The Nuclear Form of Glutathione Peroxidase 4 Is Associated with Sperm Nuclear Matrix and Is Required for Proper Paternal Chromatin Decondensation at Fertilization

Original Research Article

The nuclear form of Glutathione Peroxidase 4 is associated with sperm nuclear matrix and is required for proper paternal chromatin decondensation at fertilization

Rossella Puglisi1, Irene Maccari1,, Simona Pipolo1, Marcus Conrad3, Franco Mangia2, Carla Boitani1*

1DAHFMO, Section of Histology & Medical Embryology, University of Rome “La Sapienza”, 2Dept Psychology, Section of Neuroscience, University of Rome “La Sapienza”, Rome, Italy, 3DZNE - German Center for Neurodegenerative Diseases, Munich, and Helmholtz Zentrum München, Institute of Developmental Genetics, 85764 Neuherberg, Germany

*Corresponding author:

Carla Boitani, PhD DAHFMO- Section of Histology and Medical Embryology University of Rome "La Sapienza" Via A. Scarpa 14 00161 Roma, Italy tel:+39-06-4976-6571 FAX: +39-06-4462854 e-mail: [email protected]

Running head: nGPx4 impact on sperm chromatin assembly

Key words: nGPx4, sperm chromatin condensation, nuclear matrix, fertilization

Contract grant sponsor: Italian Ministry of Education and Institute Pasteur Cenci Bolognetti Contract grant sponsor: European Union Contract grant number: 513991

Additional Supporting information may be found in the online version of this article.

The nuclear isoform of the selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is expressed in haploid male germ cells, contains several cysteines and is able to oxidize protein thiols, besides glutathione. In this study we have investigated the subnuclear localization of this isoform in isolated mouse male germ cells at different steps of maturation. Immunoblotting and confocal microscopy analyses of subnuclear fractions showed that nGPx4 is localized to the nuclear matrix together with well known markers of this subnuclear compartment like lamin B and topoisomerase IIȕ at all stages of germ cell differentiation. The peculiar nGPx4 distribution was confirmed by both biochemical and morphological analyses of COS-1 cells overexpressing Flag- tagged nGPx4. To test the functional role of nGPx4 in the process of chromatin assembly, sperm isolated from the caput and the cauda epididymides of wild-type (WT) and genetically deficient in nGPx4 (nGPx4-KO) mice were analysed in an in vitro chromatin decondensation assay. Results showed that sperm from nGPx4-KO mice were more prone to decondense than those from WT mice at all stages of epididymal maturation, providing conclusive evidence that nGPx4 is required for a correct sperm chromatin compaction. We next addressed the issue of whether the lack of nGPx4 impacts on early events occurring at fertilization. Indeed, in vitro fertilization experiments showed an acceleration of sperm chromatin dispersion in oocytes fertilized by nGpx4-KO sperm compared with control. Overall these data indicate that the absence of nGPx4 leads to sperm nuclear matrix /chromatin instability that may negatively affect the embryo development.

2 Introduction

The selenoprotein Glutathione Peroxidase 4 (GPx4) is a monomeric enzyme having peculiar catalytic properties unlike other members of glutathione peroxidase family. This enzyme is able to reduce peroxidized membrane phospholipids and to oxidize not only the thiol group of glutathione but also those of proteins (Godeas et al., 1997; Ursini et al., 1999). Three distinct GPx4 isoforms have been identified so far, referred to as mitochondrial (mGPx4), cytosolic (cGPx4) and nuclear (nGPx4) forms depending on their intracellular localization. The Gpx4 gene is highly expressed in both murine and human testis, mainly in germ cells, and is regarded as the pivotal link between selenium, sperm quality and male fertility. Studies aimed at identifying spatial and temporal expression of various GPx4 proteins have revealed isoform-specific patterns of expression during mouse spermatogenesis (Puglisi et al., 2003). Interestingly, expression of the nuclear form occurs for the first time in haploid cells at the round spermatid stage and is thereafter maintained in sperm (Moreno et al., 2003; Puglisi et al., 2003). In line with a stringent regulation of isoform –specific expression during germ cell differentiation, elaborate genetic approaches demonstrated distinct functions for each of the three isoforms, pointing to the idea that GPx4 proteins play different roles depending on the cell type where they are expressed, the subcellular compartment where they are present and the specific molecules they interact with. The mGPx4 importance to testis function was recently demonstrated by the phenotypic analysis of knockout (KO) mice specifically lacking this isoform (Schneider et al., 2009). Male mGPx4-KO mice were infertile due to severe structural abnormalities in mature sperm, in good agreement with the abundant localization of mGPx4 in sperm tail of wild-type (WT) mice (Godeas et al., 1997), where this enzyme is inactive and forms the mitochondrial capsule by crosslinking with itself and other proteins (Ursini et al., 1999). Interestingly, the sterility of male mGPx4 –KO mice was not associated with apparent histological defects at the level of seminiferous epithelium in the testis, providing direct and conclusive evidence that the mitochondrial form is the key selenium-dependent molecule in male fertility (Schneider et al., 2009). The in vivo function of the cytosolic form of GPx4 was investigated using transgenic mice that overexpressed this isoform. In contrast with the early embryonic lethality of null mice for the whole Gpx4, mice specifically overexpressing cGPx4 but lacking the mitochondrial and nuclear ones (Liang et al., 2009) were viable, revealing that the cytosolic form of GPx4 plays a crucial role for embryonic survival and development. As expected and in agreement with the results of Schneider (Schneider et al., 2009), male sterility associated with marked sperm defects was also observed in these mice, due to mGPx4 deficiency.

3 Concerning the nuclear GPx4 form, studies reported so far have highlighted an apparent dispensability for male fertility and development in the mouse, reinforcing the concept that GPx4 isoforms play distinct and independent roles. Nevertheless a possible involvement of nGPx4 in sperm chromatin stability has been proposed on the basis that sperm isolated from the caput epididymis of KO mice specifically lacking nGPx4 showed defective chromatin condensation that, surprisingly, was not evident in sperm from the cauda epididymis (Conrad et al., 2005). Furthermore, the study of nGPx4-KO mice brought into light an interesting feature of nGPx4 related to the catalytic action as a thiol peroxidase. In fact, higher levels of free protein thiols were observed in spermatozoa obtained from the cauda epididymis of nGPx4-/- mice compared to WT mice. In keeping with these data, sperm from rats maintained under selenium deficient diet displayed both head and tail abnormalities and incompletely condensed nuclei (Pfeifer et al., 2001; Watanabe and Endo, 1991). However, no data are currently available on the subnuclear compartment (s) in which nGPx4 is localized in differentiating male germ cells and sperm, nor has it been solved which specific function (s) it may regulate. More importantly it has remained unclear whether defective chromatin condensation in sperm of nGPx4-deficient mice is limited to immature spermatozoa from caput epididymis and if the lack of nGPx4 affects paternal chromatin decondensation at fertilization. In the present study, we have addressed these fundamental questions and report here that nGPx4 is localized to the nuclear matrix of testicular haploid germ cells and epididymal sperm. We also show that spermatozoa from both caput and cauda epididymis of nGPx4-KO mice decondense earlier than those from WT mice both in vitro and following gamete fusion at fertilization, providing the first direct functional evidence that this selenoprotein is required for the structural integrity of mammalian sperm chromatin.

Materials and Methods

Animals and Genotyping

C57BL/6J and CD1 mice (Charles River Laboratories, Italy) and single/double knock-out C57Bl/6J mice specifically lacking nGPx4 were used in this study. The nGPx4 colony was maintained by intercrossing both nGPx4+/- and nGPx4 -/- mice. Genotyping of mice was performed by polymerase chain reaction (PCR) of genomic DNA extracted from tail biopsies, using the following primer pairs: I1f2/Earev1,5’-TCGGCGG CGCCTTGGCTACCGGCTC-3’ and 5’- GGATCCGCCGCGCTGTCTGCAGCGTCCC-3’, specific for the wild-type allele; and

4 I1f2/eGFPrev, 5’-TGAAGAAGTCGTGCTGCT TCATGTGG-3’, specific for the knock-out allele, as previously described (Conrad et al., 2005). All animals were housed in accordance with the Sapienza University guidelines for animal care and were sacrificed by asphyxia with CO2.

Germ cell preparation

Highly purified pachytene spermatocytes and round spermatids (steps 1–8) were obtained from 28/ 30 day-old mouse testes, as previously described (Boitani et al., 1980). Briefly, the cell suspension obtained by enzymatic digestion of testicular tissue was fractionated by velocity sedimentation at unit gravity on 0.5–3% bovine serum albumin (BSA) gradient. The purity of cell fractions was evaluated by flow cytometry and morphology of cytocentrifuged cell preparations (Puglisi et al., 2003). Round spermatids nuclei (Platz et al., 1977) were lysed, centrifuged and the pellet was resuspended in 5 mM MgCl2, 5 mM Na phosphate and then passed twice through a 25-gauge needle. Spermatozoa were collected from caput epididymis, cauda epididymis and vas deferens of adult mice by squeezing and mincing tissues in PBS. Released sperm were then washed twice in PBS by centrifugation at 1,000 g for 15 min. Nuclei of sonication-resistant testicular cell were prepared from adult mouse testes as described (Mayer, Jr. et al., 1981). Nearly pure ( >99%) elongated spermatid nuclei (steps 12-16) were obtained after sonication and purification on a discontinuous sucrose gradient. Isolated nuclei were spotted on polysine coated slides for further analysis.

Chromatin and nuclear matrix preparation

Chromatin and nuclear matrix fractions of male germ cells and in vitro transfected COS-1 cells were prepared by two different protocols depending on germ cell maturation stage. Protocol I: high salt isolation of nuclear matrix from spermatocytes, round spermatids and COS-1 cells was carried out as previously described (Reyes et al., 1997; Wu et al., 2000) with minor modifications. Cells were homogenized in cold CSK buffer (10 mM PIPES pH 6.8, 100 mM NaCl,

300 mM Sucrose, 3 mM MgCl2, 1 mM EGTA, supplemented with leupeptin, aprotinin and pepstatin, 1 mM PMSF, 1 mM DTT, 0.5% Triton X-100) for 3 min. The cytoskeletal framework was separated from soluble proteins and the chromatin fraction was then solubilized by digestion with 1 mg/ml of RNase-free DNase (Roche) for 15 min at 37°C. The pellet obtained from 0.25 M ammonium sulphate precipitation was extracted with 2 M NaCl for 5 min at 4 °C and then

5 centrifuged. The remaining pellet (the nuclear matrix fraction) was solubilized in urea buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris pH 8.0) for SDS-PAGE analysis. Protocol II: sonication resistant spermatids (SRS) and epididymal spermatozoa were resuspended and vortexed in 0.5% ATAB, 2 mM DTT, 0.5 mM PMSF as previously described (Ward et al., 1999). Sperm heads were isolated by layering the cell suspension on 1 M sucrose cushion and centrifuging at 3,000g for 10 min. Prior to biochemical analysis, SRS and sperm nuclei were resuspended in 2 M NaCl, 25 mM Tris, pH 7.4, with 2 mM DTT and incubated for 25 min for protamines removal, followed by 2 h incubation with DNase I (100.000 U/ml) at 37°C as previously described (Shaman et al., 2007). For in situ isolation of nuclear matrix, round spermatid nuclei and COS-1 cells were cytocentrifuged on polysine coated slides and subjected to in situ extraction with protocol I described above. SRS nuclei and sperm heads were spotted on polysine coated slides and extracted directly with protocol II. Nuclear matrices were fixed with 4% paraformaldehyde (PFA) for 10 min at 4°C for immunofluorescence analysis.

DNA constructs

The FLAG-tagged nGPx4 expression construct was prepared by cloning the full-length nGPx4 cDNA sequence generated by RT-PCR from mouse testis RNA, in frame with 5’ end FLAG epitope coding sequences of the pFLAG-CMVTM-2 vector (Sigma) by using Hind III and Xba I restriction sites. Nucleotide sequencing of the nGPx4 construct demonstrated 100% identity to that of the nGPx4 reference sequence AB072797.1 (Supplementary Fig. 1A). The full-length mGPx4 cDNA sequence was generated by RT-PCR from mouse testis RNA and cloned into pCMV-vector by using Hind III and Xba I restriction sites. Nucleotide sequencing of the mGPx4 cDNA demonstrated 100% identity to that of the mGPx4 reference sequence AF274027 (Supplementary Fig. 1B).

Cell culture and transfection

COS-1 cells were grown at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium with high glucose, supplemented with 10% fetal bovine serum and penicillin-streptomycin. For transient expression experiments, cells were seeded at a density of 7 x 106 cells in 100-mm Petri dishes one day before transfection. Cells were transfected with 14 µg pCMV-Flag-nGPx4 vector using 35 µl lipofectamine reagent (LipofectamineTM 2000, Invitrogen) according to manifacturer’s recommendations. After 6 h incubation, the transfection medium was replaced by the growth

6 medium without antibiotics and supplemented with Na2SeO3 (50 nM final concentration) for 48-72 h.

Generation and assessment of anti-nGPx4 polyclonal antibody specificity

Three peptides [arkrgrcrqrggspr, rgpgrqsprkrpgpr, pgpllqeppqyc] located in the mouse nGPx4 amino-terminal sequence were conjugated to keyhole limpet hemocyanin and used to immunize rabbits by subcutaneous injection (produced by Primm srl). Polyclonal antibody specificity was determined by western blot analysis of protein extracts from: (1) COS-1 cells expressing the nuclear isoform (transfected with pCMV-Flag-nGPx4); (2) COS-1 cells expressing the mitochondrial isoform (transfected with pCMV-mGPx4); (3) mock-transfected COS-1 cells (transfected with pCMV empty vector) ; (4) isolated pachytene spermatocytes, which physiologically express mGPx4 and cGPx4, but not nGPx4 (Puglisi et al., 2003) and (5) adult mouse testis, containing all GPx4 isoforms. Blots were incubated with either the primary antibody (1:100) raised against nGPx4 (anti- nGPx4) or a primary antibody (1:20) recognizing the three GPx4 isoforms (anti–GPx4), kindly provided by Drs F. Tramer and E. Panfili, (University of Trieste, Italy). Results shown in Supplementary Fig. 2 demonstrate that the anti-nGPx4 antibody revealed the presence of a 34 kDa (the expected nGPx4 MW), but not the 20 kDa band (the expected mGPx4 MW) in nGPx4- transfected COS-1 cells and in mouse testis. In contrast, the anti-GPx4 antibody revealed the presence of the 34 kDa band in nGPx4-transfected COS-1 cells and the 20 kDa in both pachytene spermatocytes and mGPx4-transfected COS-1 cells, as expected. Mock-transfected COS-1 cells were negative. Blot treatment with preimmune sera did not reveal significant 34 kDa and/or 20 kDa signals with all protein extracts.

Immunofluorescence analysis

After fixation and washing with PBS, cells were incubated for 30 min in 5% BSA or Donkey Serum (Invitrogen) and 0.1% Triton X-100. Samples were then treated for 1 h at room temperature (RT) with the following primary antibodies: rabbit anti-nGPx4 (1:100; Primm); rabbit anti-GPx4 (1:20; rabbit anti-topoisomerase IIȕ (1:100, Santa Cruz); rabbit anti-Flag (1:300; Sigma); mouse monoclonal anti-Flag (1:50; Sigma), goat anti-lamin B (1:50, Santa Cruz) and mouse anti-acrosin (1:200; Biosonda). Cells were carefully washed with PBS and then incubated for 1 h at RT with the appropriate secondary antibody diluted in PBS containing 1% BSA and 0.1% Triton X-100: anti- rabbit Alexa Fluor 488-conjugated antibody (1:400, Molecular Probes); anti-rabbit Alexa Fluor 555-conjugated antibody (1:500, Molecular Probes); anti-goat Alexa Fluor 555-conjugated antibody

7 (1:1000, Molecular Probes) and anti-mouse Cy3-conjugated antibody (1:1000, Jackson ImmunoResearch). TO-PRO-3 (Molecular Probes, 1:1000 ) was used to stain DNA. Slides were mounted with Vectashield (Vector Laboratories) and analysed under a Zeiss optical fluorescence microscope and a Leica confocal microscope.

Western blot analysis

Protein quantification was performed by the bicinchoninic acid method (Pierce Chemical Co.) using albumin as standard. Otherwise proteins were loaded per cell number. Proteins were fractionated by 12.5% SDS-PAGE (Laemmli, 1970) and then transferred onto a nitrocellulose membrane (Hybond- C extra, Amersham Pharmacia). Blotted membranes were incubated for 1 h at RT in 5% nonfat dry milk and 0.1% Tween 20 with the following primary antibodies: rabbit anti-nGPx4 (1:100; Primm), rabbit anti-GPx4 (1:1000), rabbit anti-Flag (1:2000, Sigma), goat anti-lamin B (1:250, Santa Cruz) and rabbit anti-histone H3 (1:5000, Sigma). After several washes, filters were incubated with the appropriate secondary antibody: anti-rabbit horseradish peroxidase-conjugated (1:3000, Zymed), anti-mouse peroxidase-conjugated (1:1000, DAKO) and anti-goat peroxidase conjugated (1:5000, Santa Cruz). Bands were visualized by an enhanced chemiluminescence system (Amersham Pharmacia) according to manufacturer’s recommendations. The biotin goat anti-rabbit IgG diluted 1:5000 (Zymed) with avidin-biotinylated horseradish peroxidase (Vectastain) and DAB colorimetric system (Amersham, Pharmacia) was used according to the manufacturer’s instructions. For protamine 1 detection, proteins were fractionated by 18% acid-urea polyacrylamide gel electrophoresis (AU-PAGE) (Meistrich, 1989), transferred onto polyvinylidene fluoride membrane (Immobilon PSQ, Millipore) and then stained with goat anti-protamine 1 antibody (1:150, Santa Cruz) followed by the anti-goat peroxidase conjugated antibody.

In vitro sperm nuclear decondensation assay

Spermatozoa separately collected from caput and cauda epididymis of adult nGPx4+/+, nGPx4+/- and nGPx4-/- mice were dispersed in T6 medium (Quinn et al., 1982) and then capacitated in vitro by incubation at 37 °C for 1.5 h. Nuclei decondensation was obtained by incubating cells in the presence of 5 mM glutathione (GSH) and 10 µM heparin in T6 medium supplemented with 1% Triton X-100 at 37°C for increasing times (15, 30, 50, 80 and 100 min) (Romanato et al., 2003). Controls consisted of parallel incubations with medium with 1% Triton X-100 and supplemented with heparin or GSH alone. At each time period, a sperm aliquot was removed, fixed with 4% PFA in PBS for 10 min at 4°C, cytocentrifuged on polysine coated slides and analysed by phase-

8 contrast/fluorescence microscopy after DNA staining with Hoechst 33342. Spermatozoa were classified in four classes of decondensation: no decondensation, low, medium and high decondensation, according to nucleus refringency, granular aspect and size. Experiments were run in triplicates and at least 200 cells were evaluated in each sample, by expressing frequency distribution into different decondensation classes as percentage of total cells analyzed.

Oocyte collection and in vitro fertilization

4-8 week-old female CD1 mice were induced to superovulate by intraperitoneal injections of 5 IU PMSG, followed by 5 IU hCG 46 h later. Females were sacrificed the next morning at 9:00. Metaphase II oocytes were freed from zona pellucida by treatment with Tyrode solution. After washing in HTF medium, oocytes were stained with Hoechst for 30 min at 37°C and sperm were added to a final concentration of 1x105 cells /ml. Sperm were obtained from WT and nGPx4-/- mice of 6-20 weeks. The cauda epididymides were minced in 1 ml of HTF medium and were left for 30 min at 37°C to allow swimming-out. Hereafter the sperm suspension of 2 caudae was capacitated and 10 µl added to oocytes. Embryos were washed after 1 h incubation at 37°C and fixed with 2% PFA in M2 medium, whole mounted and analysed under Zeiss microscope to monitor changes of decondensing sperm nucleus. Sperm numbers for each class of morphology were expressed as percentage of total cells examined in five separate experiments.

Results nGPx4 is associated with the nuclear matrix in haploid male germ cells

Because it had been previously proposed that nGPx4 is implicated in late spermatid nucleus formation (Pfeifer et al., 2001), we initially investigated the subnuclear localization of this enzyme in differentiating male germ cells and sperm. Pachytene spermatocytes and round spermatids were isolated from mouse testis and used as a source of chromatin and nuclear matrix protein fractions. The purity of subnuclear fractions was assessed by western blotting, using lamin B and histone H3 as markers of nuclear matrix and chromatin, respectively. In round spermatids, nGPx4 appeared to be specifically associated with nuclear matrix and absent from chromatin fraction (Fig. 1A). In contrast, no nGPx4 band was apparent in both fractions from spermatocyte nuclei (Fig. 1A), in agreement with our previous conclusion that nGPx4 is not expressed in meiotic germ cells (Puglisi et al., 2003). We next examined whether nGPx4 association with the nuclear matrix was also a

9 feature of epididymal spermatozoa (Fig. 1B). In this case nGPx4 was indeed associated with the nuclear matrix, being represented not only by the canonical 34 kDa band but also by a lower MW band(s), as previously described (Bertelsmann et al., 2007; Maiorino et al., 2005), while the chromatin fraction, identified by the presence of protamine 1, had none. To further characterize the subnuclear compartment(s) where nGPx4 is actually localized during spermiogenesis and epididymal sperm maturation, round spermatids (steps 1-8), sonication- resistant spermatids (namely late elongating spermatids, steps 12-16) and epididymal spermatozoa were spotted on a slide and then subjected to in situ sequential removal of DNA-binding proteins and DNA, resulting in full nuclear matrix unmasking. In situ preparations of nuclear matrices were then stained with an antibody specific for nGPx4, using lamin B and topoisomerase IIȕ as markers in round spermatids and late-condensing spermatids and spermatozoa, respectively (Fig. 2) to validate the preparation. nGPx4 was apparently localized to the nuclear matrix in all germ cell types analyzed, in full agreement with biochemical analyses described above. nGPx4 immunofluorescence was apparently lacking in the nuclear matrix of sperm from nGPx4-/- mice (data not shown), further indicating immmunostaining specificity. We therefore conclude that nGPx4 is a constant component of nuclear matrix and/or nuclear matrix-associated proteins throughout the complex series of changes in nuclear protein rearrangement and aggregation during spermiogenesis and sperm transit along epididymis.

Besides the nuclear matrix, nGPx4 is localized at the level of acrosome

To better characterize the subnuclear distribution of nGPx4 in sperm head, epididymal spermatozoa were analyzed by immunofluorescence at various steps of in situ nuclear component extraction (Supplementary Fig. 3). Untreated sperm displayed the presence of a thin and crescent- like nGPx4 positivity in the head apical pole, which became more evident after in situ sperm treatment with ATAB-DTT. The crescent-like labelling was absent in epididymal sperm from nGPx4-KO mice (not shown), indicating immunostaining specificity. Because nGPx4 localization at the apical pole of sperm head was reminiscent of acrosome and in light of previous observations of GPx4 presence in the acrosome (Haraguchi et al., 2003; Schneider et al., 2009; Tramer et al., 2002), we double-stained sperm with antibodies against nGPx4 and acrosin, an established marker of this sperm head compartment. Confocal microscopy analysis showed that nGPx4 largely colocalized with acrosin, indicating that nGPx4 is an acrosome component (Fig. 3A). Similar results were also obtained with other acrosome markers, including peanut agglutinin lectin (PNA) (not shown). However, when sperm were subjected to a number of treatments that solubilize

10 acrosomal proteins, including the exposure to detergents, sonication, in vitro capacitation and in vitro acrosome reaction, the crescent-like immunostaining appeared not to be significantly affected by the treatments (Fig. 3B), suggesting that acrosomal nGPx4 is not soluble, but stably associated with insoluble sperm structure(s) as acrosomal and/or perinuclear matrices. Detailed immunofluorescence analysis carried out at different steps of nuclear matrix extraction procedure using either anti-nGPx4 or anti-GPx4 antibodies (Supplementary Fig. 3) revealed that the crescent- like nGPx4 signal apparently disappeared after high-salt protamine extraction step. Because protamine extraction causes sperm nuclei to swell by extruding DNA as highly extended loop domains arranged around the nuclear matrix, it appeared that the enzyme was masked by the DNA halo at this procedure step. In fact the crescent-like immunostaining appeared again when sperm DNA was digested with DNase, a treatment that also unmasked the nuclear matrix-associated nGPx4 (Supplementary Fig. 3), in agreement with findings described for nuclear matrix preparation.

nGPx4 specifically associates with the nuclear matrix in transfected COS-1 cells

To study whether the nGPx4 targeting to nuclear matrix is a property of this protein per se or it requires a cell machinery specific to male germ cells, we overexpressed the Flag-nGPx4 DNA construct in COS-1 cells and then determined the presence of nGPx4 in the nuclear matrix by western blotting (Fig. 4A) and immunofluorescence (Fig. 4B), using lamin B and histone H3 as markers of nuclear matrix and chromatin, respectively. Results of both analyses showed that Flag- nGPx4 localized to the nuclear matrix together with lamin B also in this heterologous cell system, with features similar to those previously observed in mouse spermatids/spermatozoa. We therefore conclude that the process of nGPx4 targeting to nuclear matrix is independent of male germ cell- specific factors. nGPx4 is required for chromatin condensation in sperm from cauda epididymis

It was previously proposed that nGPx4 is involved in sperm chromatin stability (Conrad et al., 2005; Pfeifer et al., 2001). To gain more insight into this issue we initially compared the nuclear decondensation kinetics of sperm isolated from nGPx4+/+, nGPx4+/- and nGPx4-/- mice, by using an in vitro functional assay based on sperm incubation in the presence of both glutathione (the most abundant thiol group donor of mammalian egg (Sutovsky and Schatten, 1997) and heparin (a

11 glycosaminoglycan acting as protamine acceptor) (Romanato et al., 2003; Romanato et al., 2005). We classified sperm nucleus into four arbitrary classes of nuclear decondensation degree, namely none, low, medium and high, according to the change in nuclear morphology observed at increasing incubation times in the presence of decondensing reagents (Fig. 5A). In our assay, the addition of either glutathione or heparin alone resulted in sperm head inability to decondense, as previously described (Romanato et al., 2003; Romanato et al., 2005). In contrast, more than 50 % of sperm isolated from the caput epididymis of nGPx4-/- mice achieved the highest level of decondensation within 30 min of incubation under decondensing conditions, whereas most of sperm from nGPx4+/+ and nGPx4+/- mice were still at the very beginning of chromatin dispersion (Fig. 5B). These results indicate that nGPx4-deficient sperm from caput epididymis had a level of chromatin compaction lower than that of WT sperm, in agreement with previous observations based on sperm staining with acridine orange and toluidine blue (Conrad et al., 2005). Interestingly, we also found that sperm from the cauda epididymis of nGPx4-/- mice decondensed their chromatin faster than wild-type sperm, although with a kinetics slower than that of nGPx4- deficient caput epididymis sperm. In fact in our assay, approximately half of nGPx4-deficient cauda epididymis sperm were either fully or partially decondensed after 50 min incubation, while only a small fraction of cauda epididymis sperm from WT/heterozygous mice had progressed up to the initial dispersion degree, about 75 % of them still being fully condensed (Fig. 5C). Our findings demonstrate that the lack of nGPx4 causes a defective chromatin compaction at all stages of epididymal maturation. These results prompted us to analyze sperm nuclear decondensation by a more physiological approach. To this purpose we performed in vitro fertilization experiments aimed at comparing the paternal nucleus ability of wild-type and nGPx4-defective sperm to decondense inside the oocyte after gamete fusion. In these experiments, metaphase II oocytes from superovulated female mice were first pre-loaded with the DNA stain Hoechst 33342 (allowing to directly visualize the sperm-egg fusion and the stage of sperm head decondensation after fertilization, as previously performed) (Conover and Gwatkin, 1988; Tatone et al., 1994)), and were then deprived of the zona pellucida allowing sperm-egg fusion to occur in a highly synchronous manner in all fertilized eggs (Tatone et al., 1994). Fused sperm morphology was analysed 1 h after insemination and classified into four arbitrary classes of sperm head decondensation (Fig. 6). The frequency of partially/fully decondensed heads was significantly higher in sperm from nGPx4 -/- than from nGPx4+/+ mice, indicating that nuclei of nGPx4-deficient sperm decondensed significantly faster than those of wild-type sperm, in full agreement with the data obtained by the in vitro decondensation assay. We therefore conclude that the lack of nGPx4

12 during spermiogenesis and sperm epididymal transit causes a sperm chromatin instability that in turn accelerates the kinetics of paternal chromatin decondensation at fertilization.

Discussion

This study focused on the characterization of subnuclear localization and functional significance of the nuclear form of GPx4 in mouse sperm. Two main findings emerge from our data. First, nGPx4 is associated with the nuclear matrix in haploid male germ cells from round spermatids to epididymal sperm; and second, a lack of nGPx4 function during sperm chromatin condensation affects paternal genome decondensation at fertilization. Our results provide new insights into the subnuclear location of nGPx4. In fact previous immunogold labelling at the electron microscopy level showed that, besides mitochondria, GPx4 is associated with a fibrous material within the nucleus (Haraguchi et al., 2003; Tramer et al., 2002). Present biochemical, immunofluorescence and in situ nuclear protein extraction-DNA digestion analyses have conclusively (i) identified the GPx4 variant in the nucleus as the nGPx4 isoform; and (ii) demonstrated that this isoform is actually associated with the nuclear matrix, but not chromatin, throughout the haploid phase of spermatogenesis, namely from round spermatids to cauda epididymis sperm. The temporal overlapping between nGPx4 presence in the nucleus and the process of sperm chromatin condensation during spermiogenesis and passage through the epididymis strongly suggests that, along with protamines, nGPx4 is directly involved in this process. Indeed, nGPx4 contains several cysteines and is able to react with protamine thiols and with itself to form polymers under oxidizing conditions in the absence of GSH (Mauri et al., 2003). In this context it is noteworthy that nGPx4 is absent in sperm of animal species, such as trout and chicken, that do not contain cysteines in their protamines (Bertelsmann et al., 2007). On the basis of its properties and in analogy with the mitochondrial GPx4, which is responsible for crosslinking proteins in the mitochondrial capsule of spermatozoa (Ursini et al., 1999), it was proposed that nGPx4 contributes to thiol oxidation during sperm chromatin condensation, although a role of cGPx4 in this process cannot be ruled out (Conrad et al., 2005). Our findings showing that nGPx4 is specifically associated with nuclear matrix strengthen the concept that, having a part in disulfide bond formation, this isoform plays a scaffolding role relevant to the structural integrity of sperm nuclear architecture and similar to the functions played by topoisomerase II and lamin B as specific components of nuclear matrix substructures. Furthermore, it is generally accepted that the nuclear matrix/scaffold serves both organizational and dynamic roles. Transcriptional factors were

13 demonstrated to be associated with the nuclear matrix in a cell-specific context (Zeng et al., 1997), and the specific depletion of lamin B1 in Hela cells resulted in RNA synthesis inhibition and altered location of chromosomes (Tang et al., 2008). Because a proper sperm chromatin packaging and a specific gene cluster conformation during spermiogenesis and epididymal maturation are linked to paternal genome imprinting and the transmission of epigenetic information to the embryo (Brykczynska et al., 2010; Hammoud et al., 2009), it is tempting to hypothesize that a lack or a functional/structural defect in nGPx4 is relevant to sperm quality and embryo development. In vivo evidence has given strong support to the concept that nGPx4 promotes nuclear stability in sperm. Sperm of selenium-deficient rats displayed an altered head morphology associated with defective nuclear condensation (Pfeifer et al., 2001; Watanabe and Endo, 1991). Furthermore, the lack of nGPx4 in homozygous KO mice for this isoform resulted in a chromatin status more susceptible to dispersion in sperm isolated from caput epididymis, although sperm from the cauda epididymis of those mice unexpectedly appeared normally condensed (Conrad et al., 2005). In that study, however, sperm chromatin condensation was assayed by experimental approaches that may be not sensitive enough to reveal subtle changes in chromatin condensation during sperm maturation. Our findings based on a quantitative evaluation of epididymal sperm chromatin condensation have overcome that inconsistency by providing conclusive evidence that cauda epididymis spermatozoa lacking nGPx4 actually display a chromatin structure less stable and more prone to decondense than that of wild-type ones. In agreement with this conclusion, an interesting result of this study was the precocious nuclear decondensation of nGPx4 KO sperm at fertilization. This finding is consistent with the accelerated timing of nuclear disassembly observed using the in vitro assay with the mixture of glutathione and heparin, in which each of the reagents were inefficient when present alone in the incubation buffer. Indeed, the sperm decondensing ability of mammalian oocytes has been shown to be assured by the presence of both reduced GSH and heparan sulfate in the egg, being the disulfide bond reduction by GSH necessary but not sufficient for decondensation to occur in vivo (Perreault et al., 1984; Perreault et al., 1988; Romanato et al., 2005; Romanato et al., 2008; Sutovsky and Schatten, 1997). The nuclear decondensation is the first event the spermatozoon undergoes in the fertilized oocyte and is an essential prerequisite to male pronucleus formation. During this phase, the paternal chromatin remodelling occurs independently from the maternal nucleus (McLay and Clarke, 2003; van der Heijden et al., 2005), determining the ability of the male genome to participate in the formation of the diploid zygote. Our finding that in the absence of nGPx4 sperm nuclei undergo a more rapid decondensation inside the oocyte indicates that this selenoprotein is likely to be related to sperm chromatin remodelling and early embryo

14 development. In this context, it is of particular interest that nGPx4 is localized to the nuclear matrix of the male gamete. In fact, there is substantial evidence that this functional complex with specialized domains within the sperm nucleus plays a role in the formation of male pronucleus and subsequent paternal DNA replication (Johnson et al., 2011; Shaman et al., 2007; Ward et al., 1999; Ward, 2010; Yamauchi et al., 2007). Another finding of this study is the association of nGPx4 with the acrosome. Although this evidence confirms previous data and needs to be further substantiated (Haraguchi et al., 2003; Schneider et al., 2009; Tramer et al., 2002)), the observation that nGPx4 immunostaining persisted even after a variety of extraction conditions and treatments points to the idea that nGPx4 is an insoluble protein, and part of the particulate components of the acrosome that are biochemically defined as “acrosomal matrix” (Buffone et al., 2008). As such it is likely that nGpx4 plays a structural role also in this subcellular compartment of the spermatozoon, as it does when it is associated with the nuclear matrix. This result raises the possibility of another nGPx4 function specific to this localization in the fertilization process.

Acknowledgments

We are grateful to Prof.s Mario Stefanini and Maria Teresa Fiorenza for helpful discussion and advise. We are indebted to Prof.s Federica Tramer and Enrico Panfili for the generous gift of the anti-GPx4 antibody. We also thank Ms Tiziana Menna for her technical assistance and Ms Stefania De Grossi for her valuable support of the confocal microscopy analysis. This work was supported by grants from the Italian Ministry of Education and European Union to CB, and Institute Pasteur Cenci Bolognetti to FM.

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18 Figure Legends

Fig. 1. nGPx4 localizes to the nuclear matrix of round spermatids and spermatozoa. (A)Western blot analysis of nGPx4 presence in chromatin (Ch) and nuclear matrix (NM) fractions obtained from pachytene spermatocytes (PS) and round spermatids (RS). Filters were immunoblotted with the anti-nGPx4 antibody. The purity of the fractions was assessed by using lamin B and histone H3 as markers of nuclear matrix and chromatin, respectively. (B) Western blot analysis of subnuclear fractions from epididymal spermatozoa (SPZ). Chromatin and nuclear matrix proteins were fractionated by both SDS-PAGE and AU-PAGE and immunoblotted with anti-nGPx4 and anti- protamine 1 antibodies, respectively. Fraction purity was confirmed by the presence of protamine 1 in the chromatin, but not the nuclear matrix, fraction.

Fig. 2. Immunofluorescence analysis of nGPx4 in the nuclear matrix of round spermatids (steps 1-8, RS), sonication resistant spermatids (steps 12-16, SRS) and epididymal spermatozoa (SPZ). Nuclear matrices were prepared by in situ detergent/high salt extraction and DNase digestion as described under Materials and Methods, immunostained, counterstained with TO-PRO-3, and analysed by confocal microscopy. Note that nGPx4 (green) localizes to the nuclear matrix of haploid germ cells at all maturation stages. We used lamin B and topoisomerase IIȕ (TOPOIIȕ) (red) in RS and in SRS/SPZ, respectively, to assess the accuracy of nuclear matrix preparation. The removal of DNA was confirmed by the lack of any TO-PRO-3 staining (blue). In SRS and SPZ, the double staining with anti-nGPx4 and anti-TopoIIȕ was not possible, because both antibodies had been generated in rabbit. (Scale bars: 10 Pm)

Fig. 3. nGPx4 associates with an additional compartment in sperm head. Confocal microscopy analysis of epididymal spermatozoa. (A) Double staining with anti-nGPx4 (green) and anti-acrosin (red) antibodies, in combination with TO-PRO-3 (blue). The co- localization of nGPX4 with acrosin at the apical pole of sperm heads indicates that a fraction of nGPx4 is a component of the acrosome. (Scale bars: 10 Pm). (B) nGPx4 immunoreactivity (green) and TO-PRO-3 (blue) after sperm had been subjected to: a) 1% Triton X-100 treatment for 30 min at RT; b) sonication for 5 min (30 sec pulse, 15 sec pause, amplitude 40%); c) in vitro capacitation for 1.5 h at 37 °C in T6 medium; d) capacitation and then to 1.9 µM Ca 2+ionophore A 23187 treatment for 30 min at 37 °C to induce acrosome reaction. The staining pattern was retained in all conditions and similar to that of untreated sperm. (Scale bars: 5 µm )

19 Fig. 4. Analysis of subnuclear localization of nGPx4 in transfected COS-1 cells. (A) Western blot analysis of chromatin (Ch) and nuclear matrix (NM) fractions obtained from COS-1 cells transfected with pCMV-Flag-nGPx4 plasmid or the pCMV empty vector. Fractions were analysed by SDS-PAGE, followed by filter immunostaining with antibodies recognizing Flag, lamin B and histone H3. Similarly to germ cells, Flag-nGPx4 was detected as a 34 kDa band in the nuclear matrix, but not the chromatin, fraction. (B) Confocal microscopy analysis of the nuclear matrix isolated from COS-1 cells transfected with pCMV-Flag-nGPx4. Cells were spotted on a slide and subjected to in situ extraction and DNA digestion as described above for germ cells and finally triple-stained with anti-Flag antibody (green), anti-lamin B antibody (red) and TO-PRO-3 (blue). Note the presence of nGPx4 at the level of nuclear matrix as assessed by the positivity for lamin B. (Scale bars: 20 Pm)

Fig. 5. The lack of nGPx4 impairs chromatin condensation in sperm from caput and cauda epididymis. Sperm isolated from either the caput or the cauda epididymis were incubated for increasing times in the presence of a combination of GSH and heparin to induce nuclear decondensation, as described under Materials and Methods. (A) Morphological changes were monitored under phase-contrast microscopy and four classes were identified: =no decondensation, = low decondensation, = medium decondensation, = high decondensation. (B) and (C) Decondensation kinetics of capacitated spermatozoa isolated from caput (B) and cauda (C) epididymis of wild type (+/+), heterozygous (+/-) and homozygous(-/-) nGPx4 knock-out mice. Sperm numbers for each class of decondensation morphology are expressed as percentage of total cells examined in three separate experiments. (Scale bars: 10 Pm).

Fig. 6. Decondensation of cauda epididymal sperm from wild-type (WT) and nGPx4 knock-out (KO) mice 1 h after fertilization of zona pellucida free oocytes. (A) Sperm nuclear decondensation status analysed under optical fluorescence microscope after DNA staining with Hoechst. = no decondensation, = low decondensation, = medium decondensation, = high decondensation. (B) Decondensation of WT and homozygous nGPx4 KO mice, 1 h after oocyte fertilization. KO sperm nuclei decondense faster than WT counterparts. Sperm numbers for each class of decondensation morphology are expressed as percentage of total cells examined in five separate experiments. Frequencies of four classes of sperm morphology from WT mice are significantly different from those from KO at p<0.0001, as assessed by the chi-square test for frequency data. (Scale bars: 10 Pm)

20 Supplementary Fig. 1 Schematic representation of the constructs used to transfect COS-1 cells. (A) pCMV-Flag-nGPx4 construct; the full-length nGPx4 cDNA sequence was fused with a Flag epitope and cloned into the pCMV vector as described under Materials and Methods. (B) pCMV-mGPx4 construct; the full-length mGPx4 cDNA sequence was cloned into pCMV- vector.

Supplementary Fig. 2 Characterization of the nGPx4-specific antibody. Western blot analysis of proteins extracted from transfected COS-1 cells and mouse testis/germ cells using the following primary polyclonal antibodies: anti-GPx4 antibody (anti- GPx4) recognizing the three GPx4 forms and the anti-nGPx4 antibody (anti-nGPx4) specific for the nuclear form of GPx4. COS-1 cells were transfected with pCMV-Flag-nGPx4/pCMV- mGPx4 constructs, or mock-transfected with pCMV empty vector as described under Materials and Methods, according to manufacturer’s recommendations. Protein fractions from pachytene spermatocytes and adult mouse testis were prepared as described previously (Puglisi et al., 2007). A: pachytene spermatocytes; B: cells transfected with pCMV empty vector; C: cells transfected with pCMV-Flag-nGPx4 construct; D: cells transfected with pCMV-mGPx4 construct; E: adult mouse testis.

21 Supplementary Fig. 3 Immunofluorescence analysis of nGPx4 in epididymal spermatozoa subjected to in situ sequential extraction. Sperm heads were treated with ATAB/DTT and then cytocentrifuged on polysine coated slides. Cells were in situ extracted with 2M NaCl/DTT followed by DNase digestion. At each step of the extraction immunostaining was performed using either the anti-nGPx4 antibody or anti-GPx4 antibody (green), in combination with TO-PRO-3 (blue). Phase-contrast microscopy analysis revealed the morphological changes of sperm heads following each step of the extraction. Note the swollen and the empty aspect after NaCl/DTT and DNase treatment, respectively. Confocal images showed that in untreated cells and after ATAB/DTT treatment nGPx4 signal was restricted to a marginal anterior region of sperm heads. After NaCl/DTT subsequent treatment no nGPx4 staining was apparent, being masked by DNA. Following DNA digestion by DNase (as confirmed by the absence of TO-PRO-3 staining) the nGPx4 localization to the nuclear matrix was revealed. Note that the nGPx4 distribution within the nucleus was similarly detected by both antibodies. The anti-GPx4 antibody recognizing all three Gpx4 isoforms also stained some residual sperm tails at all extraction steps. (Scale bars: 20 µm)

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