Transgenerational epigenetic impacts of parental infection on offspring health and disease susceptibility

Parental environmental experiences have the potential to reprogramme offspring phenotypes via changes in germline epigenetics.While the detrimental reprogramming effects of maternal immune activation during gestation are well characterised, the epigenetic effects of paternal preconceptual exposure to infection and immune activation have not been well explored.Recent evidence suggests that both paternal preconceptual Toxoplasma gondii infection and sepsis can have deleterious health consequences for multiple generations via changes in the sperm epigenome.Given that many fertile men and women have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [the virus causing coronavirus disease 2019 (COVID-19)], the potential epigenetic effects of infection and immune activation need to be investigated in preclinical maternal and paternal models, so that we may mitigate any multigenerational health impacts of the COVID-19 pandemic. Maternal immune activation (MIA) and infection during pregnancy are known to reprogramme offspring phenotypes. However, the epigenetic effects of preconceptual paternal infection and paternal immune activation (PIA) are not currently well understood. Recent reports show that paternal infection and immune activation can affect offspring phenotypes, particularly brain function, behaviour, and immune system functioning, across multiple generations without re-exposure to infection. Evidence from other environmental exposures indicates that epigenetic inheritance also occurs in humans. Given the growing impact of the coronavirus disease 2019 (COVID-19) pandemic, it is imperative that we investigate all of the potential epigenetic mechanisms and multigenerational phenotypes that may arise from both maternal and paternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as well as associated MIA, PIA, and inflammation. This will allow us to understand and, if necessary, mitigate any potential changes in disease susceptibility in the children, and grandchildren, of affected parents. Maternal immune activation (MIA) and infection during pregnancy are known to reprogramme offspring phenotypes. However, the epigenetic effects of preconceptual paternal infection and paternal immune activation (PIA) are not currently well understood. Recent reports show that paternal infection and immune activation can affect offspring phenotypes, particularly brain function, behaviour, and immune system functioning, across multiple generations without re-exposure to infection. Evidence from other environmental exposures indicates that epigenetic inheritance also occurs in humans. Given the growing impact of the coronavirus disease 2019 (COVID-19) pandemic, it is imperative that we investigate all of the potential epigenetic mechanisms and multigenerational phenotypes that may arise from both maternal and paternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as well as associated MIA, PIA, and inflammation. This will allow us to understand and, if necessary, mitigate any potential changes in disease susceptibility in the children, and grandchildren, of affected parents. Most chronic diseases have complex aetiologies that cannot be explained by genetic factors alone [1.Blanco-Gómez A. et al.Missing heritability of complex diseases: enlightenment by genetic variants from intermediate phenotypes.BioEssays. 2016; 38: 664-673Crossref PubMed Scopus (0) Google Scholar]. Accumulating evidence suggests that both maternal and paternal environmental exposures can reprogramme offspring phenotypes via germline epigenetic modifications [2.Bale T.L. Epigenetic and transgenerational reprogramming of brain development.Nat. Rev. Neurosci. 2015; 16: 332-344Crossref PubMed Google Scholar,3.Sales V.M. et al.Epigenetic mechanisms of transmission of metabolic disease across generations.Cell Metab. 2017; 25: 559-571Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar]. Both human and animal studies show that inherited epigenetic changes can promote the development of various chronic diseases, including schizophrenia, cardiovascular disease, and type 2 diabetes [4.Watkins A.J. et al.Paternal programming of offspring health.Early Hum. Dev. 2020; 150105185Crossref PubMed Scopus (7) Google Scholar, 5.Burton N.O. Greer E.L. Multigenerational epigenetic inheritance: Transmitting information across generations.Semin. Cell Dev. Biol. 2021; 123: 1-12PubMed Google Scholar, 6.Chen Q. et al.Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder.Science. 2016; 351: 397Crossref PubMed Scopus (667) Google Scholar]. Maternal stress, dietary changes, and infection during gestation can have intergenerational (see Glossary) and transgenerational impacts on offspring, including neurodevelopmental and metabolic dysfunctions [2.Bale T.L. Epigenetic and transgenerational reprogramming of brain development.Nat. Rev. Neurosci. 2015; 16: 332-344Crossref PubMed Google Scholar,7.Weber-Stadlbauer U. Epigenetic and transgenerational mechanisms in infection-mediated neurodevelopmental disorders.Transl. Psychiatry. 2017; 7: e1113Crossref PubMed Google Scholar,8.Pembrey M. et al.Human transgenerational responses to early-life experience: potential impact on development, health and biomedical research.J. Med. Genet. 2014; 51: 563-572Crossref PubMed Scopus (144) Google Scholar]. However, due to the mature sperm’s tightly packaged chromatin and reduced transcriptional activity, the paternal epigenetic contributions to offspring (F1) and grand-offspring (F2) phenotypes have been previously disregarded [9.Le Blévec E. et al.Paternal epigenetics: mammalian sperm provide much more than DNA at fertilization.Mol. Cell. Endocrinol. 2020; 518110964Crossref PubMed Scopus (14) Google Scholar]. Nevertheless, recent discoveries show that the sperm delivers intergenerational information, particularly in the form of noncoding RNAs, to the oocytes at fertilisation [10.Sharma U. Paternal contributions to offspring health: role of sperm small RNAs in intergenerational transmission of epigenetic information.Front. Cell Dev. Biol. 2019; 7: 215Crossref PubMed Scopus (46) Google Scholar,11.Teperek M. et al.Sperm is epigenetically programmed to regulate gene transcription in embryos.Genome Res. 2016; 26: 1034-1046Crossref PubMed Scopus (73) Google Scholar]. Furthermore, the small noncoding RNA ‘payload’ of the sperm can alter embryonic development and offspring phenotypes [12.Conine C.C. et al.Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice.Dev. Cell. 2018; 46: 470-480.e3Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]. Paternal preconceptual exposure to various environmental factors, including stress, toxins, and dietary changes, can modulate the sperm epigenome, potentially increasing offspring disease susceptibility [13.Gapp K. et al.Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice.Nat. Neurosci. 2014; 17: 667-669Crossref PubMed Scopus (700) Google Scholar, 14.Gapp K. et al.Alterations in sperm long RNA contribute to the epigenetic inheritance of the effects of postnatal trauma.Mol. Psychiatry. 2020; 25: 2162-2174Crossref PubMed Scopus (46) Google Scholar, 15.Rodgers A.B. et al.Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 13699-13704Crossref PubMed Scopus (402) Google Scholar, 16.Bodden C. et al.Diet-induced modification of the sperm epigenome programs metabolism and behavior.Trends Endocrinol. Metab. 2020; 31: 131-149Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17.Anway M.D. et al.Endocrine disruptor vinclozolin induced epigenetic transgenerational adult-onset disease.Endocrinology. 2006; 147: 5515-5523Crossref PubMed Scopus (405) Google Scholar]. Although there is substantial evidence that maternal infection and immune activation have detrimental intergenerational (and even transgenerational) outcomes [18.Weber-Stadlbauer U. et al.Transgenerational transmission and modification of pathological traits induced by prenatal immune activation.Mol. Psychiatry. 2017; 22: 102-112Crossref PubMed Scopus (89) Google Scholar], these exposures are largely unexplored in paternal epigenetic inheritance models. Pathogenic infections, and associated immune activation, can impact an organism’s physiology and epigenetic profile [19.Tyebji S. et al.Pathogenic infection in male mice changes sperm small RNA profiles and transgenerationally alters offspring behavior.Cell Rep. 2020; 31107573Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,20.Silmon de Monerri N.C. Kim K. Pathogens hijack the epigenome: a new twist on host-pathogen interactions.Am. J. Pathol. 2014; 184: 897-911Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. Furthermore, maternal immune activation and infection in both humans and animals are known to increase offspring predisposition to neurodevelopmental disorders, including schizophrenia and autism spectrum disorder (ASD) [21.Knuesel I. et al.Maternal immune activation and abnormal brain development across CNS disorders.Nat. Rev. Neurol. 2014; 10: 643-660Crossref PubMed Scopus (453) Google Scholar,22.Shi L. et al.Maternal influenza infection causes marked behavioral and pharmacological changes in the offspring.J. Neurosci. 2003; 23: 297-302Crossref PubMed Google Scholar]. Intriguingly, Tyebji and colleagues recently discovered that paternal preconceptual infection with Toxoplasma gondii can alter sperm epigenetics, leading to F1 and F2 cognitive and behavioural abnormalities without re-exposure to infection [19.Tyebji S. et al.Pathogenic infection in male mice changes sperm small RNA profiles and transgenerationally alters offspring behavior.Cell Rep. 2020; 31107573Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Nevertheless, the mechanisms underlying how infection and immune activation can reprogramme the sperm epigenome are not well understood [19.Tyebji S. et al.Pathogenic infection in male mice changes sperm small RNA profiles and transgenerationally alters offspring behavior.Cell Rep. 2020; 31107573Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Children of those affected by major disasters, including survivors of the Holocaust, famines, and the World Trade Centre attacks, often display altered stress responsivity and metabolism [23.Yehuda R. et al.Transgenerational effects of posttraumatic stress disorder in babies of mothers exposed to the World Trade Center attacks during pregnancy.J. Clin. Endocrinol. Metab. 2005; 90: 4115-4118Crossref PubMed Scopus (359) Google Scholar]. Furthermore, epigenetic mechanisms, including modified DNA methylation at imprinted genes, have been associated with these changes [24.Shen L. et al.Early-life exposure to severe famine is associated with higher methylation level in the IGF2 gene and higher total cholesterol in late adulthood: the Genomic Research of the Chinese Famine (GRECF) study.Clin. Epigenetics. 2019; 11: 88Crossref PubMed Scopus (0) Google Scholar]. At the time of writing, over 500 million people worldwide have been infected with COVID-19, resulting in over 6 million reported deaths (World Health Organization, April 2022). Given the enormous physical and mental health burden associated with the COVID-19 pandemic, there is a significant possibility that long-term public health repercussions of the pandemic may arise due to epigenetic inheritance. Therefore, there is a clear imperative to investigate the potential intergenerational and transgenerational COVID-19-related health effects in preclinical studies, so that we can prevent these from arising in humans and, if they do occur, develop targeted therapeutic approaches. Henceforth, we discuss how the evidence from both MIA and paternal infection models of epigenetic inheritance can inform future research into the intergenerational and transgenerational impacts of COVID-19. Epigenetics broadly refers to the mitotically stable molecular processes that regulate gene expression independently of the DNA sequence itself [25.Portela A. Esteller M. Epigenetic modifications and human disease.Nat. Biotechnol. 2010; 28: 1057-1068Crossref PubMed Scopus (1862) Google Scholar]. The main epigenetic processes include DNA methylation (and other modifications), histone modifications, as well as the actions of small and long noncoding RNAs [25.Portela A. Esteller M. Epigenetic modifications and human disease.Nat. Biotechnol. 2010; 28: 1057-1068Crossref PubMed Scopus (1862) Google Scholar]. Importantly, these epigenetic mechanisms work together to regulate gene expression. For example, DNA methylation modifications can regulate the expression of microRNAs (miRNAs) and in turn, miRNAs can modulate the expression of DNA methyltransferases and histone deacetylases [5.Burton N.O. Greer E.L. Multigenerational epigenetic inheritance: Transmitting information across generations.Semin. Cell Dev. Biol. 2021; 123: 1-12PubMed Google Scholar]. These epigenetic processes are also dynamic and susceptible to changing environmental conditions during development [5.Burton N.O. Greer E.L. Multigenerational epigenetic inheritance: Transmitting information across generations.Semin. Cell Dev. Biol. 2021; 123: 1-12PubMed Google Scholar]. The timing and severity of particular environmental insults, including diet and stress, can also affect whether epigenetic changes occur in germ cells [2.Bale T.L. Epigenetic and transgenerational reprogramming of brain development.Nat. Rev. Neurosci. 2015; 16: 332-344Crossref PubMed Google Scholar]. Global erasure of epigenetic marks, including DNA methylation patterns and histone modifications, occurs at two stages in a mammal’s lifetime [26.Messerschmidt D.M. et al.DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos.Genes Dev. 2014; 28: 812-828Crossref PubMed Scopus (431) Google Scholar]. These are immediately after fertilisation in the zygote and when the primordial germ cells (PGCs) enter the developing gonad during gametogenesis [26.Messerschmidt D.M. et al.DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos.Genes Dev. 2014; 28: 812-828Crossref PubMed Scopus (431) Google Scholar]. These reset periods restore totipotency in the embryo and prevent many epigenetic aberrations from being inherited [2.Bale T.L. Epigenetic and transgenerational reprogramming of brain development.Nat. Rev. Neurosci. 2015; 16: 332-344Crossref PubMed Google Scholar]. Furthermore, the developing sperm undergoes extensive chromatin remodelling during maturation as shown in Box 1 [27.Larose H. et al.Chapter eight - gametogenesis: a journey from inception to conception.in: Wellik D.M. Current Topics in Developmental Biology. Academic Press, 2019: 257-310Google Scholar], which has previously led scientists to believe that paternal epigenetic inheritance is unlikely to occur. Contrastingly, these reprogramming and chromatin remodelling periods have been identified as critical periods where environmental insults can cause heritable epigenetic changes [2.Bale T.L. Epigenetic and transgenerational reprogramming of brain development.Nat. Rev. Neurosci. 2015; 16: 332-344Crossref PubMed Google Scholar]. These epigenetic marks can escape reprogramming and potentially confer increased disease risk in offspring [2.Bale T.L. Epigenetic and transgenerational reprogramming of brain development.Nat. Rev. Neurosci. 2015; 16: 332-344Crossref PubMed Google Scholar]. Moreover, the sperm and oocyte retain small and long noncoding RNAs that can guide embryonic development and subsequently affect offspring phenotypes [6.Chen Q. et al.Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder.Science. 2016; 351: 397Crossref PubMed Scopus (667) Google Scholar,12.Conine C.C. et al.Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice.Dev. Cell. 2018; 46: 470-480.e3Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,28.Svoboda P. Long and small noncoding RNAs during oocyte-to-embryo transition in mammals.Biochem. Soc. Trans. 2017; 45: 1117-1124Crossref PubMed Scopus (19) Google Scholar]. Therefore, both paternal and maternal epigenetic marks can escape erasure and be inherited.Box 1Spermatogenesis and sperm epigenome remodellingIn mammals, spermatogenesis involves the development and maturation of PGCs into mature spermatozoa (see Figure 1 in main text) [29.Griswold M.D. Spermatogenesis: the commitment to meiosis.Physiol. Rev. 2016; 96: 1-17Crossref PubMed Scopus (299) Google Scholar]. Spermatogenesis is a continuous and efficient process that spans a mammal’s reproductive lifetime [29.Griswold M.D. Spermatogenesis: the commitment to meiosis.Physiol. Rev. 2016; 96: 1-17Crossref PubMed Scopus (299) Google Scholar]. The process begins in the basement membrane and ends in the lumen of the seminiferous tubules in the testes. A full cycle of spermatogenesis typically takes approximately 35 days in mice and 74 days in humans [30.Oakberg E.F. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium.Am. J. Anat. 1956; 99: 507-516Crossref PubMed Google Scholar]. It begins with the development of PGCs into diploid, self-renewing spermatogonia, which then mitotically divide to become spermatocytes [27.Larose H. et al.Chapter eight - gametogenesis: a journey from inception to conception.in: Wellik D.M. Current Topics in Developmental Biology. Academic Press, 2019: 257-310Google Scholar,31.Hamatani T. Human spermatozoal RNAs.Fertil. Steril. 2012; 97: 275-281Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar]. Spermatocytes then undergo two meiotic divisions to become round haploid spermatids [27.Larose H. et al.Chapter eight - gametogenesis: a journey from inception to conception.in: Wellik D.M. Current Topics in Developmental Biology. Academic Press, 2019: 257-310Google Scholar]. These spermatids morphologically mature into spermatozoa in a process known as spermiogenesis [32.Kimmins S. et al.Testis-specific transcription mechanisms promoting male germ-cell differentiation.Reproduction. 2004; 128: 5-12Crossref PubMed Google Scholar]. However, the spermatozoa exiting the testes after spermatogenesis still need to gain the motility and functional maturity to be able to fertilise the oocytes [33.Sullivan R. Mieusset R. The human epididymis: its function in sperm maturation.Hum. Reprod. Update. 2016; 22: 574-587Crossref PubMed Scopus (140) Google Scholar]. As they transition from the caput to the caudal region of the epididymis during their final stages of maturation, the spermatozoa receive growth factors and extracellular vesicles from the epithelial cells bordering the tubule [34.Sharma U. et al.Small RNAs are trafficked from the epididymis to developing mammalian sperm.Dev. Cell. 2018; 46: 481-494.e6Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar,35.Sharma U. et al.Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.Science. 2016; 351: 391Crossref PubMed Google Scholar].During spermatogenesis, chromatin packaging occurs to attain nuclear compaction [27.Larose H. et al.Chapter eight - gametogenesis: a journey from inception to conception.in: Wellik D.M. Current Topics in Developmental Biology. Academic Press, 2019: 257-310Google Scholar]. Histones that package the spermatozoal DNA are mostly replaced by intermediary transition proteins, which are then replaced by protamines [36.Godmann M. et al.The dynamic epigenetic program in male germ cells: its role in spermatogenesis, testis cancer, and its response to the environment.Microsc. Res. Tech. 2009; 72: 603-619Crossref PubMed Scopus (0) Google Scholar]. Protamines are small basic proteins that can tightly pack the sperm DNA [37.Wykes S.M. Krawetz S.A. The structural organization of sperm chromatin.J. Biol. Chem. 2003; 278: 29471-29477Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar]. In humans, 15% of histones are retained in mature sperm, whereas only 1–2% of histones are retained in sperm from mice [38.Rando O.J. Intergenerational transfer of epigenetic information in sperm.Cold Spring Harb. Perspect. Med. 2016; 6a022988Crossref PubMed Scopus (75) Google Scholar,39.Yehuda R. et al.Relationship of parental trauma exposure and PTSD to PTSD, depressive and anxiety disorders in offspring.J. Psychiatr. Res. 2001; 35: 261-270Crossref PubMed Scopus (192) Google Scholar]. Sperm transcriptomic activity, including RNA synthesis, is also minimised during this process of nuclear compaction [39.Yehuda R. et al.Relationship of parental trauma exposure and PTSD to PTSD, depressive and anxiety disorders in offspring.J. Psychiatr. Res. 2001; 35: 261-270Crossref PubMed Scopus (192) Google Scholar,40.Dadoune J.-P. Spermatozoal RNAs: what about their functions?.Microsc. Res. Tech. 2009; 72: 536-551Crossref PubMed Scopus (0) Google Scholar]. This entire compaction process has led many to disregard that the sperm carries any heritable information other than the paternal haploid genome. By contrast, researchers have found that the histones retained in human sperm map onto promotors for genes that are important in embryonic development [41.Brykczynska U. et al.Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa.Nat. Struct. Mol. Biol. 2010; 17: 679-687Crossref PubMed Scopus (482) Google Scholar]. Additionally, the mature sperm contain many noncoding RNA subtypes, most notably tRNAs, miRNAs, and PIWI-interacting RNAs (piRNAs) [38.Rando O.J. Intergenerational transfer of epigenetic information in sperm.Cold Spring Harb. Perspect. Med. 2016; 6a022988Crossref PubMed Scopus (75) Google Scholar]. Rather than being a redundant remnant from spermatogenesis, these RNAs appear necessary for embryo implantation and development [12.Conine C.C. et al.Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice.Dev. Cell. 2018; 46: 470-480.e3Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,42.Yuan S. et al.Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development.Development (Cambridge, England). 2016; 143: 635-647Crossref PubMed Scopus (0) Google Scholar]. Furthermore, the extracellular vesicles delivered to the maturing sperm during epididymal transit contain noncoding RNAs that are also involved in fertility and implantation [12.Conine C.C. et al.Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice.Dev. Cell. 2018; 46: 470-480.e3Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,42.Yuan S. et al.Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development.Development (Cambridge, England). 2016; 143: 635-647Crossref PubMed Scopus (0) Google Scholar]. This highlights a mechanism by which crosstalk between somatic cells and the maturing spermatozoa can occur. Altogether, despite extensive chromatin compaction, there are multiple mechanisms whereby the sperm epigenome can reshape offspring embryonic and postnatal development.Furthermore, the noncoding RNA profiles of sperm can be altered by environmental exposures experienced during a male’s reproductive lifetime [14.Gapp K. et al.Alterations in sperm long RNA contribute to the epigenetic inheritance of the effects of postnatal trauma.Mol. Psychiatry. 2020; 25: 2162-2174Crossref PubMed Scopus (46) Google Scholar,19.Tyebji S. et al.Pathogenic infection in male mice changes sperm small RNA profiles and transgenerationally alters offspring behavior.Cell Rep. 2020; 31107573Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,43.Short A.K. et al.Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring.Transl. Psychiatry. 2016; 6: e837Crossref PubMed Google Scholar]. Strikingly, microinjection of these differentially expressed noncoding RNAs into mouse zygotes can reproduce similar offspring phenotypes to those observed in epigenetic inheritance models involving paternal stress and infection [15.Rodgers A.B. et al.Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 13699-13704Crossref PubMed Scopus (402) Google Scholar,19.Tyebji S. et al.Pathogenic infection in male mice changes sperm small RNA profiles and transgenerationally alters offspring behavior.Cell Rep. 2020; 31107573Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Although the roles of sperm noncoding RNAs in intergenerational and transgenerational inheritance are yet to be fully characterised, this evidence highlights direct links between sperm epigenetic alterations and offspring phenotypes. In mammals, spermatogenesis involves the development and maturation of PGCs into mature spermatozoa (see Figure 1 in main text) [29.Griswold M.D. Spermatogenesis: the commitment to meiosis.Physiol. Rev. 2016; 96: 1-17Crossref PubMed Scopus (299) Google Scholar]. Spermatogenesis is a continuous and efficient process that spans a mammal’s reproductive lifetime [29.Griswold M.D. Spermatogenesis: the commitment to meiosis.Physiol. Rev. 2016; 96: 1-17Crossref PubMed Scopus (299) Google Scholar]. The process begins in the basement membrane and ends in the lumen of the seminiferous tubules in the testes. A full cycle of spermatogenesis typically takes approximately 35 days in mice and 74 days in humans [30.Oakberg E.F. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium.Am. J. Anat. 1956; 99: 507-516Crossref PubMed Google Scholar]. It begins with the development of PGCs into diploid, self-renewing spermatogonia, which then mitotically divide to become spermatocytes [27.Larose H. et al.Chapter eight - gametogenesis: a journey from inception to conception.in: Wellik D.M. Current Topics in Developmental Biology. Academic Press, 2019: 257-310Google Scholar,31.Hamatani T. Human spermatozoal RNAs.Fertil. Steril. 2012; 97: 275-281Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar]. Spermatocytes then undergo two meiotic divisions to become round haploid spermatids [27.Larose H. et al.Chapter eight - gametogenesis: a journey from inception to conception.in: Wellik D.M. Current Topics in Developmental Biology. Academic Press, 2019: 257-310Google Scholar]. These spermatids morphologically mature into spermatozoa in a process known as spermiogenesis [32.Kimmins S. et al.Testis-specific transcription mechanisms promoting male germ-cell differentiation.Reproduction. 2004; 128: 5-12Crossref PubMed Google Scholar]. However, the spermatozoa exiting the testes after spermatogenesis still need to gain the motility and functional maturity to be able to fertilise the oocytes [33.Sullivan R. Mieusset R. The human epididymis: its function in sperm maturation.Hum. Reprod. Update. 2016; 22: 574-587Crossref PubMed Scopus (140) Google Scholar]. As they transition from the caput to the caudal region of the epididymis during their final stages of maturation, the spermatozoa receive growth factors and extracellular vesicles from the epithelial cells bordering the tubule [34.Sharma U. et al.Small RNAs are trafficked from the epididymis to developing mammalian sperm.Dev. Cell. 2018; 46: 481-494.e6Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar,35.Sharma U. et al.Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.Science. 2016; 351: 391Crossref PubMed Google Scholar]. During spermatogenesis, chromatin packaging occurs to attain nuclear compaction [27.Larose H. et al.Chapter eight - gametogenesis: a journey from inception to conception.in: Wellik D.M. Current Topics in Developmental Biology. Academic Press, 2019: 257-310Google Scholar]. Histones that package the spermatozoal DNA are mostly replaced by intermediary transition proteins, which are then replaced by protamines [36.Godmann M. et al.The dynamic epigenetic program in male germ cells: its role in spermatogenesis, testis cancer, and its response to the environment.Microsc. Res. Tech. 2009; 72: 603-619Crossref PubMed Scopus (0) Google Scholar]. Protamines are small basic proteins that can tightly pack the sperm DNA [37.Wykes S.M. Krawetz S.A. The structural organization of sperm chromatin.J. Biol. Chem. 2003; 278: 29471-29477Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar]. In humans, 15% of histones are retained in mature sperm, whereas only 1–2% of histones are retained in sperm from mice [38.Rando O.J. Intergenerational transfer of epigenetic information in sperm.Cold Spring Harb. Perspect. Med. 2016; 6a022988Crossref PubMed Scopus (75) Google Scholar,39.Yehuda R. et al.Relationship of parental trauma exposure and PTSD to PTSD, depressive and anxiety disorders in offspring.J. Psychiatr. Res. 2001; 35: 261-270Crossref PubMed Scopus (192) Google Scholar]. Sperm transcriptomic activity, including RNA synthesis, is also minimised during this process of nuclear compaction [39.Yehuda R. et al.Relationship of parental trauma exposure and PTSD to PTSD, depressive and anxiety disorders in offspring.J. Psychiatr. Res. 2001; 35: 261-270Crossref PubMed Scopus (192) Google Scholar,40.Dadoune J.-P. Spermatozoal RNAs: what about their functions?.Microsc. Res. Tech. 2009; 72: 536-551Crossref PubMe