© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1835-1844 doi:10.1242/jcs.202721

RESEARCH ARTICLE TSSK6 is required for γH2AX formation and the -to- transition during spermiogenesis Kula N. Jha*, Swamy K. Tripurani and Gibbes R. Johnson*

ABSTRACT sperm is closely linked to infertility in men (Evenson et al., 1999; Spermiogenesis includes transcriptional silencing, Hammoud et al., 2009). The molecular basis of nuclear condensation and extensive morphological changes as spermatids condensation is poorly understood but involves key events such transform into sperm. Chromatin condensation involves histone as expression of testis-specific histone variants, post-translational hyperacetylation, transitory DNA breaks, histone H2AX (also known modifications of , transient DNA strand breaks and as H2AFX) phosphorylation at Ser139 (γH2AX), and replacement of transcriptional silencing. Several post-translational modifications histones by . Previously, we have reported that the of histones, such as acetylation, phosphorylation, ubiquitination and spermatid kinase TSSK6 is essential for fertility in mice, but methylation, all facilitate the displacement of histones from its specific role in spermiogenesis is unknown. Here, we show that chromatin and regulation in spermatids (Govin et al., 2004; TSSK6 expression is spatiotemporally coincident with γH2AX Li et al., 2014; Rathke et al., 2014; Zhuang et al., 2014). Modified ‘ ’ formation in the nuclei of developing mouse spermatids. RNA- histones are recognized by reader , which are often sequencing analysis demonstrates that genetic ablation of Tssk6 components of chromatin-remodeling complexes that are involved does not impact gene expression or silencing in spermatids. in the histone-to-protamine transition. For example, bromo- However, loss of TSSK6 blocks γH2AX formation, even though the domain-containing proteins, such as BRDT and BRWD1, bind to timing and level of the transient DNA breaks is unaltered. Further, hyperacetylated histones before histone removal from the Tssk6-knockout sperm contained increased levels of histones H3 chromatin, and mice lacking BRWD1 or expressing mutant and H4, and protamine 2 precursor and intermediate(s) indicative of a BRDT are sterile due to impaired spermiogenesis (Pattabiraman defective histone-to-protamine transition. These results demonstrate et al., 2015; Shang et al., 2007). that TSSK6 is required for γH2AX formation during spermiogenesis, In somatic cells, one of the first responses to DNA breaks is and also link γH2AX to the histone-to-protamine transition and male phosphorylation of histone H2AX (also known as H2AFX) at γ fertility. residue Ser139 (hereafter referred to as H2AX) by PI3K-like protein kinases, such as ATM, DNA-PKcs and ATR (Bellani et al., KEY WORDS: Spermiogenesis, Protamine, Chromatin 2005; Burma et al., 2001; Stiff et al., 2004; Ward and Chen, 2001), condensation, Spermatid, TSSK6, γH2AX in order to recruit the repair machinery to these sites (Rogakou et al., 1998; Rossetto et al., 2012). γH2AX has also been implicated in INTRODUCTION chromatin remodeling that takes place during several other During mammalian , spermatogonia undergo biological processes, such as male sex inactivation several rounds of mitotic divisions to yield spermatocytes, which in germ cells, X chromosome inactivation in somatic cells, then complete two meiotic divisions and give rise to haploid round asymmetric sister chromosome segregation in stem cells and spermatids (Hess and Renato de Franca, 2008). The round cellular senescence maintenance in fibroblasts (Turinetto and spermatids develop into mature sperm by undergoing a lengthy Giachino, 2015). Targeted deletion of histone H2AX in mice and complex process of cell differentiation called spermiogenesis, results in male, but not female, infertility due to failure of sex-body which includes elongation and condensation of nuclei, formation of formation and meiotic sex chromosome inactivation (MSCI) in the acrosome and flagellum, and removal of excess cytoplasm spermatocytes (Celeste et al., 2002; Fernandez-Capetillo et al., (Cheng and Mruk, 2002; Griswold, 1995). This process includes an 2003). In leptotene spermatocytes, γH2AX is formed in response to extensive chromatin reorganization, often referred to as the histone- recombination-associated DNA double-strand breaks (Blanco- to-protamine transition, wherein histones are replaced first by Rodríguez, 2009; Carofiglio et al., 2013; Celeste et al., 2002). In transition proteins (TP) and subsequently by protamines (Prms), pachytene spermatocytes and in the absence of DNA breaks, leading to a more compact packaging of DNA in sperm (Braun, γH2AX is detected in the sex body, and thought to be produced by 2001; Govin et al., 2004; Rathke et al., 2014; Ward and Coffey, ATR (Carofiglio et al., 2013; Fernandez-Capetillo et al., 2003; 1991). Genetic ablation of Prm1 or Prm2 leads to sterility in male Mahadevaiah et al., 2001; Turner et al., 2004). In elongating mice (Cho et al., 2001), and abnormal chromatin condensation in spermatids, γH2AX foci and transitory DNA breaks occur simultaneously (Agnieszka, 2014; Govin et al., 2004; Leduc et al., 2008b; Marcon and Boissonneault, 2004; Rathke et al., Division of Biotechnology Review and Research IV, Office of Biotechnology 2014; Wojtczak et al., 2008), but the kinase responsible for the Products, Center for Drug Evaluation and Research, U.S. Food and Drug γ Administration, Silver Spring, MD 20993, USA. H2AX phosphorylation and the role of H2AX in spermiogenesis is not clearly understood. *Author for correspondence ([email protected]; [email protected]) The family of testis-specific serine/threonine kinases (TSSKs) K.N.J., 0000-0002-2218-1864 comprises six members that are present only in spermatids and sperm (Li et al., 2011). Little is known about the factors that drive

Received 14 February 2017; Accepted 4 April 2017 expression of the TSSK during spermatogenesis, with the Journal of Cell Science

1835 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1835-1844 doi:10.1242/jcs.202721 exception of TSSK6 (also known as small serine/threonine kinase, SSTK), which is highly dependent on BRWD1 (Pattabiraman et al., 2015). Heat shock protein 90 (HSP90) is essential for the stability of all TSSKs and for catalytic activation of TSSK2 and TSSK4 (Jha et al., 2013, 2010). Tssk6-knockout (KO) and Tssk1/Tssk2 double KO mice exhibit male infertility (Shang et al., 2010; Spiridonov et al., 2005; Xu et al., 2008), whereas the targeted disruption of Tssk4 causes subfertility in male mice (Wang et al., 2015). Electron microscopy analysis of testis sections from Tssk6-KO mice indicates DNA condensation defects, and KO sperm have abnormal morphology, highly reduced motility and are incapable of fusing with zona pellucida-free eggs (Sosnik et al., 2009). Furthermore, variations in the TSSK6 gene are associated with impaired spermatogenesis and infertility in men (Su et al., 2010). While numerous hallmarks of spermiogenesis have been well documented, there remain unanswered questions and an incomplete understanding of the elaborate mechanisms required to transform round spermatids into sperm. The close connection between Tssk6 expression and nuclear condensation during spermatid development, genetic ablation of Tssk6 and male infertility, and our previous observation that TSSK6 can phosphorylate histones such as H2AX in vitro (Spiridonov et al., 2005) led us to perform a systematic investigation of cellular and biochemical events during spermiogenesis in wild-type (WT) and Tssk6-KO mice. We have found that TSSK6 mediates γH2AX production and the histone-to- protamine transition required for proper nuclear condensation, and for the first time, we directly link γH2AX formation in maturing spermatids to male fertility. Based on our findings, we are now able to position TSSK6 within our current understanding of spermiogenesis and provide a model for the role of TSSK6 in male fertility.

RESULTS TSSK6 is localized in nuclei of elongating spermatids, and Fig. 1. Characterization of the TSSK6 protein during spermiogenesis. WT Tssk6 genetic deletion of does not impact the transcriptome and Tssk6-knockout (KO) testis sections were stained with periodic acid Schiff As spermatogenesis progresses, germ cells move from the basal (PAS) and hematoxylin in panels A and B, or with TSSK6 monoclonal antibody membrane towards the lumen of the seminiferous tubule, and the followed by immunofluorescence (IF) detection in C-F. Representative light cycle of spermatogenesis in mice is divided into 12 stages microscopy images from PAS-hematoxylin staining are shown. Confocal representing distinct cellular associations in serial cross-sections images of IF staining at 40× (C and D) and 63× magnification (E and F) are of seminiferous tubules (Ahmed and de Rooij, 2009; Oakberg, presented to show TSSK6 (red) in elongating spermatids at step 11-12 in stage XI-XII seminiferous tubules. Overlay of TSSK6 staining and the nuclear 1956). Spermiogenesis in the mouse is further subdivided into 16 stain DAPI are shown in the panels C,D,F. Roman numerals denote the steps based on the morphology of the nucleus and acrosome of seminiferous epithelial stage of the tubule, and elongating spermatids (es) maturing spermatids. To thoroughly characterize the expression of and spermatocytes (spc) are indicated by arrows. Scale bars: 50 µm (PAS- Tssk6 mRNA and protein during spermatogenesis, we performed hematoxylin images) and 40 µm (IF images). All analyses were performed in situ hybridization (ISH) and immunofluorescence (IF) staining on at least three WT and three KO mice, and 50-100 tubules were evaluated on testis sections. Using periodic acid Schiff (PAS) and in sections from each mouse. hematoxylin staining, we evaluated the morphology of developing germ cells in WT and Tssk6-KO seminiferous tubules but observed To determine whether TSSK6 influenced spermatid gene no obvious differences when various stages and steps of expression, we performed RNA sequencing (RNA-Seq) and spermatogenesis were compared (Fig. 1A,B and data not shown). compared the mRNA profiles of purified spermatids from WT Chromogenic ISH detected Tssk6 transcripts in both round and and Tssk6-KO mice. RNA-Seq analyses were performed on three elongating spermatids, but not in spermatocytes (Fig. S1) or in sets of purified spermatids from age-matched adult WT and KO condensed spermatids (data not shown). IF analysis demonstrated mice, wherein each set represented a pool of spermatids from 4-6 intense TSSK6 protein staining in step 11-12 spermatids in stage mice. Enriched spermatid populations with >90% purity were XI-XII tubules, as characterized by thin and compact spermatid obtained as determined by microscopic evaluation of size and nuclei (Fig. 1C,E,F). Notably, TSSK6 protein was confined to the morphological criteria described previously (Bellvé, 1993). Out of nuclei of the elongating spermatids as the TSSK6 staining 23,235 genes present in the reference genome (Mus overlapped with that of DAPI, and no IF positivity was observed musculus_UCSC_mm10), transcripts from a total of 19,885 genes in sections from Tssk6-KO mice (Fig. 1D). Post-meiotic expression were detected in WT or Tssk6-KO spermatids, and the top 500 most and nuclear localization of TSSK6 suggested that TSSK6 may have abundant transcripts were ranked by fragments per kilobases per an important role in the nuclear condensation and/or regulation of million mapped reads (FPKM) values (Table S1). Transcripts for gene expression in developing spermatids. spermatid-specific genes such as Prm1 and Prm2, Tnp1 and Tnp2 Journal of Cell Science

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(TPs), Smcp, Akap4, Ldhc, Odf and those encoding TSSKs etc. were and γH2AX generation during spermiogenesis. WT testis sections included in this list and were not significantly affected by the were co-labeled with antibodies against γH2AX and TSSK6, and targeted deletion of Tssk6 (P>0.05). Interestingly, transcripts for immunoreactivity was detected using fluorescence (Fig. 2). Strong Tssk6 and its activators Tsacc and Hsp90aa1 (Jha et al., 2013, 2010) γH2AX staining was detected in the nuclei of spermatids at step were the 242nd, 180th and 106th most abundant transcripts, 10-12 that then drastically reduced in the later steps (Fig. 2). As respectively, underscoring the importance of TSSK6 in spermatid expected, γH2AX was also detected in leptotene and pachytene development. A very small number of low-level transcripts (mean spermatocytes (Blanco-Rodríguez, 2009; Hamer et al., 2003). IF FPKM of 0.5–15 in either WT or KO) were either increased (34) or staining for TSSK6 was detected in step 10-12 elongating spermatids decreased (5) by >50% in KO versus WT cells, and exhibited a and was retained in step 13-16 condensing/condensed spermatids in differential expression at P≤0.05 (Table S2). However, none of the the lumen of seminiferous tubules at stage I-VI (Fig. 2B,E,H). proteins expressed by these transcripts are known to be important Importantly, γH2AX and TSSK6 colocalized in the nuclei of for spermiogenesis. Table S3 lists genes with less-abundant developing spermatids as demonstrated by the merged images of transcripts than those listed in Table S1 but that are relevant to γH2AX, TSSK6 and DAPI (Fig. 2C,F,I). These results demonstrated spermiogenesis, such as chromatin remodeling mediators (Chd5, that TSSK6 expression is coincident with γH2AX formation in nuclei Ctcf), bromo-domain containing proteins (Brd genes), histones of elongating spermatids, suggesting a relationship between TSSK6 (Hist genes), DNA damage response proteins (Brca genes, H2afx), and γH2AX formation during spermiogenesis. PI3K-like protein kinases (Atm, Atr, Prkdc) or other TSSK genes. The levels of these transcripts were also found to be comparable in Genetic ablation of TSSK6 blocks γH2AX formation in WT and KO spermatids (P>0.05). Thus, genetic ablation of Tssk6 spermatids did not cause significant alteration in gene expression in spermatids To assess whether TSSK6 is involved in γH2AX formation, we as the amount of transcripts for the vast majority of genes were performed IF studies on testis sections from WT and KO mice. similar in WT and KO spermatids. Remarkably, no staining was observed in spermatids from KO mice (Fig. 3Ag-Al), but in parallel experiments γH2AX was TSSK6 and γH2AX are colocalized in elongating spermatids readily detected in the nuclei of spermatids in WT sections as they undergo nuclear condensation (Fig. 3Aa-Af). These results demonstrated that TSSK6 is essential Nuclear abnormalities observed previously in electron micrographs for γH2AX formation in spermatids. Conversely and as expected, of Tssk6-KO spermatids indicate defects in chromatin condensation γH2AX staining was indistinguishable in spermatocytes from WT (Spiridonov et al., 2005). Based on that observation and nuclear and KO testis sections, demonstrating that genetic ablation of localization of TSSK6 in elongating spermatids in the present study, Tssk6 does not affect γH2AX formation that is associated with we investigated the relationship between TSSK6 protein expression either homologous recombination or sex-body formation during

Fig. 2. TSSK6 and γH2AX generation are temporally and spatially coincident. Double IF staining was performed on WT testis sections with antibodies against γH2AX (green) and TSSK6 (red). Representative confocal microscopy images of γH2AX (A,D,G), TSSK6 (B,E,H) and overlay images with DAPI (C,F,I) are presented. Tubule stages are marked with roman numerals, and elongating spermatids (es), condensed spermatids (cs) and spermatocytes (spc) are indicated by arrows. Experiments were repeated with at least three WT and three KO mice, and 50-100 tubules were evaluated from each mouse. Journal of Cell Science

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Fig. 3. γH2AX formation is not detected in Tssk6-KO spermatids. (A) WT (Aa-Af) and Tssk6-KO (Ag-Al) testis sections were probed with an antibody against γH2AX (green) and stained with DAPI (blue), and representative IF staining at different stages of spermatogenesis is presented. Stage IX-XII tubules are present in Ag-Al but labels have been omitted for clarity. (B) Cell lysates of WT and KO purified spermatids (spd) were probed by western blotting (WB) with antibodies against γH2AX, H2AX, ATR, TSSK6 or β-tubulin, and results from a representative experiment are shown. Western blotting was performed on three sets of purified spermatids from WT and KO mice, wherein each set represented a pool of spermatids from four to six mice. The histone γH2AX protein band was quantified by densitometry, and values were normalized to those for β-tubulin, and the amounts in the KO relative to in WT are presented in the graph as mean±s.e.m. of three independent experiments. *P<0.05. (C) WT and Tssk6-KO testis sections were probed with an antibody against ATR (red), and representative overlay images with DAPI are presented. IF for γH2AX and ATR was performed on at least three WT and three KO mice, and 50-100 tubules were evaluated from each mouse. spc, spermatocytes. meiosis (Fig. 3A). A drastically reduced amount of γH2AX in the versus KO testis sections (Fig. S2) consistent with the RNA-Seq lysate from purified KO spermatids was seen by western blotting, analysis (H2afx in Table S3). Finally, western blotting when compared to WT lysate (Fig. 3B). In the western blotting demonstrated that similar amounts of H2AX protein were present analyses, some γH2AX was detected due to unavoidable in the lysates of purified spermatids from WT and KO mice spermatocyte contamination of the spermatid preparation (Bellvé, (Fig. 3B). Histone H4 hyperacetylation is another early molecular 1993). To confirm that the absence of γH2AX in KO spermatids event that is believed to be essential for chromatin condensation was due to a lack of Ser139 phosphorylation and not to reduced (Bao and Bedford, 2016; Rathke et al., 2014). Histone H4 H2AX protein, we performed IF and western blotting experiments hyperacetylation was detected in step 8-12 spermatids in stage on WT and KO samples. IF staining of testis sections showed the VIII-XII tubules, but unlike γH2AX, no differences in the staining presence of H2AX protein in the nuclei of spermatocytes and pattern were observed in WT and KO testis sections (Fig. S3 and elongating spermatids, and no differences were observed in WT data not shown). Journal of Cell Science

1838 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1835-1844 doi:10.1242/jcs.202721

In our previous work, we have observed that both histone H2A protein expression and/or localization of ATR in spermatids, we and H2AX are phosphorylated to similar extents by TSSK6 in performed western blotting and IF experiments to measure ATR on vitro (Spiridonov et al., 2005). Because H2A and H2AX have WT and Tssk6-KO testis samples. Western blotting demonstrated almost identical amino acid sequences, with the exception of the that similar amounts of ATR were present in the lysates of purified extended C-terminus in H2AX that contains Ser139, it is unlikely spermatids from WT and KO mice (Fig. 3B). In IF analyses, ATR that H2AX Ser139 is a good phosphorylation site for TSSK6. was detected in the nuclei of spermatids at steps 9-12 and, Nevertheless, a temporal and spatial colocalization of TSSK6 and importantly, the staining pattern was very similar in WT and KO γH2AX in elongating spermatids led us to directly test whether spermatids (Fig. 3C). Taken together, these results demonstrate that Ser139 could be phosphorylated by TSSK6. We expressed TSSK6 is essential for γH2AX generation during spermiogenesis, Myc-tagged TSSK6 in Cos-7 cells and performed in vitro but is most likely not the kinase that phosphorylates H2AX at immunokinase reactions using peptides corresponding to the Ser139 in spermatids. H2AX C-terminal region containing the wild-type sequence or Ser to Ala changes (Table S4). Histone H2AX peptides were Transitory DNA strand breaks are indistinguishable in WT synthesized and characterized as described previously (Fan et al., and Tssk6-null spermatids 2004). No kinase activity was detected for TSSK6 with any of Due to the lack of γH2AX formation in the KO spermatids, we these H2AX peptides, whereas kinase activity was easily performed a sensitive fluorescent terminal deoxynucleotidyl detected with histone H2A protein as substrate. As a positive transferase dUTP nick-end labeling (TUNEL) assay to evaluate control, we confirmed that commercially available purified ATM DNA breaks on testis sections. As shown in Fig. 4, TUNEL kinase phosphorylated the peptides containing Ser139 (H2AX- labeling in both WT and Tssk6-KO testis was very similar, and P1 and H2AX-P2), but phosphorylation was dramatically detected as a homogeneous nuclear stain in spermatocytes and reduced when Ser139 was changed to Ala (H2AX-P3) step 9-12 elongating spermatids (stages IX-XII). Reduced (Table S4). staining in both WT and KO spermatids was observed at step Among the kinases that are known to phosphorylate Ser139 in 13 (stage I), with no detection in condensed spermatids (stages histone H2AX, ATR transcripts were most abundant in our RNA- VII-VIII). These results demonstrate that the spermiogenesis- Seq analysis of spermatids (Table S3), and ATR is also reported to associated transient DNA strand breaks occur in the absence of be responsible for H2AX Ser139 phosphorylation in spermatocytes γH2AX formation, and the extent and timing are the same in WT (Turner et al., 2004). To test whether TSSK6 was involved in the and Tssk6-KO mice.

Fig. 4. Genetic ablation of Tssk6 does not impact the timing and level of transient DNA breaks in spermatids. A sensitive fluorescent TUNEL assay was performed on mouse testis sections to detect DNA strand breaks during spermiogenesis, and representative images are presented of TUNEL-positive cells (green) with or without DAPI merge images at various stages of spermatogenesis in WT (A-H) and KO (I-P) mice. TUNEL staining was performed on at least three WT and three KO mice, and 50-100 tubules were evaluated from each mouse. spc, spermatocytes; spd, spermatids. Journal of Cell Science

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Sperm from Tssk6-KO mice contain elevated amounts of histones H3 and H4, and protamine 2 precursors and intermediates The final packaged DNA in sperm nuclei contains bound residual histones and protamines. To characterize the histone profile of KO sperm, we performed western blotting of sperm lysates with antibodies against histones H1, H2A, H2B, H3 and H4. As shown in Fig. 5A,B, loss of Tssk6 resulted in significant increases in histone H3 and H4 in sperm. Densitometry analysis revealed that histone H3 and H4 proteins were ∼3.7- and ∼2.9-fold higher in KO compared to WT sperm, respectively (Fig. 5B). Histones H1, H2A and H2B were undetectable in western blots of KO and WT sperm lysates (data not shown). Western blotting of purified spermatid lysates did not reveal any difference in the amounts of histones H1, H2A, H2B, H3 or H4, or of TP1 and TP2 proteins in WT and KO spermatids (Fig. S4A), indicating that the basic nuclear proteins profile is largely unaltered during early spermiogenesis in Tssk6-KO mice. Unlike histones, protamines are not readily extracted from sperm, and the analysis of protamine levels requires a protocol that results in quantitative extraction of proteins from the compacted chromatin followed by protein fractionation in acid-urea-polyacrylamide gels (Balhorn et al., 1977; Yu et al., 2000; Zhao et al., 2001). Proteins extracted from the chromatin of WT and KO sperm were separated in these gels and either stained with Coomassie Brilliant Blue (Fig. 5C, left) or analyzed by western blotting for Prm1 (middle) and Prm2 (right). No differences were noted in KO and WT samples for Prm1 and mature Prm2, whereas strikingly increased amounts of Fig. 5. Tssk6-null sperm possess aberrant levels of histones and Prm2 both Prm2 precursor and intermediate(s) were observed in KO precursor and intermediate(s). (A) Lysates from WT and KO sperm were sperm compared to in WT. separated in SDS-PAGE gels, transferred onto PVDF membrane and probed by Consistent with the observation in sperm, the acid-urea- western blotting for histones H3 and H4, or for β-tubulin, and results from a polyacrylamide gel analysis also revealed an enhanced presence representative experiment are shown. (B) Histones H3 and H4 protein bands of the Prm2 precursor and intermediate(s) in spermatids from the were quantified by densitometry and values were normalized to those of β KO mice (Fig. S4B). No TSSK6 protein was detected by western -tubulin, and amounts in the KO relative to in WT are presented as mean±s.e.m. blotting in these spermatid extracts from WT or KO mice (data not of three independent experiments. *P<0.05. (C) Equal amounts of chromatin- bound proteinsthat had been extracted from WT and KO sperm were analyzed in shown), indicating that TSSK6, unlike protamines, is not tightly acid-urea-polyacrylamide gel, as described in Materials and Methods. Gels were bound to chromatin. Prm1 and Prm2 were detected in condensing/ either stained with Coomassie Blue (left) or transferred and probed by western condensed spermatid nuclei by IF, however, no difference in blotting for Prm1 (middle) or Prm2 (right), and results from a representative staining was observed in WT and KO sections (data not shown). experiment are presented (n=3 experiments). The positions of the Prm2 These results demonstrated that TSSK6 is required for murine sperm precursor (Pre-Prm2), intermediate (Int-Prm2) and mature Prm2 are denoted. to possess normal quantities of histone H3, H4 and Prm2 precursor and intermediate(s). production of γH2AX, removal of histones (H3 and H4) and effective processing of the Prm2 precursor and intermediate(s). DISCUSSION Most of the core nucleosomal histones are displaced from the TSSK6 is a testis-specific protein kinase that is highly conserved in chromatin during steps 9-12 of spermiogenesis in the mouse, and mammals and essential for spermatogenesis. To determine the condensed spermatids or sperm retain only small residual amounts biological role of TSSK6, here we have performed a comparative of histones H3 and H4 (Li et al., 2014; Meistrich et al., 2003). analysis of molecular events during spermiogenesis in WT and Increased retention of histone H3 and H4 in spermatids/sperm has Tssk6-KO mice. Based on our study, the relationship of TSSK6 to been closely linked to male infertility in many gene-KO mouse key events that occur during chromatin condensation can be better models (Bell et al., 2014; Li et al., 2014; Lu et al., 2010; Zhuang understood. Fig. 6 highlights TSSK6-dependent and -independent et al., 2014). Prm2 is synthesized as a precursor protein that binds to events as round spermatids mature into condensed spermatids. the chromatin and then undergoes proteolytic processing giving rise Tssk6 mRNA is expressed in round spermatids, and the transcripts to several intermediates and finally mature Prm2 (Balhorn, 2007; are still detected in elongating spermatids, whereas TSSK6 protein Wu et al., 2000). Thus, incomplete Prm2 processing of the first appears in elongating spermatids and is retained in mature chromatin-associated precursor and intermediate(s) occurs in the sperm. Genetic ablation of Tssk6 did not have a significant impact absence of TSSK6. Numerous studies have implicated altered Prm1 on the transcriptome, indicating that TSSK6 is not involved in gene or Prm2 amounts, and aberrant processing of Prm2 precursor and expression. Histone H4 hyperacetylation, transient DNA breaks, intermediate(s) in sterility in mice (Bao and Bedford, 2016; chromatin displacement of histones H1, H2A and H2B, and the TP Hernández-Hernández et al., 2016; Li et al., 2014; Lu et al., exchange all appeared to be normal in Tssk6-KO spermatids. 2010; Rathke et al., 2014; Wu et al., 2000; Yuen et al., 2014) and Further, Prm1 and mature Prm2 levels were found to be comparable humans (Carrell et al., 2007; Oliva, 2006; Torregrosa et al., 2006). in WT and KO chromatin-bound protein extracts from spermatids We interpret the defects in the histone-to-protamine transition, as and sperm. Conversely, TSSK6 was found to be essential for the evidenced by altered histone and protamine homeostasis in the Journal of Cell Science

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Fig. 6. Role of TSSK6 in the histone-to-protamine transition. At the bottom of the diagram, developmental expression of Tssk6 mRNA and protein is shown and aligned to steps (1-16) of mouse spermiogenesis as round spermatids develop into condensed spermatids. Molecular changes involved in the histone-to-protamine transition are categorized into TSSK6-dependent (red) and -independent (yellow) events, and their occurrence has been aligned with steps of spermatid development. Alignment of histone removal, TP exchange, Prm1 and Prm2 integration, and Prm2 precursor and intermediate (P/I) processing is based on the literature (Bao and Bedford, 2016; Govin et al., 2004; Meistrich et al., 2003).

chromatin of Tssk6-KO spermatids and sperm, as the cause for H2AX at Ser139 in vitro, it is more likely that ATR, and not TSSK6, infertility in the null mice. is the kinase that phosphorylates Ser139 of H2AX during γH2AX marks sites of DNA breakage, and γH2AX foci are spermiogenesis. However, it is important to note that genetic detected in spermatocytes during meiotic recombination (Blanco- ablation of Tssk6 had no impact on ATR expression and nuclear Rodríguez, 2009; Carofiglio et al., 2013; Celeste et al., 2002). localization in spermatids and thus, there is no direct evidence that However, γH2AX can also be formed in a DNA break-independent ATR phosphorylates H2AX in this instance. Nevertheless, the manner, as in the case of sex-body-associated γH2AX in pachytene requirement of TSSK6 for γH2AX generation demonstrates that spermatocytes (Carofiglio et al., 2013; Fernandez-Capetillo et al., TSSK6 is an upstream kinase responsible for the proper localization 2003; Mahadevaiah et al., 2001). As expected, and because and/or activation of the kinase that directly produces γH2AX during expression of TSSK6 is limited to spermatids and sperm, genetic spermatid nuclear condensation. ablation of TSSK6 did not cause any defect in γH2AX formation IF studies demonstrated that TSSK6 is confined to the nuclei, but that is associated with homologous recombination or sex-body it was not detected in the chromatin-bound protein extracts derived formation in spermatocytes. Detection of γH2AX foci in step 9-12 from spermatids. Accordingly, these findings indicate that while elongating spermatids is coincident with DNA strand breaks (Govin TSSK6 is in the nucleus as chromatin is condensing, it is not tightly et al., 2004; Leduc et al., 2008a; Rathke et al., 2014), and here we bound to DNA. Based on the insights gained from the present study, have demonstrated that TSSK6 is essential for γH2AX production we postulate that TSSK6 mediates γH2AX generation to govern a during spermiogenesis. The timing and level of the DNA breaks chromatin-associated remodeling complex that is essential in the were very similar in WT and Tssk6-KO spermatids, and can be histone-to-protamine transition. Further studies are warranted to interpreted two ways. Either DNA breaks are not the driver of the identify the TSSK6 protein substrates that are required for γH2AX H2AX phosphorylation in spermiogenesis or TSSK6 is required for formation, the histone-to-protamine transition and the production of γH2AX to be generated in response to the breaks. Previous studies normal sperm in mammals. have suggested that precise control of γH2AX may be important during spermiogenesis since prolonged or increased amounts have MATERIALS AND METHODS been observed in KO mouse models with a sterile phenotype (Li Reagents Antibodies against histones H3 (96C10), H4 (L64C1), H2A (catalogue et al., 2014; Wang et al., 2016). To the best of our knowledge, our γ findings regarding TSSK6 are the first that link γH2AX formation number 2578), H2AX (catalogue number 2595) and H2AX (20E3) were purchased from Cell Signaling Technology Inc; against histones H1 (AE-4), during spermiogenesis to the production of functional sperm and Atr H2B (FL-126) and TP2 (K-18) were from Santa Cruz Biotechnology Inc; fertility. Based on the high abundance of transcripts in against TNP1 (catalogue number 17178-1-AP) were from ProteinTech spermatids, nuclear spermatid localization of ATR, a recognized Group Inc (Chicago, IL); and against Prm1 (Hup1N) and Prm2 (Hup2B) role for ATR in γH2AX generation in pachytene spermatocytes were from Briar Patch Biosciences (Livermore, CA). Recombinant human

(Turner et al., 2004) and the inability of TSSK6 to phosphorylate ATM kinase and antibody against hyperacetylated histone H4 (Penta) were Journal of Cell Science

1841 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1835-1844 doi:10.1242/jcs.202721 from EMD Millipore (Billerica, MA), and terminal deoxynucleotidyl scanning microscope (Zeiss) equipped with EC Plan-Neofluar 20×/0.8 NA, transferase (TdT), TdT buffer and biotin-16-dUTP were from Sigma 40×/1.3 NA oil or Plan-Apochromat 63×/1.4 NA oil objective lens. Images (St Louis, MO). Monoclonal antibody against mouse TSSK6 was generated were captured with AxioCam camera (Zeiss) using Zen imaging software in our lab and has been described previously (Jha et al., 2010). All reagents (Zeiss) and processed using Photoshop CS4 (Adobe). For ISH imaging, used in this study were of analytical grade. slides were scanned using Panoramic MIDI slide scanner (3DHISTECH) equipped with a Plan-Apochromat 20×/0.8 NA objective lens (Zeiss) and a Animals VCC-FC60FR19CL/4MP camera (CIS). Image analysis was performed The Tssk6-KO mouse model was generated in our lab and has been described using Panoramic Viewer software (3DHISTECH), and images were previously (Spiridonov et al., 2005). Mice were handled and killed according processed using Photoshop CS4. No gamma correction was made in IF, to the guidelines of the Animal Care and Use Committee (Center for TUNEL or ISH images, and scale bars were added. Biologics and Evaluation Research, U.S. Food and Drug Administration). Adult male Tssk6-KO mice and WT littermates were used in this study. Purification of mouse spermatids An enriched population of mouse spermatids was purified following the ISH analysis STA-PUT method as described previously (Jha et al., 2013). Briefly, testes RNA ISH was performed using the RNAScope ISH technology (Advanced from adult mice were enzymatically dissociated, and the cells were separated Cell Diagnostics, Hayward, CA) (Wang et al., 2012). Briefly, testes were by sedimentation at unit gravity in 2-4% BSA gradient in a STA-PUT harvested and fixed in 10% neutral buffered formalin for 24 h at room apparatus. Fractions were examined under a microscope, and germ cell types temperature, dehydrated and embedded in paraffin. Tissue sections were cut at were determined based on the size and morphology (Bellvé, 1993). 5 μm thickness, air-dried at room temperature and processed for RNA in situ Fractions enriched with spermatids were pooled to obtain a population of detection by using the RNAscope 2.5 HD Reagent Kit-RED (catalogue spermatids with >90% purity. number 322360). To ensure RNA integrity and assay procedure, pre-treatment conditions were optimized to 15 min heat-pre-treatment, followed by 30 min RNA-Seq analysis protease digestion. Custom RNA ISH probes were prepared by Advanced Spermatids were purified from three sets of age-matched adult WT and Cell Diagnostics to detect mouse Tssk6, and a probe to bacterial mRNA DapB Tssk6-KO mice, and total RNA was isolated using RNeasy kit (Qiagen) with served as a negative control. Slides were scanned using Panoramic MIDI slide DNase I treatment. RNA-Seq libraries were prepared using Illumina TruSeq scanner (3DHISTECH) to acquire images of ISH. Stranded mRNA sample preparation kit and sequenced on Illumina HiSeq 2500. The quality of raw data was assessed and passed by FastQC, and reads IF staining of proteins in testis sections were mapped to the reference genome (Mus musculus_UCSC_mm10). Testes were fixed either in 4% paraformaldehyde or Carnoy solution (ethanol, Cufflinks software (v2.2.1) was used to estimate transcript levels chloroform, acetic acid in the ratio 6:3:1, respectively) and embedded in represented by the FPKM, and differential expression between samples paraffin, and 5-μm thick cross-sections were mounted on glass slides. was determined using Cuffdiff software (v.2.2.1). Sections were deparaffinized with xylene, rehydrated through a graded ethanol series and washed briefly in water. Slides were then processed for Western blotting and immunoprecipitation antigen retrieval in 10 mM citrate buffer (pH 6.0) for 20 min at 100°C, Proteins were fractionated in 4-20% SDS-PAGE gels. Western blotting and washed with PBS and blocked with 1% bovine serum albumin (BSA) in PBS immunoprecipitation experiments were performed as described previously containing 0.1% Tween-20 (PBS-T). Sections were incubated with primary (Jha et al., 2013, 2010), and the primary antibody dilution used in western antibody or isotype control IgG overnight, and the antibody dilution used in blotting was 1:1000. IF studies was 1:100 with the exception of γH2AX (20E3) antibody, which was used at 1:400 dilution. Following primary antibody incubation, the slides Protein kinase assay were washed with PBS-T and incubated with Alexa-Fluor-conjugated Myc-tagged Tssk6 was expressed in Cos-7 cells (American Type Culture secondary antibody (1:200 dilution) (Molecular Probes, Eugene, OR) for 1 h. Collection, Manassas, Virginia) and immunoprecipitated with an antibody Slides were washed with PBS-T, counterstained with DAPI and mounted against Myc, and in vitro kinase reactions were performed (Jha et al., 2013). with Slow-Fade light reagent (Molecular Probes). Sections were examined Briefly, the reaction was performed at room temperature for 30 min in under a confocal microscope (LSM 710, Zeiss), and images were captured. a reaction buffer containing 25 mM HEPES (pH 7.4), 10 mM MgCl , Established morphological criteria were used to determine specific stages of 2 10 mM MnCl , 2 mM EGTA, 30 µM ATP, 10 µCi of [γ-32P]ATP, and the mouse seminiferous epithelium cycle (Ahmed and de Rooij, 2009; 2 synthetic histone H2AX peptides or histone H2A protein. The reaction was Meistrich and Hess, 2013). Experiments were repeated with testes sections terminated by acidification, and phosphorylation of the H2AX peptides or from at least three different sets of WT and Tssk6-KO mice. H2A protein was quantified by measuring incorporated radioactive 32P (Fan et al., 2004). Values are represented as mean kinase activity TUNEL assay (fmol/min)±s.e.m. (n=3). A modified TUNEL assay was performed to detect DNA breaks in testis sections as previously reported (Marcon and Boissonneault, 2004). Briefly, Isolation of mouse sperm Carnoy-fixed testis sections were deparaffinized with xylene and rehydrated Sperm from mouse cauda epididymis were collected in modified Krebs– through a graded ethanol series. Sections were washed with PBS and Ringer medium (Whitten’s-HEPES buffered medium) containing 100 mM equilibrated with TdT buffer for 30 min at 37°C. TdT buffer was removed NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, and a freshly prepared terminal transferase reaction mix (50 µl TdT buffer 1 mM pyruvic acid, 4.8 mM L(+) lactic acid hemicalcium salt in 20 mM containing 25 U terminal transferase enzyme and 0.5 nmol Biotin-16- HEPES (pH 7.3), as described previously (Jha et al., 2006, 2008). dUTP) was added. End-labeling was performed for 1 h at 37°C, followed by – washing with PBS and incubation with fluorescein avidin (1:100 dilution in Extraction and analysis of chromatin-bound proteins from mouse PBS) for 1 h at room temperature. Sections were washed with PBS, spermatids and sperm counterstained with DAPI, mounted with Slow-Fade light reagent, and Chromatin-bound proteins from spermatids and sperm were extracted and images were captured using a confocal microscope. A negative control with analyzed following the methods described previously (Balhorn et al., no TdT enzyme was included in all experiments. 1977; Yu et al., 2000; Zhao et al., 2001). Testicular germ cells were isolated by the enzymatic dissociation of WT and Tssk6-KO testes, and Image acquisition subjected to sonication followed by centrifugation through a layer of 5% Image acquisition from IF and TUNEL staining was performed at room sucrose in 0.2× concentration of MP buffer (5 mM MgCl2 and 5 mM temperature. Data analysis was performed using a LSM 710 confocal laser sodium phosphate, pH 6.5) containing 0.25% Triton X-100 and protease Journal of Cell Science

1842 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1835-1844 doi:10.1242/jcs.202721 inhibitors. The pellet fraction, which comprises sonication-resistant nuclei Bell, E. L., Nagamori, I., Williams, E. O., Del Rosario, A. M., Bryson, B. D., of condensing/condensed spermatids, was washed in MP buffer containing Watson, N., White, F. M., Sassone-Corsi, P. and Guarente, L. (2014). SirT1 0.22 M NaCl and incubated at 37°C for 10 min in 10 mM DTT and is required in the male germ cell for differentiation and fecundity in mice. Development 141, 3495-3504. protease inhibitors. DNA was precipitated with 0.5 M HCl on ice for 1 h, Bellani, M. A., Romanienko, P. J., Cairatti, D. A. and Camerini-Otero, R. D. and the supernatant was subjected to protein precipitation with 25% (2005). SPO11 is required for sex-body formation, and Spo11 heterozygosity trichloroacetic acid (TCA), followed by washing with acidified acetone rescues the prophase arrest of Atm-/- spermatocytes. J. Cell Sci. 118, 3233-3245. and dissolving the protein precipitate in acid-urea gel sample buffer (5 M Bellvé,A.R.(1993). Purification, culture, and fractionation of spermatogenic cells. urea, 5% acetic acid, 1% β-mercaptoethanol, and Methyl Green dye). Methods Enzymol. 225, 84-113. ı́ Extraction from sperm was performed as above with the exception that the Blanco-Rodr guez, J. (2009). gammaH2AX marks the main events of the spermatogenic process. Microsc. Res. Tech. 72, 823-832. nuclei pellet isolated after sonication was incubated in 5 M guanidium Braun, R. E. (2001). Packaging paternal with protamine. Nat. Genet. chloride solution (pH 8.0) for 30 min on ice, and then dissolved in a 28, 10-12. solution containing 3 M urea, 0.5 M β-mercaptoethanol and 2 M NaCl. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A. and Chen, D. J. (2001). Supernatant collected after DNA precipitation was subjected to buffer ATM phosphorylates histone H2AX in response to DNA double-strand breaks. exchange with 0.01 N HCl by centrifugal filtration (3 kDa cut-off) before J. Biol. Chem. 276, 42462-42467. Carofiglio, F., Inagaki, A., de Vries, S., Wassenaar, E., Schoenmakers, S., the proteins were precipitated with 25% TCA. Chromatin-bound proteins Vermeulen, C., van Cappellen, W. A., Sleddens-Linkels, E., Grootegoed, that had been isolated from spermatids or sperm were resolved in acid- J. A., Te Riele, H. P. et al. (2013). SPO11-independent DNA repair foci and their urea-polyacrylamide gels and subjected to either Coomassie Brilliant Blue role in meiotic silencing. PLoS Genet. 9, e1003538. staining or western blotting. Experiments were performed on three sets of Carrell, D. T., Emery, B. R. and Hammoud, S. (2007). Altered protamine sperm from WT and KO mice, wherein each set comprised sperm from expression and diminished spermatogenesis: what is the link? Hum. Reprod. three to five mice. Update 13, 313-327. Celeste, A., Petersen, S., Romanienko, P. J., Fernandez-Capetillo, O., Chen, H. T., Sedelnikova, O. A., Reina-San-Martin, B., Coppola, V., Meffre, E., Densitometry and statistical analysis Difilippantonio, M. J. et al. (2002). Genomic instability in mice lacking histone Western blots were scanned, and protein band intensities were quantified H2AX. Science 296, 922-927. using ImageJ software (National Institutes of Health, Bethesda, USA). Cheng, C. Y. and Mruk, D. D. (2002). Cell junction dynamics in the testis: Sertoli- Densitometry values of protein bands were normalized to those of β-tubulin, germ cell interactions and male contraceptive development. Physiol. Rev. 82, 825-874. and then the normalized protein level in KO relative to WT samples was Cho, C., Willis, W. D., Goulding, E. H., Jung-Ha, H., Choi, Y. C., Hecht, N. B. and calculated. Western blots from three independent experiments were used for Eddy, E. M. (2001). Haploinsufficiency of protamine-1 or -2 causes infertility in statistical analysis, and results were expressed as mean±s.e.m. Student’s mice. Nat. Genet. 28, 82-86. t-test was performed to calculate the P-values. Evenson, D. P., Jost, L. K., Marshall, D., Zinaman, M. J., Clegg, E., Purvis, K., de Angelis, P. and Claussen, O. P. (1999). Utility of the sperm chromatin structure Acknowledgement assay as a diagnostic and prognostic tool in the human fertility clinic. We are grateful to Dr Wells Wu, Core facility, Center for Biologics Evaluation and Hum. Reprod. 14, 1039-1049. Research, U.S. Food and Drug Administration, for his assistance with RNA-Seq Fan, Y.-X., Wong, L., Deb, T. B. and Johnson, G. R. (2004). Ligand regulates analysis. epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites. J. Biol. Chem. 279, 38143-38150. Competing interests Fernandez-Capetillo, O., Mahadevaiah, S. K., Celeste, A., Romanienko, P. J., The authors declare no competing or financial interests. Camerini-Otero, R. D., Bonner, W. M., Manova, K., Burgoyne, P. and Nussenzweig, A. (2003). H2AX is required for chromatin remodeling and Author contributions inactivation of sex chromosomes in male mouse meiosis. Dev. Cell 4, 497-508. Conceptualization: K.N.J., G.R.J.; Methodology: K.N.J., S.K.T., G.R.J.; Validation: Govin, J., Caron, C., Lestrat, C., Rousseaux, S. and Khochbin, S. (2004). The K.N.J., G.R.J.; Formal analysis: K.N.J.; Investigation: K.N.J., S.K.T.; Writing - role of histones in chromatin remodelling during mammalian spermiogenesis. original draft: K.N.J., G.R.J.; Writing - review & editing: K.N.J., S.K.T., G.R.J.; Eur. J. Biochem. 271, 3459-3469. Visualization: K.N.J., G.R.J.; Supervision: K.N.J., G.R.J.; Project administration: Griswold, M. D. (1995). Interactions between germ cells and Sertoli cells in the K.N.J., G.R.J. testis. Biol. Reprod. 52, 211-216. Hamer, G., Roepers-Gajadien, H. L., van Duyn-Goedhart, A., Gademan, I. S., Kal, H. B., van Buul, P. P. and de Rooij, D. G. (2003). DNA double-strand breaks Funding and gamma-H2AX signaling in the testis. Biol. Reprod. 68, 628-634. This work was supported in part by an appointment of S.K.T. to the Oak Ridge Hammoud, S., Liu, L. and Carrell, D. T. (2009). Protamine ratio and the level Institute for Science and Education (ORISE) Research Participation Program of histone retention in sperm selected from a density gradient preparation. at the Center for Drug Evaluation and Research (CDER), U.S. Food and Drug Andrologia 41, 88-94. 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