Advertisement

Characterization of the Sperm Proteome and Reproductive Outcomes with in Vitro, Fertilization after a Reduction in Male Ejaculatory Abstinence Period*

  • Author Footnotes
    ‖‖ These authors contributed equally to this work.
    Zi-Qi Shen
    Footnotes
    ‖‖ These authors contributed equally to this work.
    Affiliations
    From the ‡Center of Reproductive Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China;
    Search for articles by this author
  • Author Footnotes
    ‖‖ These authors contributed equally to this work.
    Bei Shi
    Footnotes
    ‖‖ These authors contributed equally to this work.
    Affiliations
    Department of Physiology, College of Basic Medical Science, China Medical University, Shenyang 110122, China;
    Search for articles by this author
  • Tian-Ren Wang
    Affiliations
    Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, CT 06520, USA;
    Search for articles by this author
  • Jiao Jiao
    Affiliations
    From the ‡Center of Reproductive Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China;
    Search for articles by this author
  • Xue-Jun Shang
    Affiliations
    Department of Andrology, Jinling Hospital Affiliated to Nanjing University School of Medicine, Nanjing 210002, China;
    Search for articles by this author
  • Qi-Jun Wu
    Affiliations
    Department of Clinical Epidemiology, Shengjing Hospital of China Medical University, Shenyang 110004, China;
    Search for articles by this author
  • Yi-Ming Zhou
    Affiliations
    ‡Department of Medicine, Brigham and Women's Hospital, Harvard Institutes of Medicine, Harvard Medical School, Boston, MA 02115
    Search for articles by this author
  • Tie-Feng Cao
    Affiliations
    Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, CT 06520, USA;
    Search for articles by this author
  • Qiang Du
    Affiliations
    From the ‡Center of Reproductive Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China;
    Search for articles by this author
  • Xiu-Xia Wang
    Correspondence
    To whom correspondence may be addressed:Center of Reproductive Medicine, Shengjing Hospital of China Medical University, No. 36, SanHao Street, Shenyang 110004, China, Tel.:+86-18940251898; Fax:+86-96615-44219;
    Affiliations
    From the ‡Center of Reproductive Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China;
    Search for articles by this author
  • Da Li
    Correspondence
    To whom correspondence may be addressed:Center of Reproductive Medicine, Shengjing Hospital of China Medical University, No. 36, SanHao Street, Shenyang 110004, China, Tel.:+86-18940252099; Fax:+86-96615-44219;
    Affiliations
    From the ‡Center of Reproductive Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China;
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the National Natural Science Foundation of China (81671423, 81402130, 81370750, and 81501247); Fok Ying Tung Education Foundation (151039); Campus Research Fund of China Medical University (YQ20160004); Distinguished Talent Program of Shengjing Hospital (No. ME76). None declared.
    This article contains supplemental Figures and Tables.
    ‖‖ These authors contributed equally to this work.
Open AccessPublished:August 20, 2018DOI:https://doi.org/10.1074/mcp.RA117.000541
      Semen samples from men after a short ejaculatory abstinence show improved sperm quality and result in increased pregnancy rates, but the underlying mechanisms remain unclear. Herein, we report that ejaculates from short (1–3 h) compared with long (3–7 days) periods of abstinence showed increases in motile sperm count, sperm vitality, normal sperm morphology, acrosome reaction capacity, total antioxidant capacity, sperm mitochondrial membrane potential, high DNA stainability, and a decrease in the sperm DNA fragmentation index (p, < 0.05). Sperm proteomic analysis showed 322 differentially expressed proteins (minimal fold change of ±1.5 or greater and p, < 0.05), with 224 upregulated and 98 downregulated. These differentially expressed proteins are profoundly involved in specific cellular processes, such as motility and capacitation, oxidative stress, and metabolism. Interestingly, protein trimethyllysine modification was increased, and butyryllysine, propionyllysine, and malonyllysine modifications were decreased in ejaculates from a short versus, long abstinence (p, < 0.05). Finally, the rates of implantation, clinical pregnancy, and live births from in vitro, fertilization treatments were significantly increased in semen samples after a short abstinence. Our study provides preliminary mechanistic insights into improved sperm quality and pregnancy outcomes associated with spermatozoa retrieved after a short ejaculatory abstinence.

      Graphical Abstract

      In 1952, MacLeod and Gold surveyed fertile men and suggested that the most motile spermatozoa were found among samples from men with fewer than 4 days of ejaculatory abstinence (
      • Macleod J.
      • Gold R.Z.
      The male factor in fertility and infertility. V. Effect of continence on semen quality.
      ). In 1979, Schwartz et al., using multivariate statistical techniques, were the first to evaluate the within-subject variability for semen characteristics in normal subjects who maintained an approximately normal ejaculatory frequency. It has been shown that there is very large within-subject variability in semen characteristics, but certainly one influential factor is the period of abstinence (
      • Schwartz D.
      • Laplanche A.
      • Jouannet P.
      • David G.
      Within-subject variability of human semen in regard to sperm count, volume, total number of spermatozoa and length of abstinence.
      ). Recently, Alipour et al., using standardized semen analysis, characterized the intra-individual differences in semen samples of normozoospermic men collected after 2 h versus, 4–7 days of abstinence. A higher percentage of motile spermatozoa with higher velocity and progressive motility was detected in the 2-hour semen samples (
      • Alipour H.
      • Van Der Horst G.
      • Christiansen O.B.
      • Dardmeh F.
      • Jorgensen N.
      • Nielsen H.I.
      • Hnida C.
      Improved sperm kinematics in semen samples collected after 2 h versus 4–7 days of ejaculation abstinence.
      ). Likewise, ejaculates from men with oligozoospermia exhibited a significant improvement in sperm motility, progression, and morphology when a second ejaculate was produced within only 40 min of the first (
      • Bahadur G.
      • Almossawi O.
      • Zeirideen Zaid R.
      • Ilahibuccus A.
      • Al-Habib A.
      • Muneer A.
      • Okolo S.
      Semen characteristics in consecutive ejaculates with short abstinence in subfertile males.
      ). In addition, it is noteworthy that sperm deoxyribonucleic acid (DNA) fragmentation can be significantly reduced by short-term recurrent ejaculation (
      • Gosalvez J.
      • Gonzalez-Martinez M.
      • Lopez-Fernandez C.
      • Fernandez J.L.
      • Sanchez-Martin P.
      Shorter abstinence decreases sperm deoxyribonucleic acid fragmentation in ejaculate.
      ). Higher pregnancy rates following intrauterine insemination have also been observed with semen obtained after an abstinence of less than 2 days (
      • Marshburn P.B.
      • Alanis M.
      • Matthews M.L.
      • Usadi R.
      • Papadakis M.H.
      • Kullstam S.
      • Hurst B.S.
      A short period of ejaculatory abstinence before intrauterine insemination is associated with higher pregnancy rates.
      ). Prolonged abstinence decreases pregnancy rates after intrauterine insemination, independent of sperm motility parameters, suggesting that abstinence intervals should be controlled in studies of pregnancy outcomes after using assisted reproductive technology (
      • Jurema M.W.
      • Vieira A.D.
      • Bankowski B.
      • Petrella C.
      • Zhao Y.
      • Wallach E.
      • Zacur H.
      Effect of ejaculatory abstinence period on the pregnancy rate after intrauterine insemination.
      ). However, whether semen samples after a short abstinence improve rates of implantation, pregnancy, and live births, and what associated molecular mechanisms are involved, remains largely unknown. Notably, proteomic-based approaches are being applied to the study of cellular and developmental processes of gamete cells, and this method is currently being used to study sperm maturation and function because of the low level of transcriptional and translational activity in spermatozoa and the fact that sperm functions are principally controlled at the protein level (
      • Brohi R.D.
      • Huo L.J.
      Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.
      ,
      • Zhou T.
      • Xia X.
      • Liu J.
      • Wang G.
      • Guo Y.
      • Guo X.
      • Wang X.
      • Sha J.
      Beyond single modification: Reanalysis of the acetylproteome of human sperm reveals widespread multiple modifications.
      ). In the present study, we first compared the sperm characteristics and outcomes of in vitro, fertilization (IVF)
      The abbreviations used are: IVF, In vitro, fertilization; GO, gene ontology; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine Iodide; KEGG, kyoto encyclopedia of genes and genomes; MMP, mitochondrial membrane potential; PBS, phosphate buffered saline; PCA, principal components analysis; PSA, pisum sativum agglutinin; ROS, reactive oxygen species; TAC, total antioxidant capacity; WHO, World Health Organization.
      1The abbreviations used are: IVF, In vitro, fertilization; GO, gene ontology; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine Iodide; KEGG, kyoto encyclopedia of genes and genomes; MMP, mitochondrial membrane potential; PBS, phosphate buffered saline; PCA, principal components analysis; PSA, pisum sativum agglutinin; ROS, reactive oxygen species; TAC, total antioxidant capacity; WHO, World Health Organization.
      using semen samples collected following short (1–3 h) or long (3–7 days) periods of abstinence. We then used a tandem mass tag (TMT)-based quantitative proteomic approach to investigate proteomic changes in spermatozoa after reduced male ejaculatory abstinence. Our results suggest that the molecular events occurring in sperm proteins may play an important part in sperm quality and reproductive potential after reduced periods of male ejaculatory abstinence.

      DISCUSSION

      To our knowledge, there are no published studies on the effects of a short period of male ejaculatory abstinence (1–3 h) on IVF-derived reproductive outcomes. We herein reported for the first time that the implantation and clinical pregnancy rates—and particularly the live birth rate—were significantly increased using a semen sample obtained after 1–3 h of abstinence in a frozen-thawed cycle, rather than in a fresh IVF cycle. We know from the literature that reproductive outcomes can be significantly improved in frozen-thawed cycles compared with fresh IVF cycles because of the impaired endometrial receptivity of the latter after controlled ovarian stimulation (
      • Evans J.
      • Hannan N.J.
      • Edgell T.A.
      • Vollenhoven B.J.
      • Lutjen P.J.
      • Osianlis T.
      • Salamonsen L.A.
      • Rombauts L.J.
      Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence.
      ,
      • Roque M.
      • Valle M.
      • Guimaraes F.
      • Sampaio M.
      • Geber S.
      Freeze-all policy: fresh vs. frozen-thawed embryo transfer.
      ). It appears that frozen-thawed cycles (with an appropriate endometrial environment) are more likely to show improved sperm-related embryo quality and reproductive outcomes after IVF.
      Subsequently, we used high-throughput proteomic techniques to confirm the potential molecular diversity of spermatozoa in ejaculates after 3–7 days and 1–3 h of ejaculatory abstinence. We compared 4959 identified sperm proteins and 322 differentially expressed proteins separately with 4 male reproductive tract-related tissues: testis, epididymis, seminal vesicle, and prostate, and we observed a marked bias toward the testis in both sets of proteins (supplemental Fig. S3). The testis is the primary male reproductive organ and is responsible for the production of spermatozoa as well as steroid hormones. It appears, then, that testis-related spermatogenesis may play an important role in the reproductive potential of spermatozoa after reduced periods of ejaculatory abstinence.
      It is significant that differentially expressed proteins were found to be highly involved in sperm motility and capacitation, and that the acrosome reaction capability of spermatozoa was markedly elevated after 1–3 h of abstinence. These results suggested that spermatozoa may possess a greater motility and fertilizing ability after a reduced period of male abstinence. Accumulating evidence indicates that disturbing the balance between ROS and antioxidant capacity in seminal plasma may result in male infertility through oxidative stress (
      • Dorostghoal M.
      • Kazeminejad S.R.
      • Shahbazian N.
      • Pourmehdi M.
      • Jabbari A.
      Oxidative stress status and sperm DNA fragmentation in fertile and infertile men.
      ). However, it should be noted that semen is an admixture of secretions from the epididymis, vas deferens, seminal vesicles, and the prostate gland; and the contribution from each glandular secretion may affect ROS production and scavenging (
      • Marshburn P.B.
      • Giddings A.
      • Causby S.
      • Matthews M.L.
      • Usadi R.S.
      • Steuerwald N.
      • Hurst B.S.
      Influence of ejaculatory abstinence on seminal total antioxidant capacity and sperm membrane lipid peroxidation.
      ). Therefore, we measured sperm ROS and TAC in the present study. Interestingly, although ROS did not change significantly, sperm TAC was significantly improved after reduced male abstinence; and in this regard, spermatozoa derived after a shorter abstinence interval may enhance antioxidant defense abilities against ROS damage in semen. In addition, emerging evidence has suggested a possible link among ROS, DNA fragmentation, MMP, and reproductive outcomes, including (1) a significant association in asthenozoospermic patients with high ROS levels, decreased mitochondrial DNA integrity, and low MMP (
      • Bonanno O.
      • Romeo G.
      • Asero P.
      • Pezzino F.M.
      • Castiglione R.
      • Burrello N.
      • Sidoti G.
      • Frajese G.V.
      • Vicari E.
      • D'Agata R.
      Sperm of patients with severe asthenozoospermia show biochemical, molecular and genomic alterations.
      ); (2) mitochondrial permeability transition, which is associated with MMP dissipation, increased ROS production, and DNA fragmentation (
      • Treulen F.
      • Uribe P.
      • Boguen R.
      • Villegas J.V.
      Mitochondrial permeability transition increases reactive oxygen species production and induces DNA fragmentation in human spermatozoa.
      ); (3) sperm DNA fragmentation and MMP, which when combined, may be superior to standard semen parameters for the prediction of natural conception (
      • Malic Voncina S.
      • Golob B.
      • Ihan A.
      • Kopitar A.N.
      • Kolbezen M.
      • Zorn B.
      Sperm DNA fragmentation and mitochondrial membrane potential combined are better for predicting natural conception than standard sperm parameters.
      ); and (4) ROS-induced sperm DNA damage, which is associated with male infertility, recurrent pregnancy loss, congenital malformations, and a high frequency of childhood disorders (
      • Bisht S.
      • Dada R.
      Oxidative stress: Major executioner in disease pathology, role in sperm DNA damage and preventive strategies.
      ). In agreement with these findings, our current data indicated that sperm TAC was enhanced, that the DNA fragmentation index was reduced, and that MMP was increased after a period of abstinence as short as 1–3 h. This observation, combined with embryo transfer outcomes, provided evidence of a potential role for shorter abstinence periods in improving IVF outcomes.
      Post-translational modifications—either spontaneous or physiologic/pathologic—are the crucial steps that determine how proteins work in cells (
      • Becares N.
      • Gage M.C.
      • Pineda-Torra I.
      Posttranslational modifications of lipid-activated nuclear receptors: focus on metabolism.
      ), especially in spermatozoa, as their transcriptional and translational processes are curtailed during sperm maturation (
      • Brohi R.D.
      • Huo L.J.
      Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.
      ,
      • Zhou T.
      • Xia X.
      • Liu J.
      • Wang G.
      • Guo Y.
      • Guo X.
      • Wang X.
      • Sha J.
      Beyond single modification: Reanalysis of the acetylproteome of human sperm reveals widespread multiple modifications.
      ,
      • Baker M.A.
      Proteomics of post-translational modifications of mammalian spermatozoa.
      ). There is evidence that acylation (
      • Brohi R.D.
      • Huo L.J.
      Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.
      ,
      • Sabari B.R.
      • Zhang D.
      • Allis C.D.
      • Zhao Y.
      Metabolic regulation of gene expression through histone acylations.
      ), methylation (
      • Baker M.A.
      Proteomics of post-translational modifications of mammalian spermatozoa.
      ,
      • Zheng H.
      • Huang B.
      • Zhang B.
      • Xiang Y.
      • Du Z.
      • Xu Q.
      • Li Y.
      • Wang Q.
      • Ma J.
      • Peng X.
      • Xu F.
      • Xie W.
      Resetting epigenetic memory by reprogramming of histone modifications in mammals.
      ), ubiquitination (
      • Brohi R.D.
      • Huo L.J.
      Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.
      ,
      • Gou L.T.
      • Kang J.Y.
      • Dai P.
      • Wang X.
      • Li F.
      • Zhao S.
      • Zhang M.
      • Hua M.M.
      • Lu Y.
      • Zhu Y.
      • Li Z.
      • Chen H.
      • Wu L.G.
      • Li D.
      • Fu X.D.
      • Li J.
      • Shi H.J.
      • Liu M.F.
      Ubiquitination-deficient mutations in human Piwi cause male infertility by impairing histone-to-protamine exchange during spermiogenesis.
      ), and phosphorylation (
      • Brohi R.D.
      • Huo L.J.
      Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.
      ,
      • Jenardhanan P.
      • Mathur P.P.
      Kinases as targets for chemical modulators: Structural aspects and their role in spermatogenesis.
      ) in proteins play key roles in spermatogenesis, sperm maturation, and the fertilization process. Therefore, we evaluated these 10 kinds of major protein modifications in spermatozoa after reduced male abstinence. Preliminary data indicated that global modifications in sperm butyryllysine, propionyllysine, and malonyllysine were significantly decreased, and that trimethyllysine modification was greatly increased after reduced male abstinence. We presently know little about the role(s) of butyryllysine, propionyllysine, malonyllysine, and trimethyllysine modifications in abstinence-related sperm function, and therefore our work opens new avenues in exploring post-translational events with respect to sperm proteins.
      This study provides preliminary evidence of a significant difference in sperm gene sets, molecular function, and clinical phenotypes after a reduced male ejaculatory abstinence and suggests a potential role of using spermatozoa obtained after 1–3 h of abstinence in IVF treatments. Our findings may improve our understanding of the basic molecular mechanism(s) underlying a shorter abstinence-related reproductive potential of spermatozoa.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010695. The data files can be downloaded at: https://www.ebi.ac.uk/pride/archive/.

      Acknowledgments

      We thank the PTM Biolabs, Inc. for technical assistance.

      REFERENCES

        • Macleod J.
        • Gold R.Z.
        The male factor in fertility and infertility. V. Effect of continence on semen quality.
        Fertil. Steril. 1952; 3: 297-315
        • Schwartz D.
        • Laplanche A.
        • Jouannet P.
        • David G.
        Within-subject variability of human semen in regard to sperm count, volume, total number of spermatozoa and length of abstinence.
        J. Reprod. Fertil. 1979; 57: 391-395
        • Alipour H.
        • Van Der Horst G.
        • Christiansen O.B.
        • Dardmeh F.
        • Jorgensen N.
        • Nielsen H.I.
        • Hnida C.
        Improved sperm kinematics in semen samples collected after 2 h versus 4–7 days of ejaculation abstinence.
        Hum. Reprod. 2017; 32: 1364-1372
        • Bahadur G.
        • Almossawi O.
        • Zeirideen Zaid R.
        • Ilahibuccus A.
        • Al-Habib A.
        • Muneer A.
        • Okolo S.
        Semen characteristics in consecutive ejaculates with short abstinence in subfertile males.
        Reprod. Biomed. Online. 2016; 32: 323-328
        • Gosalvez J.
        • Gonzalez-Martinez M.
        • Lopez-Fernandez C.
        • Fernandez J.L.
        • Sanchez-Martin P.
        Shorter abstinence decreases sperm deoxyribonucleic acid fragmentation in ejaculate.
        Fertil. Steril. 2011; 96: 1083-1086
        • Marshburn P.B.
        • Alanis M.
        • Matthews M.L.
        • Usadi R.
        • Papadakis M.H.
        • Kullstam S.
        • Hurst B.S.
        A short period of ejaculatory abstinence before intrauterine insemination is associated with higher pregnancy rates.
        Fertil. Steril. 2010; 93: 286-288
        • Jurema M.W.
        • Vieira A.D.
        • Bankowski B.
        • Petrella C.
        • Zhao Y.
        • Wallach E.
        • Zacur H.
        Effect of ejaculatory abstinence period on the pregnancy rate after intrauterine insemination.
        Fertil. Steril. 2005; 84: 678-681
        • Brohi R.D.
        • Huo L.J.
        Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.
        OMICS. 2017; 21: 245-256
        • Zhou T.
        • Xia X.
        • Liu J.
        • Wang G.
        • Guo Y.
        • Guo X.
        • Wang X.
        • Sha J.
        Beyond single modification: Reanalysis of the acetylproteome of human sperm reveals widespread multiple modifications.
        J. Proteomics. 2015; 126: 296-302
        • WHO
        Laboratory Manual for the Examination and Processing of Human Semen. 5th edn. WHO Press, World Health Organization, Geneva, Switzerland2010
        • Saraswat M.
        • Joenvaara S.
        • Jain T.
        • Tomar A.K.
        • Sinha A.
        • Singh S.
        • Yadav S.
        • Renkonen R.
        Human Spermatozoa Quantitative Proteomic Signature Classifies Normo- and Asthenozoospermia.
        Mol. Cell. Proteomics. 2017; 16: 57-72
        • Cox J.
        • Mann M.
        MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
        Nat. Biotechnol. 2008; 26: 1367-1372
        • Vizcaino J.A.
        • Csordas A.
        • Del-Toro N.
        • Dianes J.A.
        • Griss J.
        • Lavidas I.
        • Mayer G.
        • Perez-Riverol Y.
        • Reisinger F.
        • Ternent T.
        • Xu Q.W.
        • Wang R.
        • Hermjakob H.
        2016 update of the PRIDE database and its related tools.
        Nucleic Acids Res. 2016; 44: 11033
        • Evans J.
        • Hannan N.J.
        • Edgell T.A.
        • Vollenhoven B.J.
        • Lutjen P.J.
        • Osianlis T.
        • Salamonsen L.A.
        • Rombauts L.J.
        Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence.
        Hum. Reprod. Update. 2014; 20: 808-821
        • Roque M.
        • Valle M.
        • Guimaraes F.
        • Sampaio M.
        • Geber S.
        Freeze-all policy: fresh vs. frozen-thawed embryo transfer.
        Fertil. Steril. 2015; 103: 1190-1193
        • Dorostghoal M.
        • Kazeminejad S.R.
        • Shahbazian N.
        • Pourmehdi M.
        • Jabbari A.
        Oxidative stress status and sperm DNA fragmentation in fertile and infertile men.
        Andrologia. 2017; 49: e12762
        • Marshburn P.B.
        • Giddings A.
        • Causby S.
        • Matthews M.L.
        • Usadi R.S.
        • Steuerwald N.
        • Hurst B.S.
        Influence of ejaculatory abstinence on seminal total antioxidant capacity and sperm membrane lipid peroxidation.
        Fertil. Steril. 2014; 102: 705-710
        • Bonanno O.
        • Romeo G.
        • Asero P.
        • Pezzino F.M.
        • Castiglione R.
        • Burrello N.
        • Sidoti G.
        • Frajese G.V.
        • Vicari E.
        • D'Agata R.
        Sperm of patients with severe asthenozoospermia show biochemical, molecular and genomic alterations.
        Reproduction. 2016; 152: 695-704
        • Treulen F.
        • Uribe P.
        • Boguen R.
        • Villegas J.V.
        Mitochondrial permeability transition increases reactive oxygen species production and induces DNA fragmentation in human spermatozoa.
        Hum. Reprod. 2015; 30: 767-776
        • Malic Voncina S.
        • Golob B.
        • Ihan A.
        • Kopitar A.N.
        • Kolbezen M.
        • Zorn B.
        Sperm DNA fragmentation and mitochondrial membrane potential combined are better for predicting natural conception than standard sperm parameters.
        Fertil. Steril. 2016; 105 (e631): 637-644
        • Bisht S.
        • Dada R.
        Oxidative stress: Major executioner in disease pathology, role in sperm DNA damage and preventive strategies.
        Front. Biosci. (Schol Ed). 2017; 9: 420-447
        • Becares N.
        • Gage M.C.
        • Pineda-Torra I.
        Posttranslational modifications of lipid-activated nuclear receptors: focus on metabolism.
        Endocrinology. 2017; 158: 213-225
        • Baker M.A.
        Proteomics of post-translational modifications of mammalian spermatozoa.
        Cell Tissue Res. 2016; 363: 279-287
        • Sabari B.R.
        • Zhang D.
        • Allis C.D.
        • Zhao Y.
        Metabolic regulation of gene expression through histone acylations.
        Nat. Rev. Mol. Cell Biol. 2017; 18: 90-101
        • Zheng H.
        • Huang B.
        • Zhang B.
        • Xiang Y.
        • Du Z.
        • Xu Q.
        • Li Y.
        • Wang Q.
        • Ma J.
        • Peng X.
        • Xu F.
        • Xie W.
        Resetting epigenetic memory by reprogramming of histone modifications in mammals.
        Mol. Cell. 2016; 63: 1066-1079
        • Gou L.T.
        • Kang J.Y.
        • Dai P.
        • Wang X.
        • Li F.
        • Zhao S.
        • Zhang M.
        • Hua M.M.
        • Lu Y.
        • Zhu Y.
        • Li Z.
        • Chen H.
        • Wu L.G.
        • Li D.
        • Fu X.D.
        • Li J.
        • Shi H.J.
        • Liu M.F.
        Ubiquitination-deficient mutations in human Piwi cause male infertility by impairing histone-to-protamine exchange during spermiogenesis.
        Cell. 2017; 169 (e1013): 1090-1104
        • Jenardhanan P.
        • Mathur P.P.
        Kinases as targets for chemical modulators: Structural aspects and their role in spermatogenesis.
        Spermatogenesis. 2014; 4: e979113