RESEARCH ARTICLE

TSSK6 is required for γH2AX formation and thehistone-to-protamine transition during spermiogenesisKula N. Jha*, Swamy K. Tripurani and Gibbes R. Johnson*

ABSTRACTSpermiogenesis includes transcriptional silencing, chromatincondensation and extensive morphological changes as spermatidstransform into sperm. Chromatin condensation involves histonehyperacetylation, transitory DNA breaks, histone H2AX (also knownas H2AFX) phosphorylation at Ser139 (γH2AX), and replacement ofhistones by protamines. Previously, we have reported that thespermatid protein kinase TSSK6 is essential for fertility in mice, butits specific role in spermiogenesis is unknown. Here, we show thatTSSK6 expression is spatiotemporally coincident with γH2AXformation in the nuclei of developing mouse spermatids. RNA-sequencing analysis demonstrates that genetic ablation of Tssk6does not impact gene expression or silencing in spermatids.However, loss of TSSK6 blocks γH2AX formation, even though thetiming and level of the transient DNA breaks is unaltered. Further,Tssk6-knockout sperm contained increased levels of histones H3and H4, and protamine 2 precursor and intermediate(s) indicative of adefective histone-to-protamine transition. These results demonstratethat TSSK6 is required for γH2AX formation during spermiogenesis,and also link γH2AX to the histone-to-protamine transition and malefertility.

KEY WORDS: Spermiogenesis, Protamine, Chromatincondensation, Spermatid, TSSK6, γH2AX

INTRODUCTIONDuring mammalian spermatogenesis, spermatogonia undergoseveral rounds of mitotic divisions to yield spermatocytes, whichthen complete two meiotic divisions and give rise to haploid roundspermatids (Hess and Renato de Franca, 2008). The roundspermatids develop into mature sperm by undergoing a lengthyand complex process of cell differentiation called spermiogenesis,which includes elongation and condensation of nuclei, formation ofthe acrosome and flagellum, and removal of excess cytoplasm(Cheng and Mruk, 2002; Griswold, 1995). This process includes anextensive chromatin reorganization, often referred to as the histone-to-protamine transition, wherein histones are replaced first bytransition proteins (TP) and subsequently by protamines (Prms),leading to a more compact packaging of DNA in sperm (Braun,2001; Govin et al., 2004; Rathke et al., 2014; Ward and Coffey,1991). Genetic ablation of Prm1 or Prm2 leads to sterility in malemice (Cho et al., 2001), and abnormal chromatin condensation in

sperm is closely linked to infertility in men (Evenson et al., 1999;Hammoud et al., 2009). The molecular basis of nuclearcondensation is poorly understood but involves key events suchas expression of testis-specific histone variants, post-translationalmodifications of histones, transient DNA strand breaks andtranscriptional silencing. Several post-translational modificationsof histones, such as acetylation, phosphorylation, ubiquitination andmethylation, all facilitate the displacement of histones fromchromatin and gene regulation in spermatids (Govin et al., 2004;Li et al., 2014; Rathke et al., 2014; Zhuang et al., 2014). Modifiedhistones are recognized by ‘reader’ proteins, which are oftencomponents of chromatin-remodeling complexes that are involvedin the histone-to-protamine transition. For example, bromo-domain-containing proteins, such as BRDT and BRWD1, bind tohyperacetylated histones before histone removal from thechromatin, and mice lacking BRWD1 or expressing mutantBRDT are sterile due to impaired spermiogenesis (Pattabiramanet al., 2015; Shang et al., 2007).

In somatic cells, one of the first responses to DNA breaks isphosphorylation of histone H2AX (also known as H2AFX) atresidue Ser139 (hereafter referred to as γH2AX) by PI3K-likeprotein kinases, such as ATM, DNA-PKcs and ATR (Bellani et al.,2005; Burma et al., 2001; Stiff et al., 2004; Ward and Chen, 2001),in order to recruit the repair machinery to these sites (Rogakou et al.,1998; Rossetto et al., 2012). γH2AX has also been implicated inchromatin remodeling that takes place during several otherbiological processes, such as male sex chromosome inactivationin germ cells, X chromosome inactivation in somatic cells,asymmetric sister chromosome segregation in stem cells andcellular senescence maintenance in fibroblasts (Turinetto andGiachino, 2015). Targeted deletion of histone H2AX in miceresults in male, but not female, infertility due to failure of sex-bodyformation and meiotic sex chromosome inactivation (MSCI) inspermatocytes (Celeste et al., 2002; Fernandez-Capetillo et al.,2003). In leptotene spermatocytes, γH2AX is formed in response torecombination-associated DNA double-strand breaks (Blanco-Rodríguez, 2009; Carofiglio et al., 2013; Celeste et al., 2002). Inpachytene spermatocytes and in the absence of DNA breaks,γH2AX is detected in the sex body, and thought to be produced byATR (Carofiglio et al., 2013; Fernandez-Capetillo et al., 2003;Mahadevaiah et al., 2001; Turner et al., 2004). In elongatingspermatids, γH2AX foci and transitory DNA breaks occursimultaneously (Agnieszka, 2014; Govin et al., 2004; Leducet al., 2008b; Marcon and Boissonneault, 2004; Rathke et al.,2014; Wojtczak et al., 2008), but the kinase responsible for theH2AX phosphorylation and the role of γH2AX in spermiogenesis isnot clearly understood.

The family of testis-specific serine/threonine kinases (TSSKs)comprises six members that are present only in spermatids andsperm (Li et al., 2011). Little is known about the factors that driveexpression of the TSSK genes during spermatogenesis, with theReceived 14 February 2017; Accepted 4 April 2017

Division of Biotechnology Review and Research IV, Office of BiotechnologyProducts, Center for Drug Evaluation and Research, U.S. Food and DrugAdministration, Silver Spring, MD 20993, USA.

*Author for correspondence (kula.jha@fda.hhs.gov; gibbes.johnson@fda.hhs.gov)

K.N.J., 0000-0002-2218-1864

1835

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

Journal

ofCe

llScience

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 stabilityof all TSSKs and for catalytic activation of TSSK2 and TSSK4 (Jhaet al., 2013, 2010). Tssk6-knockout (KO) and Tssk1/Tssk2 doubleKO mice exhibit male infertility (Shang et al., 2010; Spiridonovet al., 2005; Xu et al., 2008), whereas the targeted disruption ofTssk4 causes subfertility in male mice (Wang et al., 2015). Electronmicroscopy analysis of testis sections from Tssk6-KO miceindicates DNA condensation defects, and KO sperm haveabnormal morphology, highly reduced motility and are incapableof fusing with zona pellucida-free eggs (Sosnik et al., 2009).Furthermore, variations in the TSSK6 gene are associated withimpaired spermatogenesis and infertility in men (Su et al., 2010).While numerous hallmarks of spermiogenesis have been well

documented, there remain unanswered questions and an incompleteunderstanding of the elaborate mechanisms required to transformround spermatids into sperm. The close connection betweenTssk6 expression and nuclear condensation during spermatiddevelopment, genetic ablation of Tssk6 and male infertility, andour previous observation that TSSK6 can phosphorylate histonessuch as H2AX in vitro (Spiridonov et al., 2005) led us to perform asystematic investigation of cellular and biochemical events duringspermiogenesis in wild-type (WT) and Tssk6-KO mice. We havefound that TSSK6 mediates γH2AX production and the histone-to-protamine transition required for proper nuclear condensation, andfor the first time, we directly link γH2AX formation in maturingspermatids to male fertility. Based on our findings, we arenow able to position TSSK6 within our current understanding ofspermiogenesis and provide a model for the role of TSSK6 inmale fertility.

RESULTSTSSK6 is localized in nuclei of elongating spermatids, andgenetic deletion of Tssk6 does not impact the transcriptomeAs spermatogenesis progresses, germ cells move from the basalmembrane towards the lumen of the seminiferous tubule, and thecycle of spermatogenesis in mice is divided into 12 stagesrepresenting distinct cellular associations in serial cross-sectionsof seminiferous tubules (Ahmed and de Rooij, 2009; Oakberg,1956). Spermiogenesis in the mouse is further subdivided into 16steps based on the morphology of the nucleus and acrosome ofmaturing spermatids. To thoroughly characterize the expression ofTssk6 mRNA and protein during spermatogenesis, we performedin situ hybridization (ISH) and immunofluorescence (IF) stainingon testis sections. Using periodic acid Schiff (PAS) andhematoxylin staining, we evaluated the morphology of developinggerm cells in WT and Tssk6-KO seminiferous tubules but observedno obvious differences when various stages and steps ofspermatogenesis were compared (Fig. 1A,B and data not shown).Chromogenic ISH detected Tssk6 transcripts in both round andelongating spermatids, but not in spermatocytes (Fig. S1) or incondensed spermatids (data not shown). IF analysis demonstratedintense TSSK6 protein staining in step 11-12 spermatids in stageXI-XII tubules, as characterized by thin and compact spermatidnuclei (Fig. 1C,E,F). Notably, TSSK6 protein was confined to thenuclei of the elongating spermatids as the TSSK6 stainingoverlapped with that of DAPI, and no IF positivity was observedin sections from Tssk6-KO mice (Fig. 1D). Post-meiotic expressionand nuclear localization of TSSK6 suggested that TSSK6 may havean important role in the nuclear condensation and/or regulation ofgene expression in developing spermatids.

To determine whether TSSK6 influenced spermatid geneexpression, we performed RNA sequencing (RNA-Seq) andcompared the mRNA profiles of purified spermatids from WTand Tssk6-KO mice. RNA-Seq analyses were performed on threesets of purified spermatids from age-matched adult WT and KOmice, wherein each set represented a pool of spermatids from 4-6mice. Enriched spermatid populations with >90% purity wereobtained as determined by microscopic evaluation of size andmorphological criteria described previously (Bellvé, 1993). Out of23,235 genes present in the reference genome (Musmusculus_UCSC_mm10), transcripts from a total of 19,885 geneswere detected in WT or Tssk6-KO spermatids, and the top 500 mostabundant transcripts were ranked by fragments per kilobases permillion mapped reads (FPKM) values (Table S1). Transcripts forspermatid-specific genes such as Prm1 and Prm2, Tnp1 and Tnp2

Fig. 1. Characterization of the TSSK6 protein during spermiogenesis.WTand Tssk6-knockout (KO) testis sections were stained with periodic acid Schiff(PAS) and hematoxylin in panels A and B, or with TSSK6monoclonal antibodyfollowed by immunofluorescence (IF) detection in C-F. Representative lightmicroscopy images from PAS-hematoxylin staining are shown. Confocalimages of IF staining at 40× (C and D) and 63× magnification (E and F) arepresented to show TSSK6 (red) in elongating spermatids at step 11-12 in stageXI-XII seminiferous tubules. Overlay of TSSK6 staining and the nuclearstain DAPI are shown in the panels C,D,F. Roman numerals denote theseminiferous epithelial stage of the tubule, and elongating spermatids (es)and spermatocytes (spc) are indicated by arrows. Scale bars: 50 µm (PAS-hematoxylin images) and 40 µm (IF images). All analyses were performedon at least three WT and three KO mice, and 50-100 tubules were evaluatedin sections from each mouse.

1836

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

Journal

ofCe

llScience

(TPs), Smcp, Akap4, Ldhc,Odf and those encoding TSSKs etc. wereincluded in this list and were not significantly affected by thetargeted deletion of Tssk6 (P>0.05). Interestingly, transcripts forTssk6 and its activators Tsacc andHsp90aa1 (Jha et al., 2013, 2010)were the 242nd, 180th and 106th most abundant transcripts,respectively, underscoring the importance of TSSK6 in spermatiddevelopment. A very small number of low-level transcripts (meanFPKM of 0.5–15 in either WT or KO) were either increased (34) ordecreased (5) by >50% in KO versus WT cells, and exhibited adifferential expression at P≤0.05 (Table S2). However, none of theproteins expressed by these transcripts are known to be importantfor spermiogenesis. Table S3 lists genes with less-abundanttranscripts than those listed in Table S1 but that are relevant tospermiogenesis, such as chromatin remodeling mediators (Chd5,Ctcf ), bromo-domain containing proteins (Brd genes), histones(Hist genes), DNA damage response proteins (Brca genes, H2afx),PI3K-like protein kinases (Atm, Atr, Prkdc) or other TSSK genes.The levels of these transcripts were also found to be comparable inWT and KO spermatids (P>0.05). Thus, genetic ablation of Tssk6did not cause significant alteration in gene expression in spermatidsas the amount of transcripts for the vast majority of genes weresimilar in WT and KO spermatids.

TSSK6 and γH2AX are colocalized in elongating spermatidsas they undergo nuclear condensationNuclear abnormalities observed previously in electron micrographsof Tssk6-KO spermatids indicate defects in chromatin condensation(Spiridonov et al., 2005). Based on that observation and nuclearlocalization of TSSK6 in elongating spermatids in the present study,we investigated the relationship between TSSK6 protein expression

and γH2AX generation during spermiogenesis. WT testis sectionswere co-labeled with antibodies against γH2AX and TSSK6, andimmunoreactivity was detected using fluorescence (Fig. 2). StrongγH2AX staining was detected in the nuclei of spermatids at step10-12 that then drastically reduced in the later steps (Fig. 2). Asexpected, γH2AX was also detected in leptotene and pachytenespermatocytes (Blanco-Rodríguez, 2009; Hamer et al., 2003). IFstaining for TSSK6 was detected in step 10-12 elongating spermatidsand was retained in step 13-16 condensing/condensed spermatids inthe lumen of seminiferous tubules at stage I-VI (Fig. 2B,E,H).Importantly, γH2AX and TSSK6 colocalized in the nuclei ofdeveloping spermatids as demonstrated by the merged images ofγH2AX, TSSK6 and DAPI (Fig. 2C,F,I). These results demonstratedthat TSSK6 expression is coincident with γH2AX formation in nucleiof elongating spermatids, suggesting a relationship between TSSK6and γH2AX formation during spermiogenesis.

Genetic ablation of TSSK6 blocks γH2AX formation inspermatidsTo assess whether TSSK6 is involved in γH2AX formation, weperformed IF studies on testis sections from WT and KO mice.Remarkably, no staining was observed in spermatids from KOmice (Fig. 3Ag-Al), but in parallel experiments γH2AX wasreadily detected in the nuclei of spermatids in WT sections(Fig. 3Aa-Af ). These results demonstrated that TSSK6 is essentialfor γH2AX formation in spermatids. Conversely and as expected,γH2AX staining was indistinguishable in spermatocytes from WTand KO testis sections, demonstrating that genetic ablation ofTssk6 does not affect γH2AX formation that is associated witheither homologous recombination or sex-body formation during

Fig. 2. TSSK6 and γH2AX generation are temporallyand spatially coincident. Double IF staining wasperformed on WT testis sections with antibodies againstγH2AX (green) and TSSK6 (red). Representativeconfocal microscopy images of γH2AX (A,D,G), TSSK6(B,E,H) and overlay images with DAPI (C,F,I) arepresented. Tubule stages are marked with romannumerals, and elongating spermatids (es), condensedspermatids (cs) and spermatocytes (spc) are indicatedby arrows. Experiments were repeated with at leastthree WT and three KO mice, and 50-100 tubuleswere evaluated from each mouse.

1837

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

Journal

ofCe

llScience

meiosis (Fig. 3A). A drastically reduced amount of γH2AX in thelysate from purified KO spermatids was seen by western blotting,when compared to WT lysate (Fig. 3B). In the western blottinganalyses, some γH2AX was detected due to unavoidablespermatocyte contamination of the spermatid preparation (Bellvé,1993). To confirm that the absence of γH2AX in KO spermatidswas due to a lack of Ser139 phosphorylation and not to reducedH2AX protein, we performed IF and western blotting experimentson WT and KO samples. IF staining of testis sections showed thepresence of H2AX protein in the nuclei of spermatocytes andelongating spermatids, and no differences were observed in WT

versus KO testis sections (Fig. S2) consistent with the RNA-Seqanalysis (H2afx in Table S3). Finally, western blottingdemonstrated that similar amounts of H2AX protein were presentin the lysates of purified spermatids from WT and KO mice(Fig. 3B). Histone H4 hyperacetylation is another early molecularevent that is believed to be essential for chromatin condensation(Bao and Bedford, 2016; Rathke et al., 2014). Histone H4hyperacetylation was detected in step 8-12 spermatids in stageVIII-XII tubules, but unlike γH2AX, no differences in the stainingpattern were observed in WT and KO testis sections (Fig. S3 anddata not shown).

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 againstγH2AX (green) and stained with DAPI (blue), and representative IF staining at different stages of spermatogenesis is presented. Stage IX-XII tubules are presentin 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 antibodiesagainst γH2AX, H2AX, ATR, TSSK6 or β-tubulin, and results from a representative experiment are shown.Western blotting was performed on three sets of purifiedspermatids from WT and KO mice, wherein each set represented a pool of spermatids from four to six mice. The histone γH2AX protein band was quantifiedby 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. ofthree independent experiments. *P<0.05. (C) WT and Tssk6-KO testis sections were probed with an antibody against ATR (red), and representative overlayimages 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 eachmouse. spc, spermatocytes.

1838

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

Journal

ofCe

llScience

In our previous work, we have observed that both histone H2Aand H2AX are phosphorylated to similar extents by TSSK6 invitro (Spiridonov et al., 2005). Because H2A and H2AX havealmost identical amino acid sequences, with the exception of theextended C-terminus in H2AX that contains Ser139, it is unlikelythat H2AX Ser139 is a good phosphorylation site for TSSK6.Nevertheless, a temporal and spatial colocalization of TSSK6 andγH2AX in elongating spermatids led us to directly test whetherSer139 could be phosphorylated by TSSK6. We expressedMyc-tagged TSSK6 in Cos-7 cells and performed in vitroimmunokinase reactions using peptides corresponding to theH2AX C-terminal region containing the wild-type sequence orSer to Ala changes (Table S4). Histone H2AX peptides weresynthesized and characterized as described previously (Fan et al.,2004). No kinase activity was detected for TSSK6 with any ofthese H2AX peptides, whereas kinase activity was easilydetected with histone H2A protein as substrate. As a positivecontrol, we confirmed that commercially available purified ATMkinase phosphorylated the peptides containing Ser139 (H2AX-P1 and H2AX-P2), but phosphorylation was dramaticallyreduced when Ser139 was changed to Ala (H2AX-P3)(Table S4).Among the kinases that are known to phosphorylate Ser139 in

histone H2AX, ATR transcripts were most abundant in our RNA-Seq analysis of spermatids (Table S3), and ATR is also reported tobe responsible for H2AX Ser139 phosphorylation in spermatocytes(Turner et al., 2004). To test whether TSSK6 was involved in the

protein expression and/or localization of ATR in spermatids, weperformed western blotting and IF experiments to measure ATR onWT and Tssk6-KO testis samples. Western blotting demonstratedthat similar amounts of ATR were present in the lysates of purifiedspermatids from WT and KO mice (Fig. 3B). In IF analyses, ATRwas detected in the nuclei of spermatids at steps 9-12 and,importantly, the staining pattern was very similar in WT and KOspermatids (Fig. 3C). Taken together, these results demonstrate thatTSSK6 is essential for γH2AX generation during spermiogenesis,but is most likely not the kinase that phosphorylates H2AX atSer139 in spermatids.

Transitory DNA strand breaks are indistinguishable in WTand Tssk6-null spermatidsDue to the lack of γH2AX formation in the KO spermatids, weperformed a sensitive fluorescent terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) assay to evaluateDNA breaks on testis sections. As shown in Fig. 4, TUNELlabeling in both WT and Tssk6-KO testis was very similar, anddetected as a homogeneous nuclear stain in spermatocytes andstep 9-12 elongating spermatids (stages IX-XII). Reducedstaining in both WT and KO spermatids was observed at step13 (stage I), with no detection in condensed spermatids (stagesVII-VIII). These results demonstrate that the spermiogenesis-associated transient DNA strand breaks occur in the absence ofγH2AX formation, and the extent and timing are the same in WTand Tssk6-KO mice.

Fig. 4. Genetic ablation of Tssk6 does notimpact the timing and level of transient DNAbreaks in spermatids. A sensitive fluorescentTUNEL assay was performed on mouse testissections to detect DNA strand breaks duringspermiogenesis, and representative images arepresented of TUNEL-positive cells (green) withor without DAPI merge images at various stagesof spermatogenesis in WT (A-H) and KO (I-P)mice. TUNEL staining was performed on atleast three WT and three KO mice, and 50-100tubules were evaluated from each mouse. spc,spermatocytes; spd, spermatids.

1839

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

Journal

ofCe

llScience

Sperm from Tssk6-KO mice contain elevated amountsof histones H3 and H4, and protamine 2 precursorsand intermediatesThe final packaged DNA in sperm nuclei contains bound residualhistones and protamines. To characterize the histone profile of KOsperm, we performed western blotting of sperm lysates withantibodies against histones H1, H2A, H2B, H3 and H4. As shown inFig. 5A,B, loss of Tssk6 resulted in significant increases in histoneH3 and H4 in sperm. Densitometry analysis revealed that histone H3and H4 proteins were ∼3.7- and ∼2.9-fold higher in KO comparedto WT sperm, respectively (Fig. 5B). Histones H1, H2A and H2Bwere undetectable in western blots of KO and WT sperm lysates(data not shown). Western blotting of purified spermatid lysates didnot 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 islargely 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 resultsin quantitative extraction of proteins from the compacted chromatinfollowed by protein fractionation in acid-urea-polyacrylamide gels(Balhorn et al., 1977; Yu et al., 2000; Zhao et al., 2001). Proteinsextracted from the chromatin of WT and KO sperm were separatedin these gels and either stained with Coomassie Brilliant Blue(Fig. 5C, left) or analyzed by western blotting for Prm1 (middle) andPrm2 (right). No differences were noted in KO and WT samples forPrm1 and mature Prm2, whereas strikingly increased amounts ofboth Prm2 precursor and intermediate(s) were observed in KOsperm compared to in WT.Consistent with the observation in sperm, the acid-urea-

polyacrylamide gel analysis also revealed an enhanced presenceof the Prm2 precursor and intermediate(s) in spermatids from theKO mice (Fig. S4B). No TSSK6 protein was detected by westernblotting in these spermatid extracts from WT or KO mice (data notshown), indicating that TSSK6, unlike protamines, is not tightlybound to chromatin. Prm1 and Prm2 were detected in condensing/condensed spermatid nuclei by IF, however, no difference instaining was observed in WT and KO sections (data not shown).These results demonstrated that TSSK6 is required for murine spermto possess normal quantities of histone H3, H4 and Prm2 precursorand intermediate(s).

DISCUSSIONTSSK6 is a testis-specific protein kinase that is highly conserved inmammals and essential for spermatogenesis. To determine thebiological role of TSSK6, here we have performed a comparativeanalysis of molecular events during spermiogenesis in WT andTssk6-KO mice. Based on our study, the relationship of TSSK6 tokey events that occur during chromatin condensation can be betterunderstood. Fig. 6 highlights TSSK6-dependent and -independentevents as round spermatids mature into condensed spermatids.Tssk6 mRNA is expressed in round spermatids, and the transcriptsare still detected in elongating spermatids, whereas TSSK6 proteinfirst appears in elongating spermatids and is retained in maturesperm. Genetic ablation of Tssk6 did not have a significant impacton the transcriptome, indicating that TSSK6 is not involved in geneexpression. Histone H4 hyperacetylation, transient DNA breaks,chromatin displacement of histones H1, H2A and H2B, and the TPexchange all appeared to be normal in Tssk6-KO spermatids.Further, Prm1 and mature Prm2 levels were found to be comparablein WT and KO chromatin-bound protein extracts from spermatidsand sperm. Conversely, TSSK6 was found to be essential for the

production of γH2AX, removal of histones (H3 and H4) andeffective processing of the Prm2 precursor and intermediate(s).Most of the core nucleosomal histones are displaced from thechromatin during steps 9-12 of spermiogenesis in the mouse, andcondensed spermatids or sperm retain only small residual amountsof histones H3 and H4 (Li et al., 2014; Meistrich et al., 2003).Increased retention of histone H3 and H4 in spermatids/sperm hasbeen closely linked to male infertility in many gene-KO mousemodels (Bell et al., 2014; Li et al., 2014; Lu et al., 2010; Zhuanget al., 2014). Prm2 is synthesized as a precursor protein that binds tothe chromatin and then undergoes proteolytic processing giving riseto several intermediates and finally mature Prm2 (Balhorn, 2007;Wu et al., 2000). Thus, incomplete Prm2 processing of thechromatin-associated precursor and intermediate(s) occurs in theabsence of TSSK6. Numerous studies have implicated altered Prm1or Prm2 amounts, and aberrant processing of Prm2 precursor andintermediate(s) in sterility in mice (Bao and Bedford, 2016;Hernández-Hernández et al., 2016; Li et al., 2014; Lu et al.,2010; Rathke et al., 2014; Wu et al., 2000; Yuen et al., 2014) andhumans (Carrell et al., 2007; Oliva, 2006; Torregrosa et al., 2006).We interpret the defects in the histone-to-protamine transition, asevidenced by altered histone and protamine homeostasis in the

Fig. 5. Tssk6-null sperm possess aberrant levels of histones and Prm2precursor and intermediate(s). (A) Lysates from WT and KO sperm wereseparated in SDS-PAGE gels, transferred onto PVDFmembrane and probed bywestern blotting for histones H3 and H4, or for β-tubulin, and results from arepresentative experiment are shown. (B) Histones H3 and H4 protein bandswere quantified by densitometry and values were normalized to those ofβ-tubulin, andamounts in theKO relative to inWTare presentedasmean±s.e.m.of three independent experiments. *P<0.05. (C) Equal amounts of chromatin-boundproteins that had beenextracted fromWTandKOspermwereanalyzed inacid-urea-polyacrylamidegel, as described inMaterials andMethods.Gelswereeither stained with Coomassie Blue (left) or transferred and probed by westernblotting for Prm1 (middle) or Prm2 (right), and results from a representativeexperiment are presented (n=3 experiments). The positions of the Prm2precursor (Pre-Prm2), intermediate (Int-Prm2) and mature Prm2 are denoted.

1840

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

Journal

ofCe

llScience

chromatin of Tssk6-KO spermatids and sperm, as the cause forinfertility in the null mice.γH2AX marks sites of DNA breakage, and γH2AX foci are

detected in spermatocytes during meiotic recombination (Blanco-Rodríguez, 2009; Carofiglio et al., 2013; Celeste et al., 2002).However, γH2AX can also be formed in a DNA break-independentmanner, as in the case of sex-body-associated γH2AX in pachytenespermatocytes (Carofiglio et al., 2013; Fernandez-Capetillo et al.,2003; Mahadevaiah et al., 2001). As expected, and becauseexpression of TSSK6 is limited to spermatids and sperm, geneticablation of TSSK6 did not cause any defect in γH2AX formationthat is associated with homologous recombination or sex-bodyformation in spermatocytes. Detection of γH2AX foci in step 9-12elongating spermatids is coincident with DNA strand breaks (Govinet al., 2004; Leduc et al., 2008a; Rathke et al., 2014), and here wehave demonstrated that TSSK6 is essential for γH2AX productionduring spermiogenesis. The timing and level of the DNA breakswere very similar in WT and Tssk6-KO spermatids, and can beinterpreted two ways. Either DNA breaks are not the driver of theH2AX phosphorylation in spermiogenesis or TSSK6 is required forγH2AX to be generated in response to the breaks. Previous studieshave suggested that precise control of γH2AX may be importantduring spermiogenesis since prolonged or increased amounts havebeen observed in KO mouse models with a sterile phenotype (Liet al., 2014; Wang et al., 2016). To the best of our knowledge, ourfindings regarding TSSK6 are the first that link γH2AX formationduring spermiogenesis to the production of functional sperm andfertility. Based on the high abundance of Atr transcripts inspermatids, nuclear spermatid localization of ATR, a recognizedrole for ATR in γH2AX generation in pachytene spermatocytes(Turner et al., 2004) and the inability of TSSK6 to phosphorylate

H2AX at Ser139 in vitro, it is more likely that ATR, and not TSSK6,is the kinase that phosphorylates Ser139 of H2AX duringspermiogenesis. However, it is important to note that geneticablation of Tssk6 had no impact on ATR expression and nuclearlocalization in spermatids and thus, there is no direct evidence thatATR phosphorylates H2AX in this instance. Nevertheless, therequirement of TSSK6 for γH2AX generation demonstrates thatTSSK6 is an upstream kinase responsible for the proper localizationand/or activation of the kinase that directly produces γH2AX duringspermatid nuclear condensation.

IF studies demonstrated that TSSK6 is confined to the nuclei, butit was not detected in the chromatin-bound protein extracts derivedfrom spermatids. Accordingly, these findings indicate that whileTSSK6 is in the nucleus as chromatin is condensing, it is not tightlybound to DNA. Based on the insights gained from the present study,we postulate that TSSK6 mediates γH2AX generation to govern achromatin-associated remodeling complex that is essential in thehistone-to-protamine transition. Further studies are warranted toidentify the TSSK6 protein substrates that are required for γH2AXformation, the histone-to-protamine transition and the production ofnormal sperm in mammals.

MATERIALS AND METHODSReagentsAntibodies against histones H3 (96C10), H4 (L64C1), H2A (cataloguenumber 2578), H2AX (catalogue number 2595) and γH2AX (20E3) werepurchased from Cell Signaling Technology Inc; against histones H1 (AE-4),H2B (FL-126) and TP2 (K-18) were from Santa Cruz Biotechnology Inc;against TNP1 (catalogue number 17178-1-AP) were from ProteinTechGroup Inc (Chicago, IL); and against Prm1 (Hup1N) and Prm2 (Hup2B)were from Briar Patch Biosciences (Livermore, CA). Recombinant humanATM kinase and antibody against hyperacetylated histone H4 (Penta) were

Fig. 6. Role of TSSK6 in the histone-to-protaminetransition. At the bottom of the diagram, developmentalexpression of Tssk6 mRNA and protein is shown andaligned to steps (1-16) of mouse spermiogenesis asround spermatids develop into condensed spermatids.Molecular changes involved in the histone-to-protaminetransition are categorized into TSSK6-dependent (red)and -independent (yellow) events, and their occurrencehas been aligned with steps of spermatid development.Alignment of histone removal, TP exchange, Prm1 andPrm2 integration, and Prm2 precursor and intermediate(P/I) processing is based on the literature (Bao andBedford, 2016; Govin et al., 2004; Meistrich et al., 2003).

1841

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

Journal

ofCe

llScience

from EMD Millipore (Billerica, MA), and terminal deoxynucleotidyltransferase (TdT), TdT buffer and biotin-16-dUTP were from Sigma(St Louis, MO). Monoclonal antibody against mouse TSSK6 was generatedin our lab and has been described previously (Jha et al., 2010). All reagentsused in this study were of analytical grade.

AnimalsThe Tssk6-KOmouse model was generated in our lab and has been describedpreviously (Spiridonov et al., 2005). Micewere handled and killed accordingto the guidelines of the Animal Care and Use Committee (Center forBiologics and Evaluation Research, U.S. Food and Drug Administration).Adult male Tssk6-KO mice and WT littermates were used in this study.

ISH analysisRNA ISH was performed using the RNAScope ISH technology (AdvancedCell Diagnostics, Hayward, CA) (Wang et al., 2012). Briefly, testes wereharvested and fixed in 10% neutral buffered formalin for 24 h at roomtemperature, dehydrated and embedded in paraffin. Tissue sections were cut at5 μm thickness, air-dried at room temperature and processed for RNA in situdetection by using the RNAscope 2.5 HD Reagent Kit-RED (cataloguenumber 322360). To ensure RNA integrity and assay procedure, pre-treatmentconditions were optimized to 15 min heat-pre-treatment, followed by 30 minprotease digestion. Custom RNA ISH probes were prepared by AdvancedCell Diagnostics to detect mouse Tssk6, and a probe to bacterial mRNADapBserved as a negative control. Slides were scanned using PanoramicMIDI slidescanner (3DHISTECH) to acquire images of ISH.

IF staining of proteins in testis sectionsTesteswere fixed either in 4%paraformaldehyde or Carnoy solution (ethanol,chloroform, acetic acid in the ratio 6:3:1, respectively) and embedded inparaffin, and 5-μm thick cross-sections were mounted on glass slides.Sections were deparaffinized with xylene, rehydrated through a gradedethanol series and washed briefly in water. Slides were then processed forantigen retrieval in 10 mM citrate buffer (pH 6.0) for 20 min at 100°C,washed with PBS and blocked with 1% bovine serum albumin (BSA) in PBScontaining 0.1% Tween-20 (PBS-T). Sections were incubated with primaryantibody or isotype control IgG overnight, and the antibody dilution used inIF studies was 1:100 with the exception of γH2AX (20E3) antibody, whichwas used at 1:400 dilution. Following primary antibody incubation, the slideswere washed with PBS-T and incubated with Alexa-Fluor-conjugatedsecondary antibody (1:200 dilution) (Molecular Probes, Eugene, OR) for 1 h.Slides were washed with PBS-T, counterstained with DAPI and mountedwith Slow-Fade light reagent (Molecular Probes). Sections were examinedunder a confocal microscope (LSM 710, Zeiss), and images were captured.Established morphological criteria were used to determine specific stages ofthe mouse seminiferous epithelium cycle (Ahmed and de Rooij, 2009;Meistrich and Hess, 2013). Experiments were repeated with testes sectionsfrom at least three different sets of WT and Tssk6-KO mice.

TUNEL assayA modified TUNEL assay was performed to detect DNA breaks in testissections as previously reported (Marcon and Boissonneault, 2004). Briefly,Carnoy-fixed testis sections were deparaffinized with xylene and rehydratedthrough a graded ethanol series. Sections were washed with PBS andequilibrated with TdT buffer for 30 min at 37°C. TdT buffer was removedand a freshly prepared terminal transferase reaction mix (50 µl TdT buffercontaining 25 U terminal transferase enzyme and 0.5 nmol Biotin-16-dUTP) was added. End-labeling was performed for 1 h at 37°C, followed bywashing with PBS and incubation with fluorescein–avidin (1:100 dilution inPBS) for 1 h at room temperature. Sections were washed with PBS,counterstained with DAPI, mounted with Slow-Fade light reagent, andimages were captured using a confocal microscope. A negative control withno TdT enzyme was included in all experiments.

Image acquisitionImage acquisition from IF and TUNEL staining was performed at roomtemperature. Data analysis was performed using a LSM 710 confocal laser

scanning microscope (Zeiss) equipped with EC Plan-Neofluar 20×/0.8 NA,40×/1.3 NA oil or Plan-Apochromat 63×/1.4 NA oil objective lens. Imageswere captured with AxioCam camera (Zeiss) using Zen imaging software(Zeiss) and processed using Photoshop CS4 (Adobe). For ISH imaging,slides were scanned using Panoramic MIDI slide scanner (3DHISTECH)equipped with a Plan-Apochromat 20×/0.8 NA objective lens (Zeiss) and aVCC-FC60FR19CL/4MP camera (CIS). Image analysis was performedusing Panoramic Viewer software (3DHISTECH), and images wereprocessed using Photoshop CS4. No gamma correction was made in IF,TUNEL or ISH images, and scale bars were added.

Purification of mouse spermatidsAn enriched population of mouse spermatids was purified following theSTA-PUT method as described previously (Jha et al., 2013). Briefly, testesfrom adult micewere enzymatically dissociated, and the cells were separatedby sedimentation at unit gravity in 2-4% BSA gradient in a STA-PUTapparatus. Fractions were examined under a microscope, and germ cell typeswere determined based on the size and morphology (Bellvé, 1993).Fractions enriched with spermatids were pooled to obtain a population ofspermatids with >90% purity.

RNA-Seq analysisSpermatids were purified from three sets of age-matched adult WT andTssk6-KOmice, and total RNAwas isolated using RNeasy kit (Qiagen) withDNase I treatment. RNA-Seq libraries were prepared using Illumina TruSeqStranded mRNA sample preparation kit and sequenced on Illumina HiSeq2500. The quality of raw data was assessed and passed by FastQC, and readswere mapped to the reference genome (Mus musculus_UCSC_mm10).Cufflinks software (v2.2.1) was used to estimate transcript levelsrepresented by the FPKM, and differential expression between sampleswas determined using Cuffdiff software (v.2.2.1).

Western blotting and immunoprecipitationProteins were fractionated in 4-20% SDS-PAGE gels. Western blotting andimmunoprecipitation experiments were performed as described previously(Jha et al., 2013, 2010), and the primary antibody dilution used in westernblotting was 1:1000.

Protein kinase assayMyc-tagged Tssk6 was expressed in Cos-7 cells (American Type CultureCollection, Manassas, Virginia) and immunoprecipitated with an antibodyagainst Myc, and in vitro kinase reactions were performed (Jha et al., 2013).Briefly, the reaction was performed at room temperature for 30 min ina reaction buffer containing 25 mM HEPES (pH 7.4), 10 mM MgCl2,10 mM MnCl2, 2 mM EGTA, 30 µM ATP, 10 µCi of [γ-32P]ATP, andsynthetic histone H2AX peptides or histone H2A protein. The reaction wasterminated by acidification, and phosphorylation of the H2AX peptides orH2A protein was quantified by measuring incorporated radioactive 32P(Fan et al., 2004). Values are represented as mean kinase activity(fmol/min)±s.e.m. (n=3).

Isolation of mouse spermSperm from mouse cauda epididymis were collected in modified Krebs–Ringer medium (Whitten’s-HEPES buffered medium) containing 100 mMNaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose,1 mM pyruvic acid, 4.8 mM L(+) lactic acid hemicalcium salt in 20 mMHEPES (pH 7.3), as described previously (Jha et al., 2006, 2008).

Extraction and analysis of chromatin-boundproteins frommousespermatids and spermChromatin-bound proteins from spermatids and sperm were extracted andanalyzed following the methods described previously (Balhorn et al.,1977; Yu et al., 2000; Zhao et al., 2001). Testicular germ cells wereisolated by the enzymatic dissociation of WT and Tssk6-KO testes, andsubjected to sonication followed by centrifugation through a layer of 5%sucrose in 0.2× concentration of MP buffer (5 mM MgCl2 and 5 mMsodium phosphate, pH 6.5) containing 0.25% Triton X-100 and protease

1842

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

Journal

ofCe

llScience

inhibitors. The pellet fraction, which comprises sonication-resistant nucleiof condensing/condensed spermatids, was washed inMP buffer containing0.22 M NaCl and incubated at 37°C for 10 min in 10 mM DTT andprotease inhibitors. DNA was precipitated with 0.5 M HCl on ice for 1 h,and the supernatant was subjected to protein precipitation with 25%trichloroacetic acid (TCA), followed by washing with acidified acetoneand dissolving the protein precipitate in acid-urea gel sample buffer (5 Murea, 5% acetic acid, 1% β-mercaptoethanol, and Methyl Green dye).Extraction from sperm was performed as above with the exception that thenuclei pellet isolated after sonication was incubated in 5 M guanidiumchloride solution (pH 8.0) for 30 min on ice, and then dissolved in asolution containing 3 M urea, 0.5 M β-mercaptoethanol and 2 M NaCl.Supernatant collected after DNA precipitation was subjected to bufferexchange with 0.01 N HCl by centrifugal filtration (3 kDa cut-off ) beforethe proteins were precipitated with 25% TCA. Chromatin-bound proteinsthat had been isolated from spermatids or sperm were resolved in acid-urea-polyacrylamide gels and subjected to either Coomassie Brilliant Bluestaining or western blotting. Experiments were performed on three sets ofsperm from WT and KO mice, wherein each set comprised sperm fromthree to five mice.

Densitometry and statistical analysisWestern blots were scanned, and protein band intensities were quantifiedusing ImageJ software (National Institutes of Health, Bethesda, USA).Densitometry values of protein bands were normalized to those of β-tubulin,and then the normalized protein level in KO relative to WT samples wascalculated. Western blots from three independent experiments were used forstatistical analysis, and results were expressed as mean±s.e.m. Student’st-test was performed to calculate the P-values.

AcknowledgementWe are grateful to Dr Wells Wu, Core facility, Center for Biologics Evaluation andResearch, U.S. Food and Drug Administration, for his assistance with RNA-Seqanalysis.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: K.N.J., G.R.J.; Methodology: K.N.J., S.K.T., G.R.J.; Validation:K.N.J., G.R.J.; Formal analysis: K.N.J.; Investigation: K.N.J., S.K.T.; Writing -original draft: K.N.J., G.R.J.; Writing - review & editing: K.N.J., S.K.T., G.R.J.;Visualization: K.N.J., G.R.J.; Supervision: K.N.J., G.R.J.; Project administration:K.N.J., G.R.J.

FundingThis work was supported in part by an appointment of S.K.T. to the Oak RidgeInstitute for Science and Education (ORISE) Research Participation Programat the Center for Drug Evaluation and Research (CDER), U.S. Food and DrugAdministration, administered by ORISE through an agreement between theUnited States Department of Energy and CDER.

Data availabilityFull RNA-Seq data associated with this study are deposited in the Dryad repository:http://dx.doi.org/10.5061/dryad.95s1k

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.202721.supplemental

ReferencesAgnieszka, W. (2014). Etoposide interferes with the process of chromatincondensation during alga Chara vulgaris spermiogenesis. Micron 65, 45-50.

Ahmed, E. A. and de Rooij, D. G. (2009). Staging of mouse seminiferous tubulecross-sections. Methods Mol. Biol. 558, 263-277.

Balhorn, R. (2007). The protamine family of sperm nuclear proteins. Genome Biol.8, 227.

Balhorn, R., Gledhill, B. L. and Wyrobek, A. J. (1977). Mouse sperm chromatinproteins: quantitative isolation and partial characterization. Biochemistry 16,4074-4080.

Bao, J. and Bedford, M. T. (2016). Epigenetic regulation of the histone-to-protamine transition during spermiogenesis. Reproduction 151, R55-R70.

Bell, E. L., Nagamori, I., Williams, E. O., Del Rosario, A. M., Bryson, B. D.,Watson, N., White, F. M., Sassone-Corsi, P. and Guarente, L. (2014). SirT1is required in the male germ cell for differentiation and fecundity in mice.Development 141, 3495-3504.

Bellani, M. A., Romanienko, P. J., Cairatti, D. A. and Camerini-Otero, R. D.(2005). SPO11 is required for sex-body formation, and Spo11 heterozygosityrescues the prophase arrest of Atm-/- spermatocytes. J. Cell Sci. 118, 3233-3245.

Bellve, A. R. (1993). Purification, culture, and fractionation of spermatogenic cells.Methods Enzymol. 225, 84-113.

Blanco-Rodrıguez, J. (2009). gammaH2AX marks the main events of thespermatogenic process. Microsc. Res. Tech. 72, 823-832.

Braun, R. E. (2001). Packaging paternal chromosomes with protamine. Nat. Genet.28, 10-12.

Burma, S., Chen, B. P., Murphy, M., Kurimasa, A. and Chen, D. J. (2001).ATM phosphorylates histone H2AX in response to DNA double-strand breaks.J. Biol. Chem. 276, 42462-42467.

Carofiglio, F., Inagaki, A., de Vries, S., Wassenaar, E., Schoenmakers, S.,Vermeulen, C., van Cappellen, W. A., Sleddens-Linkels, E., Grootegoed,J. A., Te Riele, H. P. et al. (2013). SPO11-independent DNA repair foci and theirrole in meiotic silencing. PLoS Genet. 9, e1003538.

Carrell, D. T., Emery, B. R. and Hammoud, S. (2007). Altered protamineexpression and diminished spermatogenesis: what is the link? Hum. Reprod.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.,Difilippantonio, M. J. et al. (2002). Genomic instability in mice lacking histoneH2AX. Science 296, 922-927.

Cheng, C. Y. and Mruk, D. D. (2002). Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development. Physiol. Rev. 82,825-874.

Cho, C., Willis, W. D., Goulding, E. H., Jung-Ha, H., Choi, Y. C., Hecht, N. B. andEddy, E. M. (2001). Haploinsufficiency of protamine-1 or -2 causes infertility inmice. Nat. Genet. 28, 82-86.

Evenson, D. P., Jost, L. K., Marshall, D., Zinaman, M. J., Clegg, E., Purvis, K., deAngelis, P. and Claussen, O. P. (1999). Utility of the sperm chromatin structureassay as a diagnostic and prognostic tool in the human fertility clinic.Hum. Reprod. 14, 1039-1049.

Fan, Y.-X., Wong, L., Deb, T. B. and Johnson, G. R. (2004). Ligand regulatesepidermal growth factor receptor kinase specificity: activation increasespreference for GAB1 and SHC versus autophosphorylation sites. J. Biol. Chem.279, 38143-38150.

Fernandez-Capetillo, O., Mahadevaiah, S. K., Celeste, A., Romanienko, P. J.,Camerini-Otero, R. D., Bonner, W. M., Manova, K., Burgoyne, P. andNussenzweig, A. (2003). H2AX is required for chromatin remodeling andinactivation of sex chromosomes in male mouse meiosis. Dev. Cell 4, 497-508.

Govin, J., Caron, C., Lestrat, C., Rousseaux, S. and Khochbin, S. (2004). Therole of histones in chromatin remodelling during mammalian spermiogenesis.Eur. J. Biochem. 271, 3459-3469.

Griswold, M. D. (1995). Interactions between germ cells and Sertoli cells in thetestis. 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 breaksand gamma-H2AX signaling in the testis. Biol. Reprod. 68, 628-634.

Hammoud, S., Liu, L. and Carrell, D. T. (2009). Protamine ratio and the levelof histone retention in sperm selected from a density gradient preparation.Andrologia 41, 88-94.

Hernandez-Hernandez, A., Lilienthal, I., Fukuda, N., Galjart, N. and Hoog, C.(2016). CTCF contributes in a critical way to spermatogenesis and male fertility.Sci. Rep. 6, 28355.

Hess, R. A. and Renato de Franca, L. (2008). Spermatogenesis and cycle of theseminiferous epithelium. Adv. Exp. Med. Biol. 636, 1-15.

Jha, K. N., Salicioni, A. M., Arcelay, E., Chertihin, O., Kumari, S., Herr, J. C. andVisconti, P. E. (2006). Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm capacitation.Mol. Hum. Reprod. 12, 781-789.

Jha, K. N., Shumilin, I. A., Digilio, L. C., Chertihin, O., Zheng, H., Schmitz, G.,Visconti, P. E., Flickinger, C. J., Minor, W. and Herr, J. C. (2008). Biochemicaland structural characterization of apolipoprotein A-I binding protein, a novelphosphoprotein with a potential role in sperm capacitation. Endocrinology 149,2108-2120.

Jha, K. N., Wong, L., Zerfas, P. M., De Silva, R. S., Fan, Y.-X., Spiridonov, N. A.and Johnson, G. R. (2010). Identification of a novel HSP70-binding cochaperonecritical to HSP90-mediated activation of small serine/threonine kinase.J. Biol. Chem. 285, 35180-35187.

Jha, K. N., Coleman, A. R.,Wong, L., Salicioni, A. M., Howcroft, E. and Johnson,G. R. (2013). Heat shock protein 90 functions to stabilize and activate the testis-specific serine/threonine kinases, a family of kinases essential for male fertility.J. Biol. Chem. 288, 16308-16320.

Jha, K. N., Tripurani, S. K., Johnson, G. R. (2017) Data from: TSSK6 is required forγH2AX formation and the histone-to-protamine transition during spermiogenesis.Dryad Digital Repository. http://dx.doi.org/10.5061/dryad.95s1k

1843

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

Journal

ofCe

llScience

Leduc, F., Maquennehan, V., Nkoma, G. B. and Boissonneault, G. (2008a). DNAdamage response during chromatin remodeling in elongating spermatids of mice.Biol. Reprod. 78, 324-332.

Leduc, F., Nkoma, G. B. and Boissonneault, G. (2008b). Spermiogenesis andDNA repair: a possible etiology of human infertility and genetic disorders. Syst.Biol. Reprod. Med. 54, 3-10.

Li, Y., Sosnik, J., Brassard, L., Reese, M., Spiridonov, N. A., Bates, T. C.,Johnson, G. R., Anguita, J., Visconti, P. E. and Salicioni, A. M. (2011).Expression and localization of five members of the testis-specific serine kinase(Tssk) family in mouse and human sperm and testis. Mol. Hum. Reprod. 17,42-56.

Li, W., Wu, J., Kim, S. Y., Zhao, M., Hearn, S. A., Zhang, M. Q., Meistrich, M. L.and Mills, A. A. (2014). Chd5 orchestrates chromatin remodelling during spermdevelopment. Nat. Commun. 5, 3812.

Lu, L.-Y., Wu, J., Ye, L., Gavrilina, G. B., Saunders, T. L. and Yu, X. (2010). RNF8-dependent histone modifications regulate nucleosome removal duringspermatogenesis. Dev. Cell 18, 371-384.

Mahadevaiah, S. K., Turner, J. M. A., Baudat, F., Rogakou, E. P., de Boer, P.,Blanco-Rodrıguez, J., Jasin, M., Keeney, S., Bonner, W. M. and Burgoyne,P. S. (2001). Recombinational DNA double-strand breaks in mice precedesynapsis. Nat. Genet. 27, 271-276.

Marcon, L. and Boissonneault, G. (2004). Transient DNA strand breaks duringmouse and human spermiogenesis: new insights in stage specificity and link tochromatin remodeling. Biol. Reprod. 70, 910-918.

Meistrich, M. L. and Hess, R. A. (2013). Assessment of spermatogenesis throughstaging of seminiferous tubules. Methods Mol. Biol. 927, 299-307.

Meistrich, M. L., Mohapatra, B., Shirley, C. R. and Zhao, M. (2003). Roles oftransition nuclear proteins in spermiogenesis. Chromosoma 111, 483-488.

Oakberg, E. F. (1956). Duration of spermatogenesis in the mouse and timing ofstages of the cycle of the seminiferous epithelium. Am. J. Anat. 99, 507-516.

Oliva, R. (2006). Protamines andmale infertility.Hum. Reprod. Update 12, 417-435.Pattabiraman, S., Baumann, C., Guisado, D., Eppig, J. J., Schimenti, J. C. andDe La Fuente, R. (2015). Mouse BRWD1 is critical for spermatid postmeiotictranscription and female meiotic chromosome stability. J. Cell Biol. 208, 53-69.

Rathke, C., Baarends, W. M., Awe, S. and Renkawitz-Pohl, R. (2014). Chromatindynamics during spermiogenesis. Biochim. Biophys. Acta 1839, 155-168.

Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. and Bonner, W. M. (1998).DNA double-stranded breaks induce histone H2AX phosphorylation on serine139. J. Biol. Chem. 273, 5858-5868.

Rossetto, D., Avvakumov, N. and Cote, J. (2012). Histone phosphorylation: achromatin modification involved in diverse nuclear events. Epigenetics 7,1098-1108.

Shang, E., Nickerson, H. D.,Wen, D., Wang, X. andWolgemuth, D. J. (2007). Thefirst bromodomain of Brdt, a testis-specific member of the BET sub-family ofdouble-bromodomain-containing proteins, is essential for male germ celldifferentiation. Development 134, 3507-3515.

Shang, P., Baarends, W. M., Hoogerbrugge, J., Ooms, M. P., van Cappellen,W. A., de Jong, A. A. W., Dohle, G. R., van Eenennaam, H., Gossen, J. A. andGrootegoed, J. A. (2010). Functional transformation of the chromatoid body inmouse spermatids requires testis-specific serine/threonine kinases. J. Cell Sci.123, 331-339.

Sosnik, J., Miranda, P. V., Spiridonov, N. A., Yoon, S.-Y., Fissore, R. A.,Johnson, G. R. and Visconti, P. E. (2009). Tssk6 is required for Izumorelocalization and gamete fusion in the mouse. J. Cell Sci. 122, 2741-2749.

Spiridonov, N. A., Wong, L., Zerfas, P. M., Starost, M. F., Pack, S. D., Paweletz,C. P. and Johnson, G. R. (2005). Identification and characterization of SSTK, aserine/threonine protein kinase essential for male fertility. Mol. Cell. Biol. 25,4250-4261.

Stiff, T., O’Driscoll, M., Rief, N., Iwabuchi, K., Lobrich, M. and Jeggo, P. A.(2004). ATM and DNA-PK function redundantly to phosphorylate H2AX afterexposure to ionizing radiation. Cancer Res. 64, 2390-2396.

Su, D., Zhang, W., Yang, Y., Zhang, H., Liu, Y.-Q., Bai, G., Ma, Y.-X., Peng, Y. andZhang, S.-Z. (2010). c.822+126T>G/C: a novel triallelic polymorphism of theTSSK6 gene associated with spermatogenic impairment in a Chinese population.Asian J. Androl. 12, 234-239.

Torregrosa, N., Domınguez-Fandos, D., Camejo, M. I., Shirley, C. R., Meistrich,M. L., Ballesca, J. L. and Oliva, R. (2006). Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients.Hum. Reprod. 21, 2084-2089.

Turinetto, V. and Giachino, C. (2015). Multiple facets of histone variant H2AX: aDNA double-strand-break marker with several biological functions. Nucleic AcidsRes. 43, 2489-2498.

Turner, J. M. A., Aprelikova, O., Xu, X., Wang, R., Kim, S., Chandramouli,G. V. R., Barrett, J. C., Burgoyne, P. S. and Deng, C.-X. (2004). BRCA1,histone H2AX phosphorylation, and male meiotic sex chromosome inactivation.Curr. Biol. 14, 2135-2142.

Wang, F., Flanagan, J., Su, N., Wang, L.-C., Bui, S., Nielson, A.,Wu, X., Vo, H.-T.,Ma, X.-J. and Luo, Y. (2012). RNAscope: a novel in situ RNA analysis platformfor formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22-29.

Wang, X., Wei, Y., Fu, G., Li, H., Saiyin, H., Lin, G., Wang, Z., Chen, S. and Yu, L.(2015). Tssk4 is essential for maintaining the structural integrity of spermflagellum. Mol. Hum. Reprod. 21, 136-145.

Wang, H., Zhao, R., Guo, C., Jiang, S., Yang, J., Xu, Y., Liu, Y., Fan, L., Xiong,W.,Ma, J. et al. (2016). Knockout of BRD7 results in impaired spermatogenesisand male infertility. Sci. Rep. 6, 21776.

Ward, I. M. and Chen, J. (2001). Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276,47759-47762.

Ward, W. S. and Coffey, D. S. (1991). DNA packaging and organization inmammalian spermatozoa: comparison with somatic cells. Biol. Reprod. 44,569-574.

Wojtczak, A., Popłonska, K. and Kwiatkowska, M. (2008). Phosphorylation ofH2AX histone as indirect evidence for double-stranded DNA breaks related to theexchange of nuclear proteins and chromatin remodeling in Chara vulgarisspermiogenesis. Protoplasma 233, 263-267.

Wu, J. Y., Ribar, T. J., Cummings, D. E., Burton, K. A., McKnight, G. S. andMeans, A. R. (2000). Spermiogenesis and exchange of basic nuclear proteins areimpaired in male germ cells lacking Camk4. Nat. Genet. 25, 448-452.

Xu, B., Hao, Z., Jha, K. N., Zhang, Z., Urekar, C., Digilio, L., Pulido, S., Strauss,J. F., III, Flickinger, C. J. and Herr, J. C. (2008). Targeted deletion of Tssk1and 2 causes male infertility due to haploinsufficiency. Dev. Biol. 319, 211-222.

Yu, Y. E., Zhang, Y., Unni, E., Shirley, C. R., Deng, J. M., Russell, L. D., Weil,M. M., Behringer, R. R. and Meistrich, M. L. (2000). Abnormal spermatogenesisand reduced fertility in transition nuclear protein 1-deficient mice.Proc. Natl. Acad.Sci. USA 97, 4683-4688.

Yuen, B. T. K., Bush, K. M., Barrilleaux, B. L., Cotterman, R. andKnoepfler, P. S.(2014). HistoneH3.3 regulates dynamic chromatin states during spermatogenesis.Development 141, 3483-3494.

Zhao, M., Shirley, C. R., Yu, Y. E., Mohapatra, B., Zhang, Y., Unni, E., Deng, J. M.,Arango, N. A., Terry, N. H. A., Weil, M. M. et al. (2001). Targeted disruption of thetransition protein 2 gene affects sperm chromatin structure and reduces fertilityin mice. Mol. Cell. Biol. 21, 7243-7255.

Zhuang, T., Hess, R. A., Kolla, V., Higashi, M., Raabe, T. D. and Brodeur, G. M.(2014). CHD5 is required for spermiogenesis and chromatin condensation.Mech. Dev. 131, 35-46.

1844

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

Journal

ofCe

llScience