Purpose
To analyze the presence of various histone modifications in ejaculated human spermatozoa
Methods
In this prospective study, seminal ejaculates from 39 normozoospermic individuals were evaluated for semen analysis and the presence of histone modifications in isolated nuclei.
Results
We observed heterogeneous presence of histone methylation in normal mature human sperm. We observed that 12 to 30 % of the nuclei of normal sperm contain a heterogeneous distribution of the marks H3K4Me1, H3K9Me2, H3K4Me3, H3K79Me2, and H3K36Me3. Moreover, the presence of these marks is higher in the poor motile fraction of the ejaculate, which is associated with poor morphology and functional quality. In contrast, we did not observe histone acetylation (H3K4Ac and H4K5Ac) in normal or abnormal mature human sperm
Conclusions
Defects in the process of spermatogenesis may alter the correct epigenetic programing in mature sperm. Further studies are required to evaluate the impact of these findings in human infertilityFil: la Spina, Florenza Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Romanato, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Brugo Olmedo, Santiago. Centro Médico Seremas; ArgentinaFil: de Vicentiis, Sabrina. Centro Médico Seremas; ArgentinaFil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Rivera, Rocío M.. University Of Missouri; Estados UnidosFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Brugo Olmedo, Santiago

Buffone, Mariano Gabriel

de Vicentiis, Sabrina

Julianelli, Vanina Laura

la Spina, Florenza Antonella

Rivera, Rocío M.

Romanato, Marina

Journal of Assisted Reproduction and Genetics

PubMed

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

EPIGENETICSHeterogeneous distribution of histone methylationin mature human spermFlorenza A. La Spina & Marina Romanato & Santiago Brugo-Olmedo &Sabrina De Vincentiis & Vanina Julianelli & Rocio M. Rivera & Mariano G. BuffoneReceived: 2 October 2013 /Accepted: 29 October 2013 /Published online: 13 November 2013# Springer Science+Business Media New York 2013AbstractPurpose To analyze the presence of various histone modifi-cations in ejaculated human spermatozoaMethods In this prospective study, seminal ejaculates from 39normozoospermic individuals were evaluated for semen anal-ysis and the presence of histone modifications in isolatednuclei.Results We observed heterogeneous presence of histone meth-ylation in normal mature human sperm. We observed that 12 to30 % of the nuclei of normal sperm contain a heterogeneousdistribution of the marks H3K4Me1, H3K9Me2, H3K4Me3,H3K79Me2, and H3K36Me3. Moreover, the presence of thesemarks is higher in the poor motile fraction of the ejaculate,which is associated with poor morphology and functionalquality. In contrast, we did not observe histone acetylation(H3K4Ac and H4K5Ac) in normal or abnormal mature humanspermConclusions Defects in the process of spermatogenesis mayalter the correct epigenetic programing in mature sperm.Further studies are required to evaluate the impact of thesefindings in human infertilityKeywords Epigenetic . Human sperm . Histonemodifications . Sperm subpopulationsIntroductionThe establishment and maintenance of the germ cells requires afine regulation of epigenetic marks starting in the primordialgerm cell population [1]. During spermiogenesis, most of thecanonical histones are exchanged for protamines that tightlypack DNA in mature sperm [2]. It was reported that about 4–15 % of the human sperm genome is packed as nucleosomes[3], consisting of canonical or variant histone proteins.Historically, their presence in mature sperm has been associatedto an insufficient replacement of histones by protamines with noimpact in future embryo development. However, recent evi-dence using high-resolution genomic approaches has clearlydemonstrated a programmatic retention in particular genes/locithat are important for embryo development [4]. This discoveryopens the possibility of other potential sources of male infertilitylargely unexplored. Hammoud and coworkers [5] recently re-ported that some alterations in histone retention and epigeneticmodifications at developmental and imprinted gene loci existedin the sperm of infertile men when compared to fertile donors.Most of the causes of male infertility still remain undiag-nosed or classified as idiopathic because of an incompleteevaluation of sperm function due to the lack of the appropriatetests. For example, the evaluation of the sperm nucleus andDNA is limited to the analysis of its protein composition(amount of protamines or histones) or the integrity of theDNA [6]. There are almost no reports in the literature thataddress the potential effects of sperm histone content and post-translational modifications on fertility and embryo develop-ment. Histone modifications are one of the most thoroughlystudied areas in epigenetics, mainly because of their impor-tance in transcriptional regulation of gene expression. Becauselittle is known about the presence and distribution of differenthistone post-translational modifications in ejaculated humansperm, we aimed to characterize this event in normal andabnormal spermatozoa.Capsule Histone modifications in human sperm.F. A. La Spina :M. Romanato :V. Julianelli :M. G. Buffone (*)Instituto de Biología y Medicina Experimental, CONICET,Vuelta de Obligado 2490, 1428 Buenos Aires, Argentinae-mail: mgbuffone@gmail.comS. Brugo-Olmedo : S. De VincentiisCentro Médico Seremas, Buenos Aires, ArgentinaR. M. RiveraAnimal Science Research Center, Division of Animal Sciences,University of Missouri, Columbia, MO, USAJ Assist Reprod Genet (2014) 31:45–49DOI 10.1007/s10815-013-0137-4Materials and methodsSemen specimens and sample processingNormozoospermic semen specimens according to WorldHealth Organization [7] were obtained from 39 normalhealthy volunteers after informed consent. The study protocolwas approved by the IBYME Institutional Review Board.Specimens were used solely for research and not for clinicalpurposes. Donor data were kept confidential. Samples werecollected by masturbation after 36–48 h of abstinence,allowed to liquefy at room temperature and processed within1 h of collection. For nuclei isolation, samples were washedtwice by centrifugation at 300 g for 10 min in human tubalfluid (HTF) and resuspended in 1 ml PBS. In other experi-ments, semen samples were loaded onto a 45 and 90 %discontinuous Isolate gradient (Irvine, USA) gradient, as pre-viously described [8]. The resulting interfaces between thelayers of 45 and 90 % (L45) and the 90 % pellet (L90) wereaspirated and transferred to separate tubes. Sperm suspensionswere then diluted with PBS and processed for nuclei isolation.In other sets of experiments, mouse germ cells, were isolatedas previously described [9]Sperm nuclei isolationHuman sperm nuclei were isolated according to the protocolmodified fromYebra and Oliva [10]. Semen was washed threetimes by centrifugation at 1600 g for 10 min in 50 mmol/lTris–HCl, pH 7.2, and 0.15 mol/l NaCl (10× sample volume).The sperm pellet was resuspended in 2.6 ml of the same buffercontaining 1 % SDS, incubated for 15 min at room tempera-ture and the spermatozoa sonicated (6×15 s at 200 W) with aBranson sonifier cell disruptor, model W140 (Branson SonicPower Co., Plainview, NY, USA). The sonicated cells wereseparated in two equal aliquots, each of which was placed ontop of 4 ml 1.1 mol/l sucrose in 50 mmol/l Tris–HCl, pH 7.2,and centrifuged at 3500 g for 1 h. Pellets were recovered andwashed twice by centrifugation at 1600 g for 10 min in50 mmol/l Tris–HCl, pH 7.2. Lack of contamination of thenuclear fraction obtained with sperm tails was tested by mi-croscopical observation.Immunocytochemistry of isolated sperm nucleiImmunocytochemistry was performed according to the proto-col described by Zahn and coworkers [11]. Isolated spermnuclei were washed in PBS and air-dyed onto a glass slide.Cells were fixed with 4 % paraformaldehyde in PBS during20 min and permeabilize with 0.1 % Triton X-100 during20 min at room temperature. Cells were then blocked with1% goat serum during 45min at room temperature. The slideswere incubated overnight at 4 °C with primary antibodiesagainst H3K4Me, H3K4Me3, H3K9Me2, H3K36Me3 (CellSignaling) H3K79Me2 and H3K4Ac (Active Motif) orH4K5Ac (Abcam), diluted 1:100 (50 mg/ml) in blockingbuffer in a humidified chamber. After washing tree times withPBS, slides were incubated with goat anti-rabbit IgG conju-gated with Alexa488 (Molecular Probes), diluted 1:300 inblocking buffer during 60 min at room temperature in ahumidified chamber. Following incubation, slides werewashed three times with PBS, air-dried and mounted withVectashield (Vector, USA). Sperm were examined using aconfocal microscope Nikon C1 (USA). At least 200 cells werecounted in different fields and the percentage of nuclei show-ing fluorescence was calculated. Negative controls were per-formed by omitting the primary antibody.Statistical analysisStatistical analysis was carried out by a paired Student’s t testafter the data were normalized by an arcsin transformationusing the GraphPad Prism program (GraphPad software). Alltests were two-tailed with statistical significance assessed atthe P <0.05 level.ResultsTo analyze histone post-translational modifications persistingin nucleosomes of mature human sperm, we performed im-munocytochemistry using isolated nuclei. All antibodies usedto immunolocalize the modifications (H3K4Me, H3K4Me3,H3K9Me2, H3K79Me2, H3K36Me3, H3K4Ac andH4K5Ac) were previously validated for specificity ofchromatin-associated staining using Hela cells (data notshown). After nuclei isolation and treatment with Triton X-100, it was not necessary to decondense sperm nuclei usingDTT or other agents to allow the antibodies to gain access tothe specific epitopes since by using immunofluorescenceanalysis we obtained similar results with or withoutdecondensation (not shown). As a control, we also validatedour procedure using anti-histone H3 and anti protamine 1 and2 which are well known components of the mature spermnuclei (data not shown).First, we evaluated the presence of histone acetylation(H3K4Ac and H4K5Ac) in ejaculated human sperm. In thisset of experiments, we did not observe histone acetylation inejaculated human sperm (Fig.1a). As a control, we performedimmunofluorescence using the aforementioned anti-acetylated histone antibodies in isolated mouse spermatids atdifferent stages of differentiation to validate our protocolbecause of the difficulties in obtaining these cells from humantestis. As observed by other authors, we observed histoneacetylation H4K5Ac in round spermatids but not in elongatedspermatids (Fig.1b).46 J Assist Reprod Genet (2014) 31:45–49Next, we analyzed the presence of histone methylation inmature human sperm (Fig.1a). Histone methylation is a well-characterized modification that has been associated with bothtranscriptionally permissive and restrictive chromatin config-urations. Surprisingly, we observed that 12 to 30 % of thenuclei in normal sperm contain a heterogeneous distributionFig. 1 a Immunofluorescence staining of human sperm nuclei for thedifferent histone marks assessed in this study. Right panel , bright fieldimages; left panel , fluorescence images. b Immunofluorescence usinganti-acetylated histone H4K5ac antibodies in isolated mouse round andelongated spermatids. In negative controls, the primary antibodies werereplaced by rabbit IgG. PS/RS pachytene spermatocyte/round spermatids,ES elongated spermatidFig. 2 Quantification of the percentage of human sperm nuclei withpositive staining for the different histone marks. Results are expressedas mean ± S.E.MFig. 3 Quantification of the percentage of human sperm nuclei withpositive staining for the different histone marks in the different subpop-ulations of isolated sperm after discontinuous gradient centrifugation(L45 and L90). Results are expressed as mean ± S.E.M. Asterisks denotesstatistical significance compared with L45 (P <0.05). (n =6)J Assist Reprod Genet (2014) 31:45–49 47of the marks H3K4Me1, H3K9Me2, H3K4Me3, H3K79Me2,and H3K36Me3 (Fig. 2).In order to evaluate whether the heterogeneous distributionof these histone marks in mature human sperm is associatedwith the functional and morphological quality of sperm, wenext performed a discontinuous gradient separation of sper-matozoa with high and low motility [7] and proceeded toperform immmunostaining for all the histone marks previous-ly mentioned. As shown in Fig. 3, the percentage of spermwith histone marks increased significantly (P <0.05) in thepopulation with low motility (L45) compared to the highmotility population (L90). As indicated above, we did notobserve histone acetylation in either sperm subset.DiscussionHistone modifications are one of the most thoroughly studiedareas in epigenetics due to their importance in transcriptionalregulation of gene expression. Normally, around 10–15 % ofthe nuclear proteins in mature human sperm are histones.Because certain subfertile or infertile individuals displayedabnormal levels of histones due to an abnormal protamine tohistone exchange, we decided to include only samples thatdisplayed normal levels of protamines and histones in theirnuclei as evaluated by aniline blue staining [8]. The composi-tion of the histone post-translational modifications that persistin the nucleosomes of mature human sperm is largely un-known. In this study we assessed the levels of several histonemethylations (H3K4Me, H3K4Me3, H3K9Me2, H3K79Me2,H3K36Me3) and acetylations (H3K4Ac and H4K5Ac) in nor-mal and abnormal human sperm. In this regard, we evaluate in agroup of normal individuals (according to the WHO criteria)the presence of these histone marks in isolated sperm nuclei.Histone acetylation occurs during spermiogenesis prior to theexchange of histone by protamines. The massive acetylationcreates a less stable chromatin that facilitates the accessibilityof proteins that participate in the exchange [12]. This transientacetylation is lost after completion of the exchange. Thus, wehypothesize that this massive histone acetylation is not presentin mature sperm but may remain present in those situationswhere the normal spermiogenesis process is perturbed resultingin the presence of abnormal cells in the ejaculate. In this regard,it has been shown that in the human ejaculate coexist differentsperm subsets that vary in terms of sperm function (i.e., in theircapacity to undergo protein tyrosine phosphorylation duringcapacitation), maturational stage and morphology [12]. Thesesperm subsets can be separated and isolated by discontinuousgradient centrifugation, where the highest quality sperm arerecovered from the pellet of the gradient (L90), while the lowestquality sperm with poor motility and morphology remain in theupper layers of the gradient (L45). However, we did not observehistone acetylation (H3K4Ac and H4K5Ac) in normal orabnormalmature human sperm. This result indicates that histoneacetylation does not persist in human sperm after ejaculation.Histone methylation is one of the most studied epigeneticmodification. Unexpectedly, we observed an heterogeneousdistribution of the modifications H3K4Me1, H3K9Me2,H3K4Me3, H3K79Me2, and H3K36Me3. In this case, whenwe compared the best quality and the worst quality sperma-tozoa (represented by the high and low motility fraction of thediscontinuous density gradient respectively), we observedremarkable differences between both fractions. This differ-ence may be related to abnormal levels of histone in the poormotility fractions due to defective exchange by protaminesduring spermiogenesis, as observed in certain sperm patho-logical conditions.Methylation of histones in lysines 3 or 4 have been associ-ated with either activation or repression of transcription, respec-tively [13]. Some of these histone marks have been shown toparticipate in several processes in the male reproductive tract,such as gene expression during spermatogenesis, differentiationof spermatogonia into spermatocytes [14], and X-chromosomeinactivation [15] among others. A question that remains to beanswered is whether the histone methylation present in maturesperm remains associated with the paternal genome after theprotamine-histone exchange that takes place immediately afterfertilization and if these have a function during earlypremiplantation embryo development. After fertilization all theprotamines and almost all the paternal histones are degraded andreplaced by oocyte histones. However, based on ethicalgrounds, the fate of these marks in human preimplantationembryos cannot be assessed unless these embryos are discardedbecause of multinucleation, fragmentation or developmentalarrest. Nevertheless, these are pathological conditions wherethese events may be altered.In summary, we report the heterogeneous presence of his-tone modifications in human sperm. The presence of thesemarks is higher in poor functional quality sperm, suggestingthat defects in the process of spermatogenesis may alter thecorrect epigenetic programing in mature sperm. Further stud-ies are required to evaluate the impact of these observations ininfertile individuals.Acknowledgments We would like to thank Drs. Paula Stein, Juan G.Alvarez, Juan C. Calvo and Lucrecia Calvo and Alejandra Vitale for theircomments and technical assistance. RMR is supported by F21C Repro-ductive Biology Group, University of Missouri. MGB, VJ, MR aresupported by CONICET, Argentina.References1. Sasaki H, Matsui Y. Epigenetic events in mammalian germ-celldevelopment: reprogramming and beyond. Nat Rev Genet.2008;9(2):129–40.48 J Assist Reprod Genet (2014) 31:45–492. Oliva R, Dixon GH. Vertebrate protamine gene evolution I.Sequence alignments and gene structure. J Mol Evol. 1990;30(4):333–46.3. Kasinsky HE, Eirín-López JM, Ausió J. Protamines: structural com-plexity, evolution and chromatin patterning. Protein Pept Lett. 2011;18(8):755–71.4. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR.Distinctive chromatin in human sperm packages genes for embryodevelopment. Nature. 2009;460(7254):473–8.5. Hammoud SS, Nix DA, Hammoud AO, Gibson M, Cairns BR,Carrell DT. Genome-wide analysis identifies changes in histoneretention and epigenetic modifications at developmental andimprinted gene loci in the sperm of infertile men. Hum Reprod.2011;26(9):2558–69.6. Barratt C, Aitken RJ, Bjorndahl L, Carrell DT, de Boer P, Kvist U,et al. Sperm DNA: organization, protection and vulnerability: frombasic science to clinical applications, a position report. Hum Reprod.2010;25(4):824–38.7. Buffone MG, Doncel GF, Calamera JC, Verstraeten SV. Capacitation-associated changes in membrane fluidity in asthenozoospermic humanspermatozoa. Int J Androl. 2009;32(4):360–75.8. Yebra L, Oliva R. Rapid analysis of mammalian sperm nuclearproteins. Anal Biochem. 1993;209(1):201–3.9. Bedford-Guaus SJ, McPartlin LA, Xie J, Westmiller SL, BuffoneMG, Roberson MS. Molecular cloning and characterization of phos-pholipase C zeta in equine sperm and testis reveals species-specificdifferences in expression of catalytically active protein. Biol Reprod.2011 Jul;85(1):78–88.10. Zahn A, Furlong LI, Biancotti JC, Ghiringhelli PD, Marijn-BriggilerCI, Vazquez-Levin MH. Evaluation of the proacrosin/acrosin systemand its mechanism of activation in human sperm extracts. J ReprodImmunol. 2002;54(1–2):43–63.11. Oliva R, Bazett-Jones DP, Locklear L, Dixon GH. Histonehyperacetylation can induce unfolding of the nucleosome core parti-cle. Nucleic Acids Res. 1990;18(9):2739–47.12. Buffone MG, Doncel GF, Marín Briggiler CI, Vazquez-Levin MH,Calamera JC. Human sperm subpopulations: relationship betweenfunctional quality and protein tyrosine phosphorylation. HumReprod. 2004;19(1):139–46.13. Glaser S, Lubitz S, et al. The histone 3 lysine 4 methyltransferase,Mll2, is only required briefly in development and spermatogenesis.Epigenetics Chromatin. 2009;2(1):5.14. Turner JM, Mahadevaiah SK, et al. Silencing of unsynapsed meioticchromosomes in the mouse. Nat Genet. 2005;37(1):41–7.15. WHO laboratory manual for the examination and processing ofhuman semen, Cambridge University Press. 1999.J Assist Reprod Genet (2014) 31:45–49 49

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Heterogeneous distribution of histone methylation in mature human sperm

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http://dx.doi.org/10.1007/s10815-013-0137-4

Heterogeneous distribution of histone methylation in mature human sperm

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