condensation of nuclei was predominantly observed ( Figure 4B). Notably, TEM analysis revealed the detachment of the acrosomes from the nuclei of Tssk3 KO sperm, which has been previously observed in mice containing deletions of genes coding actin-related proteins, such as actin-like 7a (Actl7a) and actin-like 9 (Actl9). 47,48 Taken together, our findings indicate that oligoteratozoospermia in Tssk3 KO males results from defective spermiogenesis and spermiation that leads to degeneration and reabsorption of nearly the entire population of mature spermatozoa.

Phosphoproteomics
To investigate TSSK3 substrates in vivo, we conducted quantitative phosphoproteomic profiling of testis germ cells from Tssk3 HET and KO  49 In our present data, we did not find this motif as highly phosphorylated.

F I G U R E 3 Histological and ultrastructural analysis of testis sections from control and
To further explore the implications of our data, we manually curated proteins within our lists that have been previously reported to be critical for male fertility. In Figure 5D, we present top fertility-related proteins that are either highly-expressed in HET (downregulated in KO), highly-phosphorylated in HET (decreased in phosphorylation in KO, that may or may not be due to a change in total protein level), or highly-phosphorylated in HET with unchanged total protein levels in HET (truly decreased in phosphorylation in KO). In the highlyexpressed group, which could be due to the apparent degradation and reabsorption of sperm in the testes, OAZ3, TPPP2, GAPDHS, PGK2, and TCP11 were found ( Figure 5D). In our highly-phosphorylated list, we discovered ACTL7A, FAM71D, GAPDHS, SMCP, SPATA19, and TPPP2; and, in our highly-phosphorylated list with unchanged protein expression, we found UBE3B, CFAP57, ACTL9, and REEP6.
These data strongly suggest that TSSK3 works as a crucial kinase to directly or indirectly phosphorylate multiple proteins responsible for spermatogenesis and sperm function.

DISCUSSION
Herein, by generating a Tssk3 KO mouse model and demonstrating an altered proteome and phosphoproteome, we have demonstrated that TSSK3 is an essential kinase for male fertility. Tssk3 KO mice are sterile due to abnormal drastic reduction in sperm production and abnormal sperm morphology (oligoteratozoospermia). The abnormalities in spermatogenesis due to the absence of TSSK3 did not show an obvious change until late spermatogenesis in histological and ultrastructural analysis ( Figures 3A,B and 4B). This observation and expression analysis of the mouse Tssk3 gene ( Figure 1A-D) 47,48 Our data that show localization of TSSK3 to the tail suggests that it interacts with and/or results in the phosphorylation of the cytoskeleton, 59 as we observed significant changes in the phosphorylation state of ACTL7A and ACTL9, which are "actin-like" constituents of the cytoskeleton ( Figure 5D). The interaction of TSSK3 and cytoskeletal proteins was also suggested in the previous report. 24  To date, TSSK3 has been relatively unexplored in terms of its substrates. Only a potential phosphosite has been postulated through an in vitro assay. 49 In our global phosphoproteomics study, we found that TSSK3 phosphorylates, or at least participates in the phosphorylation of a large number of proteins. Interestingly, GAPDHS showed upregulation in both quantitative profiling and phosphorylation ( Figure 5D).
KO male mice lacking Gapdhs do not show abnormalities in spermatogenesis but have abnormalities in metabolism related to regulation of sperm motility, which in turn results in male infertility. 57 Since Tssk3 KO mice in this study displayed degeneration and rapid absorption of elongating/elongated spermatids in testes, normal sperm at the cauda epididymis could not be obtained. Thus, although we confirmed the importance of TSSK3 for proper sperm development during spermiogenesis, the function of TSSK3 in mature spermatozoa could not be examined. Our findings show that TSSK3 is predominantly localized to the tail of mature spermatozoa in WT ( Figure 1G).
Whether TSSK3 regulates GAPDHS in mature spermatozoa remains to be determined. Because Tssk3 is expressed predominantly in spermatids ( Figure 1D), we speculate that the bulk of the phenotypic response and proteomic/phosphoproteomic results come from spermatids. The testicular germ cell enrichment strategy that we used to perform our proteomic and phosphoproteomic analyses depleted interstitial cells but did not enrich for spermatids or deplete Sertoli cells; however, because germ cell-specific proteins were mainly altered in phosphoproteomic analysis, our KO phenotype is cell autonomous.
Our data are the first report to approach TSSK3 substrates in vivo using Tssk3 KO mice and a global proteomics analysis approach, which is an unavoidable and necessary step to identifying kinase substrates to understand the function(s) of a protein kinase. We here show that generating KO mice using the CRISPR/Cas9 system is a powerful gateway to exploring the function of causative genes for male infertility in vivo. We may, however, have overlooked other TSSK3 substrates, which were not trapped in our procedure. For example, we did not recognize the phosphosite previously found for TSSK3 through in vitro studies. 49 To conclude identifying kinase substrates, we may need to compare phenotypic or biological test data with proteomics data.
Future studies will investigate the potential relevance of TSSK3 and infertile patients as well. Polymorphisms in human TSSK3 have been reported in the dbSNP database (www.ncbi.nlm.nih.gov/snp), suggesting that this gene could be a cause of idiopathic infertility in men displaying oligoteratozoospermia. Simultaneously, TSSK3 and its substrates could be strong candidates as male contraceptive targets. The findings presented here hold promise for new knowledge on the diagnosis and management of male infertility as well as the development of nonhormonal contraceptives.

CONCLUSION
In conclusion, our studies using Tssk3 KO mice and proteomics have revealed that TSSK3 is essential for male fertility and is a critical kinase involved in the phosphorylation of multiple infertility-related proteins. These findings provide novel insights into the treatment of the infertile male and identify a vulnerable target for non-hormonal male contraception.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available in the supplementary material of this article.