A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance

Alexandre Champroux; Julie Cocquet; Joëlle Henry-Berger; Joël R. Drevet; Ayhan Kocer

doi:10.3389/fcell.2018.00050

A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance

Champroux, Alexandre; Cocquet, Julie; Henry-Berger, Joëlle; Drevet, Joël R.; Kocer, Ayhan 2018-05-15 00:00:00 REVIEW published: 15 May 2018 doi: 10.3389/fcell.2018.00050 A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance 1 2 1 1 Alexandre Champroux , Julie Cocquet *, Joëlle Henry-Berger , Joël R. Drevet and Ayhan Kocer * GReD, Laboratoire “Génétique, Reproduction and Développement,” UMR Centre National de la Recherche Scientiﬁque 6293, INSERM U1103, Université Clermont Auvergne, Clermont-Ferrand, France, INSERM U1016, Institut Cochin, Centre National de la Recherche Scientiﬁque UMR8104, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Edited by: The past decade has seen a tremendous increase in interest and progress in the ﬁeld of Reinhard Stöger, sperm epigenetics. Studies have shown that chromatin regulation during male germline University of Nottingham, United Kingdom development is multiple and complex, and that the spermatozoon possesses a unique Reviewed by: epigenome. Its DNA methylation proﬁle, DNA-associated proteins, nucleo-protamine Johannes Beckers, distribution pattern and non-coding RNA set up a unique epigenetic landscape which Helmholtz Zentrum München - Deutsches Forschungszentrum für is delivered, along with its haploid genome, to the oocyte upon fertilization, and therefore Gesundheit und Umwelt, Germany can contribute to embryogenesis and to the offspring health. An emerging body of Chris Murgatroyd, compelling data demonstrates that environmental exposures and paternal lifestyle can Manchester Metropolitan University, United Kingdom change the sperm epigenome and, consequently, may affect both the embryonic *Correspondence: developmental program and the health of future generations. This short review will Julie Cocquet attempt to provide an overview of what is currently known about sperm epigenome and julie.cocquet@inserm.fr Ayhan Kocer the existence of transgenerational epigenetic inheritance of paternally acquired traits that ayhan.kocer@uca.fr may contribute to the offspring phenotype. Specialty section: Keywords: sperm, paternal epigenome, chromatin, histone post-translational modiﬁcations, protamines, embryo This article was submitted to development, DNA methylation, sperm non-coding RNA Epigenomics and Epigenetics, a section of the journal Frontiers in Cell and Developmental INTRODUCTION Biology The principal function of spermatozoa is to deliver the haploid paternal genome to an oocyte Received: 19 December 2017 Accepted: 18 April 2018 during fertilization. Spermatozoa are highly specialized cells generated in the testis through a Published: 15 May 2018 diﬀerentiation process called spermatogenesis. They are equipped with speciﬁc structures to achieve fertilization, such as a ﬂagellum which confers mobility and a very compact nucleus which Citation: Champroux A, Cocquet J, ensures protection of the paternal genome. Their chromatin organization diﬀers signiﬁcantly from Henry-Berger J, Drevet JR and that of somatic cells: while, in the latter, the chromatin is packed into nucleosomes containing Kocer A (2018) A Decade of Exploring 146 bp of DNA wrapped around an octamer of basic proteins named histones (Kornberg, the Mammalian Sperm Epigenome: 1974), in mammalian sperm cells, the chromatin is organized in a very compact structure Paternal Epigenetic and described as a “doughnut loop” or “toroid” in which the DNA is wrapped around even more Transgenerational Inheritance. basic, smaller proteins, called protamines. As a result, the sperm nucleus is about seven times Front. Cell Dev. Biol. 6:50. doi: 10.3389/fcell.2018.00050 smaller than that of an interphase somatic cell (Ward and Coﬀey, 1991). During the last step of Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance spermatogenesis, histones are progressively replaced by DNA METHYLATION AND BEYOND protamines through a series of complex chromatin remodeling DNA methylation is the most studied epigenetic modiﬁcation events which have been the focus of recent studies (see for in the literature, probably owing to its robustness compared to instance, Gaucher et al., 2012; Montellier et al., 2013; Barral et al., histone post-translational modiﬁcation (PTM) or RNA. 2017; Liu et al., 2017). The key steps of histone-to-protamine DNA methylation is an important regulator of gene expression transitions are (i) opening of the chromatin, owing to the demonstrated to be globally involved in gene regulation, and, combination of post translational modiﬁcations of histones, more speciﬁcally, in transposon silencing, genomic imprinting, in particular histone hyperacetylation, and incorporation of maintenance of genome integrity or X chromosome inactivation histone variants, (ii) eviction of most histones while transition (for reviews, see Bird, 2002; Hackett et al., 2012; Messerschmidt proteins and some late expressed histone variants are transiently et al., 2014). It consists in the addition of a methyl group of incorporated, (iii) removal of transition proteins and of most S-adenosyl-1-methionine to carbon ﬁve of cytosine resulting remaining histones, while protamines are incorporated (For in the formation of 5-methylcytosine (5mC). In mammals, reviews on this topic, see Rathke et al., 2014; Hoghoughi et al., cytosine methylation occurs mostly in a context of CpG 2017). The majority of histones are replaced by protamines dinucleotides. CpG islands (CGIs), which possess high CpG during spermatogenesis but some do persist in mature sperm density (i.e., ∼1 kb-long regions with greater than 50% CpG), (Figure 1; Tanphaichitr et al., 1978; Gatewood et al., 1990; Oliva are predominantly unmethylated. Other CpGs (in a non- and Dixon, 1991; Wykes and Krawetz, 2003). First seen as CGI context) are usually methylated (Deaton and Bird, 2011; histone remnants of an ineﬃcient replacement process, it is now Messerschmidt et al., 2014). Overall, DNA methylation at rather clear to the scientiﬁc community that sperm persisting promoter regions is associated with gene repression (except for histones contribute to the paternal information brought to promoters with low CpG content, for review see Messerschmidt the oocyte and the developing embryo. There is nevertheless et al., 2014). DNA methylation is carried out by DNA an ongoing debate regarding the genomic location of those methyltransferases (DNMTs) that are divided in two classes: persistent histones (Hammoud et al., 2009; Brykczynska et al., DNMT3A, DNMT3B, and DNMT3L (Bestor T., 1988; Bestor 2010; Carone et al., 2014; Samans et al., 2014; Kocer et al., T. H., 1988) are responsible for de novo methylation, while 2015; Royo et al., 2016). Interestingly, it has also been recently DNMT1 is involved in methylation maintenance (for review, demonstrated that spermatozoa do not only transmit their DNA see Chen and Li, 2004). A fourth de novo DNMT has been and chromatin to the embryo. Along with the haploid genome recently identiﬁed in rodents (Barau et al., 2016; Jain et al., and various epigenetic marks carried by the DNA and associated 2017). chromatin proteins, spermatozoa bring along a complex array of In gametes, DNMTs also establish a diﬀerential DNA RNAs (both coding and non-coding). Recent reports have shown methylation on ICR (Imprinting Control Regions) a that some of these RNAs may aﬀect the developing embryo phenomenon known as paternal or maternal imprinting and/or the progeny (Chen et al., 2016a; Sharma et al., 2016). depending on whether it takes place in male or female germ cells Therefore, from now on, the issues of male fertility, successful (Kaneda et al., 2004; Kato et al., 2007). These ICR are maintained reproduction, optimal embryonic development, oﬀspring’s health in the embryo meaning that somatic cells present, at a few and that of the subsequent generations cannot be solely attributed loci, paternal or maternal mono-allelic methylation patterns to sperm DNA integrity. The picture is now more complex and which results in a maternal or paternal mono-allelic expression, beside an optimal paternal genetic code one has to consider respectively (Hanna and Kelsey, 2014). This phenomenon only the highly dynamic and environmentally susceptible epigenetic concerns a minor part of the genome (to date there are fewer than information carried by the paternal nucleus. 30 ICR in the mouse genome) but has an important signiﬁcation In the present review, we focus on sperm-speciﬁc chromatin in term of development and health. Most of them are maternal features and epigenome. Subsequently, with the literature ICR and only 3 of them are established in male gametes. available to date we illustrate the impact that the sperm Transposon silencing is also mediated by DNMTs and epigenome may have on early embryo development and the established in the male gametes with a high eﬃciency. consequences of environmental eﬀects on paternal epigenome All these distinct sex-dependent DNA methylation proﬁles inheritance. are essential for male gametogenesis as demonstrated by reports that loss of function of DNMT3A and 3B leads to THE MAMMALIAN SPERM EPIGENOME an arrest in meiosis, an overexpression of repeated elements such as LINEs (long interspersed nuclear elements) and Epigenetics (from “epi” in Greek which means above/upon/near) IAP (intracisternal A particles) and, a loss in spermatocytes is the study of heritable changes in gene expression that are via apoptosis; while loss of function of DNMT3L leads not caused by modiﬁcations in the primary DNA sequence to male sterility because de novo methylation is absent in (Goldberg et al., 2007). Epigenetic information is cell speciﬁc, germ cells (Walsh et al., 1998; Bourc’his and Bestor, 2004; dynamic and responsive to environmental inﬂuences. Epigenetic Kaneda et al., 2004; Kato et al., 2007). In human, a recent regulation can occur via at least three main processes in study shows that single-nucleotide polymorphisms (SNPs) mammals: (i) DNA methylation and associated modiﬁcations, in diﬀerent DNMT genes induce idiopathic male infertility (ii) the histone/chromatin code which consists mainly in histone associated with abnormal semen parameters (Tang et al., variants and their post-translational modiﬁcations, and (iii) 2017). Coding and non-coding RNA. Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance FIGURE 1 | The sperm epigenome. During spermatogenesis, most histones are replaced by protamines (PRM). However, few histones remain in chromosomal domains called ≪ solenoid ≫ as well as in the short DNA segments connecting two adjacent toroids which are also attached to the sperm nuclear matrix (Matrix Attachment Regions = MAR, red bar). The sperm DNA is thus mainly packaged into toroids (protamine rich regions) and, for a minor proportion, into solenoids (histone rich regions) overall allowing a great condensation of the chromatin (A). This condensed state of the sperm nucleus is further enhanced during epididymal maturation via intra- and inter-protamine disulﬁde bonds (B). The sperm nucleus also harbors epigenetic marks at various levels: for example, on the sperm DNA there are complex methylation proﬁles with regions rich in 5-methylcytosine (5 mC) as well as in 5-hydroxymethylcytosine (5hmC). At the level of the sperm chromatin, persisting histones are concerned by a vast array of post-translational modiﬁcations (PTM; A). Finally, a third epigenetic information is associated with the sperm cell it is represented by a complex pool of RNA (mRNA and several classes of non-coding RNA). This RNA-mediated epigenetic information is acquired both during spermatogenesis and the post-testicular maturation processes (i.e., during epididymal transit; black arrow) with non-coding RNA being transferred from the epididymal epithelium toward sperm cell via lipid-rich exosomes named, epididymosomes (B). C, cytosine; G, guanine; SH, thiol group; S-S, disulﬁde bridge. Overall, depending on the cellular type and development During the formation and migration of PGC at E12.5 to E13.5 period, the genome is more or less methylated with two major days in mouse, DNA methylation is erased up to a point that reprogramming time points during mammalian development: in solely 10% of total CpG remain methylated. This is required primordial germ cells (PGC) and pre-implantation embryos. because PGC are derived from embryo cells that have already Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance acquired a somatic fate (Hajkova et al., 2002; Guibert et al., 2012; IAP (Intracisternal A-particle) is resistant to this genome-wide Seisenberger et al., 2012). The erasure of CpG methylation (5mC) reprogramming with potential for transgenerational inheritance is a key component of PGC speciﬁcation, but the dynamics (see below). and underlying mechanisms of this process remained unclear Numerous studies have correlated abnormal sperm DNA (Hackett et al., 2012). Recent studies have shown that conversion modiﬁcation patterns with male infertility (for review, see of 5mC to 5hmC (5-hydroxymethylcytosine) is instrumental to Cui et al., 2016), starting with defects in DNA methylation the DNA demethylation process and involves enzymes belonging on ICR that are associated with failures in spermatogenesis to the ten-eleven translocation (Tet) family (Tahiliani et al., (Hartmann et al., 2006; Denomme et al., 2017). In clinic, patients 2009; Gan et al., 2013; Hackett et al., 2013). Further down, showing overall low DNA methylation or a non-methylated 5hmC can itself be oxidized by Tet into 5fC (standing for 5- Igf2/H19 locus present a reduced sperm quality, a decrease formylcytosine) and 5caC (standing for 5-carboxycytosine; He in sperm count and mobility when compared to fertile men et al., 2011; Ito et al., 2011). In mammals, the Tet family contains (Niemitz and Feinberg, 2004; Boissonnas et al., 2010; Zama and three members (Tet 1–3) that are expressed diﬀerently according Uzumcu, 2010). Other abnormal proﬁles in DNA methylation to tissue and stage of development (Tahiliani et al., 2009). Tet 1 on imprinting gene regions such as Mest (mesoderm speciﬁc and 2 are present in embryonic stem (ES) cells and PGC (Hackett transcript) or on spermatogenesis implicated genes such as for et al., 2012) while Tet3 is expressed in the oocyte, spermatozoon example Dazl (deleted in azoospermia like) were shown to be and in the preimplantation embryo. In mouse, PGC, at around associated with oligozoospermia, i.e. low sperm concentration E15.5, DNA methylation is rapidly re-acquired, and the germ (Marques et al., 2008; Poplinski et al., 2010). More recently, it cell-speciﬁc proﬁle is fully established after birth in pachytene was also reported that modiﬁcation in 5hmC pattern in sperm spermatocytes through the action of DNMT 3A, 3B, 3L, and 3C is associated with male infertility (Wang et al., 2015; Eﬁmova (Guibert et al., 2012; Seisenberger et al., 2012; Barau et al., 2016). et al., 2017). In these reports, infertile males were shown to At the end of gametogenesis, spermatozoa are extensively contain higher rate of 5 hmC than fertile males and infertility methylated with ∼90% of methylated CpG, a situation that correlated with defects in sperm morphology and a high sperm is diﬀerent from that of the oocyte where only 40% at CpG DNA fragmentation rate (Eﬁmova et al., 2017). Several studies are methylated (Popp et al., 2010). DNA modiﬁcations in have also reported that a link exists between assisted reproductive spermatozoa are mostly in transposons and intergenic regions, technologies (ART) and loss of imprinting resulting in an while gene bodies and CGI are sparsely methylated (Smith et al., increased incidence of genomic imprinting disorders in children 2012). In many species, the sperm DNA methylation pattern conceived via ART (for review, see Ventura-Juncá et al., 2015). appears to be diﬀerent from that of somatic cells but similar These include Beckwith-Wiedemann syndrome (Chang et al., to that of ES cells (Weber and Schübeler, 2007; Farthing et al., 2005), Angelman syndrome, Silver-Russel syndrome (Chopra 2008). For instance promoters of transcription and signaling et al., 2010) and retinoblastoma (Marees et al., 2009). factors controlling early development such as Sox-, Fox-, Hox Nevertheless, a study shows that mice produced by or Gata- family are equally hypomethylated in ES cells and intracytoplasmic sperm injection (ICSI) present altered spermatozoa (Hammoud et al., 2014). Interestingly, these same DNA methylation on imprinted genes (de Waal et al., 2012) regions apparently contain persisting histones (Hammoud et al., but oﬀspring of ICSI-derived males exhibited normal epigenetic 2009; Brykczynska et al., 2010; Erkek et al., 2013) suggesting a role proﬁles in their somatic tissues, suggesting correction of the of the sperm epigenome in the regulation of gene expression after observed altered epigenome by germ-line speciﬁc epigenetic fertilization. reprogramming (de Waal et al., 2012). These results suggested In the oocyte, zygote and early embryo, the DNA methylation that ART procedures can lead to epigenome alterations that pattern is the opposite, i.e. methylation restricted to CGI and are normally corrected in the germ line through epigenetic gene bodies (Smith et al., 2012). Other cytosine modiﬁcations, reprogramming and thus not propagated to subsequent such as 5hmC, 5fC, and 5caC are minor compared to 5mC generations. but despite representing only a small fraction of modiﬁed CpG, their location could be biologically meaningful. In their study of 5hmC at diﬀerent stages of male germ cell diﬀerentiation, Gan SPERM CHROMATIN et al. observed that 5hmC represents less than 2.5% of 5mC in male germ cells but is very dynamic as it changes in genomic The eukaryotic genome is compacted into the nucleus via a regions related to the regulation of gene expression (such as multi-layer structure, the chromatin. The association of a histone transposons and piRNA clusters) and correlates positively with octamer with ∼146 bp of DNA forms the basal chromatin genes expressed during spermatogenesis, with high level of 5hmC structure of all somatic cells, the nucleosome. The organization found in coding exons of highly expressed genes (Gan et al., of the sperm chromatin is unique compared to that of other cells, 2013). as its basal unit is the nucleoprotamine. During the last phase In mouse, a few days after fertilization, DNA methylation of spermatogenesis takes place an extensive remodeling of male is erased, with substantial diﬀerences in timing between the germ chromatin that results in the replacement of most histones paternal and maternal pronuclei (Messerschmidt et al., 2014). by smaller and more basic proteins than histones, the protamines. However, a small portion of DNA methylation, principally This leads to a chromatin structure known as “doughnut loop” or imprinted regions and a sub-category of transposons called “toroid” containing 50–100 Kb of DNA (depending on the species Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance analyzed). This structure is ∼6–10 times more condensed than Gene expression and chromatin organization in somatic cells a classical somatic nucleosome (Ward and Coﬀey, 1989, 1991; is also inﬂuenced by nucleosome remodeling, i.e. incorporation Balhorn, 2007). In mammals, including human, two protamines, of histone variants that have diﬀerent properties and functions Prm1 and Prm2 exist. The ratio between the two Prm is 1:1 compared to that of canonical histones leading to subtle changes in human sperm but shows a wide range of variability between in chromatin organization. The incorporation of histone variants species (Corzett et al., 2002). However, the protamine ratio depends on cellular types and cell cycle (for review, see Talbert is tightly controlled and an aberrant ratio is associated with and Henikoﬀ, 2017). male infertility (Balhorn et al., 1988; Aoki et al., 2006). In Histone PTMs and histone variants also play a major role order to study the structure and composition of the mammalian in the reorganization of sperm chromatin. First, because they sperm chromatin, two main approaches have been used: either are involved in the extensive chromatin remodeling process that DNA digestion by endonucleases, such as DNase I or Mnase takes place in elongating spermatids and leads to the replacement (Pittoggi et al., 1999; Zalenskaya et al., 2000; Hammoud et al., of histones by protamines. Secondly, because a small portion 2009; Brykczynska et al., 2010; Erkek et al., 2013), or high of histones (∼1–10% depending on species) is maintained in salt treatments to disrupt proteins-DNA associations coupled spermatozoa, and those persisting histones bear PTMs and to digestion of free DNA typically by using EcoRI and BamHI include non-canonical histones (so called histone “variants”). It is endonucleases (Wykes and Krawetz, 2003; Arpanahi et al., 2009). worth noting that a majority of histone variants are encountered Taken together, these studies showed that the sperm DNA is at one point during spermatogenesis; they are involved in mainly associated with protamines and that only few regions establishing a male germ cell-speciﬁc gene expression program remain associated with histones both canonical and variant ones and/or in histone-to-protamine chromatin remodeling process (Hammoud et al., 2009; Brykczynska et al., 2010; Erkek et al., (see Table 1). Consequently, deﬁciency in histone variants is 2013). From these studies, it was found that about 2% histones often associated with defective spermatogenesis (see Table 1). persist in the sperm nucleus of mouse, hamster and bull, while in Prior to histone eviction, in spermatids, histone variants human around 5–10% of histones remain (Balhorn et al., 1977; are incorporated and many histone residues, modiﬁed. This Gatewood et al., 1990; Bench et al., 1996; Tovich and Oko, 2003; leads to nucleosome destabilization and chromatin opening (for Hammoud et al., 2009; Erkek et al., 2013). Persisting histones reviews, see Rathke et al., 2014; Hoghoughi et al., 2017). Histones were reported to be organized into solenoid structures within are replaced by a transitory structure composed of transition the protamine embedded chromatin and also, associated with proteins (TPs) that regulate protamine processing and assembly linker DNA segments connecting adjacent toroids, a chromatin (Goudarzi et al., 2014). Interestingly, one particular spermatid- structure formed by protamines (Sotolongo et al., 2003; Ward, expressed histone variant H2A.L2 is incorporated along with 2010; Noblanc et al., 2013) as schematized in Figure 1. transition proteins after most histones have been removed, Chromatin organization in somatic cells depends on the and facilitate nucleoprotamine assembly (Barral et al., 2017). composition of nucleosomes in histone variants and histone In addition, many modiﬁcations of histone residues have been PTMs. To date, the most extensively studied histone PTMs reported to occur prior to histone removal; the most striking and are lysine acetylation (which often correlates with nucleosome described in the literature being histone H4 hyperacetylation (see destabilization and transcriptional activity) and methylation, Goudarzi et al., 2014). It is the molecular signal recognized by which depending on the residue can impact on gene expression the testis-speciﬁc bromodomain protein BRDT which controls either by activation or repression. For example, histone H3 the replacement of histones by transition proteins (Gaucher trimethylated at lysine 27 (termed H3K27me3) deposited by et al., 2012). Other chromatin remodeling proteins and enzymes the histone-lysine N-methyltransferase enzyme EZH2, leads to have been described to be involved in histone to protamine chromatin compaction and thus to inhibition of transcription. transition. This is the case of CHD5 (Chromodomain helicase This mark is notably found in the constitutive heterochromatin. DNA binding protein 5; Li et al., 2014; Zhuang et al., 2014), In contrast, H3K4me3 (i.e., trimethylation at lysine 4 of histone CDYL (Chromodomain Y-like transcription corepressor) which 3) is associated with actively transcribed genes (Barski et al., 2007; acts as a crotonyl-coA-hydratase (Liu et al., 2017), the histone for review, see Bernstein et al., 2007). The recent development acetyl transferase TIP60 (Dong et al., 2017), the ubiquitin ligase of mass spectrometry-based analyses of histone PTM has RNF8 (Lu et al., 2010) or PIWI (Gou et al., 2017). In human, low immensely complicated the picture with 20 diﬀerent types of germ cell expression of CHD5 and BRDT is associated with male modiﬁcations identiﬁed to date resulting in several hundred infertility (Steilmann et al., 2010; Li et al., 2014; Zhuang et al., diﬀerent histone PTMs (for review, see Huang et al., 2015; 2014). Andrews et al., 2016). Also, a recent study identiﬁed 11 PTMs Along with histone H4 acetylation at multiple sites, on protamines both in mouse and human sperm including other PTMs occur on histones prior to their turn-over, phosphorylation and acetylation (Brunner et al., 2014). The such as methylation, phosphorylation, crotonylation and authors suggested that PTMs of protamines are involved in the ubiquitinylation of histone H3 and H2B residues (Wendt and process of deposition of protamines in sperm and their eviction Shilatifard, 2006; Govin et al., 2010; Lu et al., 2010; Tan et al., after fertilization. These concurred with earlier observations that 2011; Brunner et al., 2014; Dottermusch-Heidel et al., 2014a,b; phosphorylation of Prm was necessary for its deposition on DNA Pentakota et al., 2014; Rathke et al., 2014; Mishra et al., 2015; during spermatogenesis (Dadoune, 2003). Hada et al., 2017; Hoghoughi et al., 2017). Some of them are Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance TABLE 1 | Variant histones in mammalian sperm nucleus. Acronym Description Phenotype of knockout mice References H1t Testis-speciﬁc variant of histone H1 No data Drabent et al., 2003 H1t2 H1 histone family, member N, testis-speciﬁc Male infertility due to sperm elongation and Martianov et al., 2005; Tanaka (H1fnt) nucleus condensation defects et al., 2005 γH2A.X Speciﬁcally present at DNA breaks during Male infertility due to failure of sex-body Celeste et al., 2002; sperm chromatin remodeling formation and defects in pairing meiotic sex Fernandez-Capetillo et al., 2003; chromosome in spermatocytes Blanco-Rodríguez, 2009; Turinetto and Giachino, 2015 H2A.Z Involved in transcription, DNA repair, chromatin Early embryo lethality Suto et al., 2000; Meneghini et al., cohesion, centromeres structures and 2003; Greaves et al., 2007; Xu eu/heterochromatin boudaries maintainance et al., 2012; Sharma et al., 2013 MacroH2A Or Associated to heterochromatin and interaction Reduced litter Pehrson and Fried, 1992; Pehrson mH2A with histones deacetylases, silencing of sex Perinatal death in the absence of two et al., 2014 chromosomes during spermatogenesis MacroH2A isoforms TH2A Testis-speciﬁc variant of histone H2A No data When both genes Trostle-Weige et al., 1982 are knocked-out: male infertility due to male infertility due to sperm TH2B Testis-speciﬁc variant of histone H2B No phenotype nucleus Montellier et al., 2013; Shinagawa condensation Canonical H2B et al., 2015 compensation defects H2A.L1 & 2 Found in condensed spermatids on Male infertility associated with sperm chromatin Govin et al., 2007; Barral et al., pericentromeric heterochromatin decondensation 2017 H3.3 Variant of histone H3 Male infertility due to sperm chromatin Szenker et al., 2011; Yuen et al., decondensation associated lack of Prm protein 2014 H3t Testis-speciﬁc variant of histone H3 Male infertility associated with loss of Govin et al., 2004; Ueda et al., spermatocytes 2017 H3.5 Testis-speciﬁc variant of histone H3 in human No data Schenk et al., 2011 CENP-A Centromeres-speciﬁc variant of histone H3 Embryos die at preimplantation stage Palmer et al., 1991 Beside the names (acronyms and extended names) of the histone variants found in the mouse sperm nucleus, the table reports the effects recorded when the corresponding gene was knocked-out. Prm, Protamine. transmitted to the embryo via the persisting histones; their role compartments) that are enriched in persisting histones (Ke et al., and eﬀect remain to be determined. 2017), as measured by Micrococcal nuclease (Mnase) digestion Finally, chromatin organization and gene regulation also assays (see below). depend on a higher order (3D) chromatin structure, in which A burning question related to the sperm epigenome is the megabase-size genomic regions form topologically associated location, function and contribution to the embryo of the small domains (TADs; Dixon et al., 2012; Sexton et al., 2012). portion of histones that persist in spermatozoa. To address Those domains of higher order chromatin structure are stable this question, several groups have undertaken studies consisting during cell diﬀerentiation and conserved through evolution in Mnase digestion of sperm chromatin followed by high (Dixon et al., 2015). They can be partitioned into diﬀerent throughput sequencing (Hammoud et al., 2009; Brykczynska compartments (A/B compartments; Lieberman-Aiden et al., et al., 2010; Erkek et al., 2013; Carone et al., 2014; Samans et al., 2009) which present good correlation with active and repressive 2014), but the answer is not clear to date. While it is clear that chromatin regions, as deﬁned by the composition in histone the positioning of sperm remaining histones is not random, PTMs. Quite surprisingly in view of the diﬀerent architectures there is an ongoing debate about the location in the sperm of sperm and somatic cell chromatin, somatic TADs appear to genome of these histones (Arpanahi et al., 2009; Hammoud be partially conserved in spermatozoa. High resolution maps et al., 2009, 2011; Brykczynska et al., 2010; Erkek et al., 2013; nevertheless showed an elevated number of additional long- Noblanc et al., 2013; Carone et al., 2014; Samans et al., 2014; range intra-chromosomal interactions (>2 Mb) many of which Royo et al., 2016). Some studies have shown that persisting occurring between diﬀerent TADs (inter-TADs), as well as inter histones are enriched at promoters of genes involved in early chromosomal contacts (Battulin et al., 2015; Jung et al., 2017; embryonic development (e.g., transcription factors, HOX genes, Ke et al., 2017). Interestingly, the organization in A/B TADs signaling proteins, etc.), microRNAs clusters, genes subjected compartments in sperm correlates with the presence of persisting to genomic imprinting and binding sites of the chromatin histones. B compartment are “repressive” domains (genes in insulator protein CCCTC-binding factor (CTCF; Arpanahi et al., B compartments showing lower expression than those in A 2009; Hammoud et al., 2009). Besides, nucleosomes were also Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance found to be preferentially retained at sparse methylated DNA enzymes of the RNA interference machinery including DROSHA sequences enriched in CpG (Hammoud et al., 2011; Erkek et al., and DICER (Subramanyam and Blelloch, 2011; Yang and Lai, 2013). When focusing on histone PTMs, those groups observed 2011) and participate to the RNA-induced silencing complex enrichment of H3K4me2/3 and H3K27me3 bivalent marks at (RISC) involved in gene silencing (for review, see Martinez promoters of developmental genes, while H3K4me2/3 alone was et al., 2002; Holoch and Moazed, 2015). Piwi-interacting RNA found at the promoter of genes with a role in spermatogenesis (termed piRNA) are another signiﬁcant part of the sncRNA and H3K27me3 alone marks promoters of genes repressed during found in spermatozoa. They are DICER-independent and are gametogenesis and early embryo development (Hammoud et al., 26–31 nucleotides long. As their name implies, piRNA interact 2009; Brykczynska et al., 2010). However, histone variant TH2B with PIWI family proteins MIWI, MIWI2 and MILI (Malone was found in gene promoters involved in sperm maturation, and Hannon, 2009). piRNA are germ cell speciﬁc and comprise fertilization and capacitation while H2A.Z was detected at peri- two subgroups according to their expression stage arbitrarily centromeric regions (Greaves et al., 2006; Hammoud et al., 2009). separated into pre-pachytene piRNA and pachytene piRNA (for These data suggest that the organization of the paternal genome review Luo et al., 2016). These piRNA are derived from repeated could inﬂuence early embryonic development (also see below). sequences and act on the silencing and DNA methylation Other reports have, however, found that persisting histones are of transposable elements (Deng and Lin, 2002; Kuramochi- predominantly associated with intergenic sequences outside of Miyagawa et al., 2004, 2008; Aravin et al., 2007; Carmell et al., gene regulatory regions (Carone et al., 2014; Samans et al., 2007; Luo et al., 2016). 2014; Kocer et al., 2015). Carone et al. (2014) reported that Recent studies have discovered that small tRNA, a novel nucleosomes are mainly distributed in gene-poor regions in class of RNA, are in fact more abundant than miRNA as they the mouse sperm, and that a subcategory of nucleosomes is constitute the majority of small ncRNA in sperm (Peng et al., retained at CTCF binding sites (Carone et al., 2014). Samans 2012; Chen et al., 2016a; Sharma et al., 2016). The sperm et al., showed nucleosome enrichment within distal intergenic small tRNA (stRNA) are mainly fragments of the 5 end of regions and introns as well as with centromere repeats and tRNA, and range in size from 29 to 34 nt. Although details of retrotransposons including LINE1 and SINEs, (Samans et al., their biogenesis remain unknown, they could have a potential 2014). But the computational approach used in this study was role in transgenerational eﬀects. This will be discussed below. contested as it appeared to induce a bias toward repetitive Otherwise, sperm tRNA harbor numerous RNA modiﬁcations elements (Royo et al., 2016). These rather conﬂicting data may or RNA-editing that contribute to their stability such as 5- be also partly explained by the diﬀerent protocols that were used methylcytidine and N -methylguanosine and has been recently in order to recover histone-bound sperm DNA domains (Kocer summarized by Chen et al., (Chen et al., 2016b). Beside stRNA, et al., 2015). In ﬁne, to date, there is no strong consensus as 28s rRNA-derived small RNAs were also recently shown enriched to which speciﬁc DNA sequences in sperm are associated with in mature sperm and possibly associated with inﬂammatory nucleosomes. situations (Chu et al., 2017). Recent studies provide evidences that beside the spermatogenetic importance of sncRNA, there is a post- SPERM RNA testicular transfer of sncRNA to sperm during post-testicular Another category of epigenetic regulators of gene expression is sperm maturation. It was shown that epididymosomes, a the vast and heterogeneous family of non-coding RNAs (ncRNA). heterogeneous population of small membrane bound vesicles They can be grouped according to their size in long or small non- that are released from the epididymal epithelium (for review, see coding RNA. In this review, we will focus on small non-coding Sullivan and Saez, 2013) bring to transiting sperm cells a load RNA (sncRNA) as they are the most described in male germ of sncRNA (Reilly et al., 2016; Sharma et al., 2016). In line with cells. In spermatozoa, sncRNA could be the basis of epigenetic these works, Dixon’s group published recently that the content information transmitted to the embryo as they enter the oocyte of sncRNA spermatozoa changed during the post-testicular upon fertilization. At the end of spermatogenesis, most of the maturation which takes place in the epididymis. The authors cytoplasm and RNA content of spermatozoa are ejected. Only a show that microRNAs (miRNAs) are highly represented in the limited number of RNA molecules remain, and they have been spermatozoa of the proximal epididymis decreased during the the focus of several studies. In addition to mRNA fragments, epididymal transit, inversely the piRNA are enriched in the several small non-coding RNA (sncRNA) were shown to be mature spermatozoa collected from cauda epididymis. These present in sperm (Kramer and Krawetz, 1997; Wykes et al., 1997, results demonstrate the complexity and dynamic nature of 2000; Ostermeier et al., 2004; Miller et al., 2005; Rassoulzadegan sncRNA proﬁle of spermatozoa (Pantano et al., 2015; Hutcheon et al., 2006; Krawetz et al., 2011; Yuan et al., 2016). Initially, the et al., 2017). To add to this complexity, a recent study, in which main classes of sncRNA found in sperm were miRNA, endo- the miRNA proﬁle of spermatozoa was investigated at the single siRNA and piRNA, the most abundant being piRNA (associated cell level, suggests that spermatozoa from the same individual with repeated sequences Krawetz et al., 2011; Pantano et al., 2015; have diﬀerent miRNA contents (Yu et al., 2017). Future studies Hutcheon et al., 2017). MicroRNA (miRNA) and endogenous are needed to shed light on the role of these post-testicular small interfering RNA (endo-siRNA) are sncRNA of about 20– transferred sncRNA especially in the way they might inﬂuence 24 nucleotides. They are synthetized in the nucleus and they embryo development, therefore contributing to another level of maturate in the cytoplasm compartment. They are catalyzed by paternal inheritance (Figure 1). Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance CONTRIBUTION OF THE SPERM genome (Probst et al., 2010; van de Werken et al., 2014). The pathway involved in this process is completely diﬀerent EPIGENOME TO THE EMBRYO between human and mouse embryos. In the human embryo, Paternal Pronucleus constitutive heterochromatin (cHC) of paternal pronucleus is As described above, the paternal nucleus is tightly compacted established by the H3K9/HP1 maternal chromatin modiﬁers due to protamines. Upon fertilization, it is remodeled via the which recognize the canonical cHC (H3K9me3 and H4K20me3) replacement of protamines with maternally derived histones carried by paternal genome. Whereas in the mouse embryo, (McLay and Clarke, 2003). The decondensed paternal DNA the sperm paternal heterochromatin is devoid of canonical then expand to approximately three times the size of the heterochromatin marks and is mainly established by PRC1/2 mature sperm nucleus, resulting in the formation of the paternal protein complexes (van der Heijden et al., 2006; Probst et al., pronucleus, compatible with DNA replication and the fusion 2010; Casanova et al., 2013; van de Werken et al., 2014). of the maternal and paternal pronuclei. Its timing has been Paternally-derived modiﬁed histones also seem to play a placed by many studies within the ﬁrst hours after fertilization critical role in establishing a totipotent embryo. For a correct depending on the species concerned (Nonchev and Tsanev, development, the paternal pronucleus is hyperacetylated shortly 1990). In studies on human sperm, protamine removal was after fertilization with acetylation of lysines 5 and 16 of H4 and observed to be completed in the hour following intracytoplasmic lysines 9, 14, 18, and 27 of histone H3 (Adenot et al., 1997; sperm injection (Jones et al., 2011). In studies in which porcine Santenard et al., 2010). sperm was used for in vitro fertilization (IVF), it was shown that Beside remaining histones and protamines, the sperm nucleus 80% of the protamines were removed within 3 h post-fertilization contain Matrix Attachment Region (MAR) that allow the (Shimada et al., 2000). On a mechanistic side, sperm nucleus formation of the male pronucleus and the replication of the protamine removal was shown to rely on an antioxidant activity zygote DNA following fertilization (Linnemann et al., 2009). mediated by maternal glutathione allowing for the reduction of Indeed, intracytoplasmic injection of sperm-DNA alone without disulﬁde bonds between protamines thus facilitating the paternal MAR into oocyte or co-injected with an isolated sperm nuclear chromatin decondensation (Perreault et al., 1988; Figure 2). matrix does not allow the formation of the male pronucleus Considering the fact that the sperm nucleus brings along or its replication. While injection of sperm nuclear matrix only persisting histones (canonical and variants) and associated associated with short DNA fragments at the base of the toroids PTMs, the question about the contribution of those persisting (so DNA fragments associated with sperm MAR) allows the histones to the embryo was asked (i.e., are they replaced by formation of the male pronucleus and DNA replication, despite maternal histones or not?). Of course, in view of the ongoing sperm DNA degradation ranging from 20 to 50% (Shaman et al., debate regarding the genomic location of sperm histones, their 2007a,b). contribution and functional role in the embryo cannot be fully understood. Most data regarding the location of sperm histones Paternal DNA Demethylation/Methylation after fecundation are based on immunodetection. It was reported Before the fusion of the two pronuclei, a demethylation step that some sperm persisting histones variants are removed after takes place. The removal of the majority of 5mC in both fertilization. For example, H2AL1/2 rapidly disappear after genomes is crucial to establish the pluripotency of the inner fertilization in the mouse (Wu et al., 2008): First detected in cell mass of the blastocyst. For both the maternal and paternal the centromeres of spermatids, these variants remain enriched pronuclei, DNA demethylation is mediated by (i) an active DNA in heterochromatin domains until displaced from paternal DNA demethylation process dependent on Tet enzymes, including shortly after fertilization (Wu et al., 2008). In contrast, histone Tet3, and the successive formation of 5 hmC, 5 caC, and 5 fC H3 replication-dependent variants H3.1 and H3.2 (Tagami et al., (Tahiliani et al., 2009). and (ii) a passive replication-dependent 2004) were detected in the male pronucleus after fertilization demethylation process (Guo et al., 2014; Shen et al., 2014; Wang and prior to DNA synthesis, though in a much lower abundance et al., 2014). Most of the DNA inherited from the father appears than in maternal chromatin (van der Heijden et al., 2005, to be demethylated before DNA replication (Mayer et al., 2000; 2008; Hajkova et al., 2010). These sperm-derived proteins are Oswald et al., 2000; Santos et al., 2002). On the contrary, the detected until the zygotic S phase initiates, at which point maternal genome undergoes slower, mainly passive, replication- they become indistinguishable from their newly incorporated dependent DNA demethylation. Only a small but signiﬁcant maternal counterparts (van der Heijden et al., 2008). H3.3 plays portion of its DNA appears to be actively demethylated by a an essential role during zygotic S-phase in the transcription of Tet3-dependent process (Guo et al., 2014; Shen et al., 2014; peri-centromeric domains that trigger their silencing following Wang et al., 2014). Tet3 is predominantly located in the male the ﬁrst cell cycle by acquisition of H3K27 methylation in pronucleus and results in an important accumulation of the the male pronucleus (Santenard et al., 2010). Four hours after 5 hmC mark in the male pronucleus (Gu et al., 2011; Iqbal fertilization, the newly formed male pronucleus appears to et al., 2011; Amouroux et al., 2016). In zygotes deﬁcient for carry only nucleosomes containing H3.3, while the female Tet3, 5mC level remains constant on the paternal genome pronucleus exists in a relatively zero-H3.3 state (Santenard et al., leading to a delay in the activation of paternal allele of genes 2010; Akiyama et al., 2011). Moreover, after fertilization, the essential for embryonic development, such as Nanog and Oct4. embryo has to rearrange paternal pronucleus heterochromatin Interestingly, it is the maternally derived Tet3 enzyme which is and euchromatin regions to form a functional embryonic responsible for the conversion of 5mC to 5 hmC in the paternal Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance FIGURE 2 | Dynamics of epigenome reprogramming of the male pronucleus. After fertilization, in one cell embryo, the sperm nucleus undergoes many remodeling events. First, protamines are replaced by maternally-derived histones (A). In addition, in some areas of the sperm nucleus where persisting histones remain, some will be replaced by canonical or other variants. Furthermore, the majority of 5-methylcytosine residues (5 mC) will be erased by two processes: a TET-TDG-BER repair process that will introduce non-methylated cytosines (C) or the AID/APOBEC/BER pathway that will change mC in thymine (T) then will repair the T-G mismatches in non-methylated cytosines (B). Some regions will be protected from DNA demethylation including repeats elements, transposons, and imprinting control regions (ICR) thanks to the presence of the Stella protein and H3K9me2 histone modiﬁcation (C). AID/APOBEC, cytidine deaminase; APE, Apurinic/Apyrimidic endonuclease; BER, Base excision repair; C, cytosine; G, guanine; GSH, Glutathione; 5m, 5-methylcytosine; 5hm, 5-hydroxymethylcytosine; 5f, 5-formylcytosine; 5caC, 5-carboxycytosine; Lig, Ligase; MAR, matrix attachment region; Pol, Polymerase; Prm, Protamine; PTM, post-translational modiﬁcations; S-S, disulﬁde bridge, TDG, Thymine DNA glycosylase; TET, Ten eleven translocation; XRCC1, X-ray repair cross-complementing protein 1. genome (Gu et al., 2011). A combined deﬁciency of Tet1 and The diﬀerent methylated-cytosine derivatives (5hmC, 5fC, Tet3 results in an increase in the 5mC mark and a loss of and 5caC) are ultimately transformed into non-methylated 5hmC at the eight-cell stage. In addition, these embryos show a cytosine by the DNA repair pathway TDG-BER (standing decrease in the expression of genes involved in the biosynthesis for thymidine DNA glycosylase-base excision repair) that is of cholesterol; the few embryos that survived showed signs of a independent of replication (Kohli and Zhang, 2013; Figure 2). holoprosencephaly associated with neurological disorders (Kang TDG enzymes recognize only the 5fC and 5caC forms whereas et al., 2015). 5hmC undergoes a deamination in 5hmU by the intervention of Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance cytosine deaminase AID/APOBEC (Zhang et al., 2012; Xue et al., embryo, as injection of a miR-34c inhibitor into zygotes was 2016). An abasic site is thus generated bringing in the players of shown to induce an arrest in embryo cell division (Liu et al., the BER repair pathways including APE1 and XRCC1 (Weber 2012). This results from the inhibiting role of miR-34c on Bcl2 et al., 2016). XRCC1 is located on the male pronucleus but absent (or B-cell leukemia/lymphoma 2) gene expression and on its from the female pronucleus (Hajkova et al., 2010; Wossidlo anti-proliferative function. et al., 2010). In addition, the DNA methyltransferase DNMT1 is It is worth noting that several studies have shown a link excluded from the nucleus of preimplantation embryos (Howell between DNA methylation and non-coding RNA. Dnmt3A and et al., 2001; Hirasawa et al., 2008), thus inducing the absence of 3B mRNA expression was shown to be negatively regulated re-methylation of the DNA immediately after fertilization. by miR-29b in mouse early embryos and alterations of miR- Importantly, some regions of the paternal sperm nucleus will 29b activity was shown to change the DNA methylation level escape demethylation after fertilization, that is the case of some in mouse preimplantation embryos leading to developmental categories of transposable elements (IAP) and imprinted genes arrest at the morula stage (Zhang et al., 2015). In another (Lane et al., 2003). Recently, studies have shown that a few study, when oocytes were fertilized using sperm derived from single copy genes (which are not imprinted) also escape the mice knocked-out for DICER or DROSHA, thus showing demethylation wave (Hackett et al., 2013; Tang et al., 2015). defective miRNAs/endo-siRNAs biogenesis, a deregulation in the To explain the absence of active DNA demethylation in expression of the embryo preimplantation genes was seen (Yuan speciﬁc regions of the male pronucleus a mechanism has been et al., 2016). Interestingly, the phenotype of these embryos could evoked. It involves the Stella protein (also known as PGC7 be saved by injecting a pool of sperm RNA from WT mice (Yuan or DPPA3) initially identiﬁed in the PGC. It is assumed that et al., 2016). Stella protects from DNA demethylation, at particular paternal Altogether these studies show the importance of all the facets imprinted loci (such as Rasgfr1) by preventing the binding of sperm epigenome not only for sperm quality and fertilization of the Tet3 protein and maintaining the presence of the abilities but also for subsequent embryo development. In histone methylation mark H3K9me2 (Nakamura et al., 2007, addition, recent studies suggest that the sperm epigenome could 2012; Bian and Yu, 2014). Interestingly, the persisting histone go beyond that and have an impact on several generations. repressive marks are associated with imprinting repression such as H3K27me3, H3K9me3, and H4K20me3 (McEwen and Ferguson-Smith, 2010). The loss of function of the Stella protein leads to an arrest in embryo development associated with a TRANSGENERATIONAL EFFECTS OF THE loss of 5mC both in male and female pronuclei (Nakamura PATERNAL EPIGENOME et al., 2007). Two other proteins have been identiﬁed to protect ICR from demethylation: Zfp57, a zinc ﬁnger protein Recent studies in animal models together with epidemiological of the KRAB family and Trim28. The interaction of these two data have suggested that epigenetic factors are responsible proteins, which speciﬁcally target ICR, induces the recruitment for the transmission of pathologies across generations. of repressor complexes such as NuRD (Nucleosome Remodesling This phenomenon was called epigenetic inheritance or Deacetylase), Setdb1 (a histone methyltransferase) and DNMTs transgenerational epigenetic inheritance. Epigenetic inheritance (Quenneville et al., 2011; Zuo et al., 2012). The loss of Trim28 involves the transmission of non-DNA base sequence is embryonic lethal due in part to defects in the expression information to the oﬀspring over multiple generations via of imprinted genes (Messerschmidt et al., 2012) while the loss the germline. Epigenetic inheritance can occur when parents of Setdb1 leads to a de-repression of retrotransposons and an (F0 generation) are exposed to multiple environmental insults increase in DNA double strand breaks (Kim et al., 2016). including nutritional stress, psychological stress, toxins and drugs exposure (Figure 3). Beside environmental origins, epigenetic Paternally Derived RNA inheritance might come from parents carrying mutations Since the discovery of sperm mRNA and non-coding RNA in epigenetic regulators (such as enzymes involved in the transmission to the embryo after fertilization, studies were establishment of DNA methylation or chromatin modiﬁcations, conducted to determine the impact(s) and role(s) of these etc.; Sharma et al., 2016). Both paternal and maternal gametes RNAs in embryonic development (Kramer and Krawetz, 1997; have been shown to transmit epigenetic information to the next Ostermeier et al., 2004; Miller et al., 2005; Jodar et al., 2013). generation (i.e., intergenerational transmission; Huypens et al., Regarding the sperm mRNA cargo, it was estimated that ∼18,000 2016). If epigenetic inheritance eﬀects due to the mother lifestyle mRNA are delivered to the embryo (Ostermeier et al., 2004). or/and environmental exposures particularly during oogenesis The same group identiﬁed six of these sperm mRNA (mRNA and during pregnancy (Gluckman et al., 2008; Jimenez-Chillaron for clusterin, AKAP4, Prm2, Cdh13, Foxg1b, and Wnt5a) and et al., 2009; Radford et al., 2014) have been widely studied and are suspected them to participate to paternal pronucleus formation now well established, only recently has it become apparent that and to control the events of early embryo development. paternal epigenetics eﬀects may also greatly inﬂuence oﬀspring Regarding sperm-borne non-coding small RNA, studies have health (Schaefer and Nadeau, 2015). This part of the review aims shown their impact in preimplantation embryo development. at summarizing recent progresses made on paternal epigenetic A striking example is that of miR-34c, a male germ line- inheritance and how environmental factors and paternal lifestyle speciﬁc miRNA that is transmitted to the embryo. Mir-34c was can alter the paternal epigenetic information, in particular its demonstrated to have a key role in the ﬁrst division of the mouse DNA methylation/modiﬁcation. Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance FIGURE 3 | Paternal epigenetic transmission to subsequent generations. Environmental exposures of male mice (F0) can modify the sperm epigenome. Epigenetic information about these exposures can be transmitted to the next F1 generation and also to the F2 generation or more F3. The offspring of subsequent generations can be subject to transgenerational phenotypes. Paternal Experiences Transmitted to the diet] could result in the appearance of metabolic disorders in the oﬀspring that were directly linked to changes in sperm Future Generation via Sperm Epigenome epigenetics (Carone et al., 2010; Ng et al., 2010; Fullston Examples of Sperm Epigenome Modiﬁcations and et al., 2013; Mejos et al., 2013; Wei J. et al., 2014; Schaefer Consequences on the Offspring and Nadeau, 2015; de Castro Barbosa et al., 2016; Schuster Several research groups using rodent models have recently et al., 2016). The detected epigenetic alterations in the oﬀspring shown that feeding male mice with unbalanced diets [either a low protein diet, a high-fat diet (HFD) or a Folate-deﬁcient concerned changes in DNA methylation at regulatory regions of Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance genes involved in metabolic processes such as glucose–insulin may increase the level of reactive oxygen species in testes, homeostasis, cholesterol biosynthesis (Carone et al., 2010; Ng which has a severe impact on DNA methylation proﬁles in et al., 2010; Fullston et al., 2013). the sperm and this germline epigenetic information can be For example, Ng et al. (2010) reported that feeding male mice transmitted from generation to generations (Anway et al., with a HFD promoted in the female oﬀspring a state of impaired 2006; Guerrero-Bosagna et al., 2012; Skinner et al., 2015). glucose-insulin homeostasis. This was evidenced by alterations Moreover, the authors demonstrate that epigenetic mechanisms in the female oﬀspring transcriptome of retroperitoneal adipose can inﬂuence and promote the occurrence of a number of DNA and pancreatic islet tissues. Changes in the methylation status sequence mutations. Indeed, by analyzing the diﬀerential DNA of the female oﬀspring Il13ra2 gene that is part of the Jak–Stat methylation regions (epimutations) and genetic mutations (copy signaling pathway was also detected in that model (Ng et al., number variation: CNV), they demonstrate an increase in copy 2010, 2014). In another study, it was reported that male mice number variants associated with altered DNA methylation in consuming a low-protein diet produced oﬀspring with increased the F3 generation (Skinner et al., 2015). Besides alterations in level of cytosine methylation in trans-acting regulatory sequences DNA methylation, exposure to environmental factors can lead of key genes involved in lipid and cholesterol biosynthetic to changes in sperm non-coding RNA and histone retention in pathways such as Pparα (Carone et al., 2010). These changes sperm of DDT-exposed male mice (Skinner et al., 2018). were correlated with the down-regulation of the expression Since changes in the methylation status of the paternal of the corresponding genes in the oﬀspring (Carone et al., genome were not always observed, other causative factors of 2010). Interestingly, in that particular model, the authors did paternal epigenetic inheritance have been suspected and looked not show any signiﬁcant change in cytosine methylation at for. Changes in chromatin packaging via sperm nuclear protein classical imprinted loci in the oﬀspring. In addition, the global modiﬁcations and non-coding RNA content were brought cytosine methylation proﬁle of the paternal sperm was not forward and explored. As discussed above, sperm chromatin is signiﬁcantly diﬀerent between male mice that were fed or mainly made of protamines which will be expelled shortly after not fed the diet. This suggested that paternal sperm DNA fertilization; the so-called persisting histones, despite its limited methylation modiﬁcations were probably not at the origin of amount, has therefore the most potential for a contribution the phenotypical trait recorded in the oﬀspring in that particular to the next generation(s). Any modiﬁcation of these persisting model. sperm histones may also be considered as part of the epigenetic In diﬀerent models, it was however shown that a diet-induced information received by the future embryo. Very few studies have stress indeed provokes changes in the DNA methylation proﬁle of succeeded to establish a relation between sperm histone changes the father’s sperm cells (Fullston et al., 2013). In particular, Wei and transgenerational defects. In one particular study, Vassoler Y. et al. (2014) reported that a situation of diet-induced paternal et al. (2013) showed that the administration of cocaine to male prediabetes modiﬁes the sperm methylation status of several rats resulted in an increased level of brain-derived neurotrophic genes such as the phosphatidylinositol (PI) 3-kinase subunits factor protein (BDNF) in the prefrontal cortex of their sons. This Pik3ca and Pik3r1 of the insulin pathway. It was proposed that was shown to be associated with an increased level of acetylated these altered sperm methylation proﬁles were at the origin of histone H3 within the Bdnf promoter in the medial prefrontal the metabolic disorders recorded in the next generations (Wei Y. cortex (Vassoler et al., 2013). This epigenetic modiﬁcation was et al., 2014). also found in the promoter of the Bdnf gene in the paternal In humans, the association between a paternal condition sperm. Although interesting, these results are not suﬃcient to and epigenetic changes in their children has been described in ascertain a transgenerational eﬀect related to sperm histone many instances. For example, Soubry et al. explored putative modiﬁcation because they did not show eﬀect across several correlations between paternal obesity and children epigenetic generations. proﬁles (Soubry et al., 2013, 2015). They reported that children of A more convincing association was shown via the use obese fathers showed low methylation at diﬀerentially methylated of a mutant mice for a protein involved in chromatin regions (so-called DMRs) of several imprinted genes such as modiﬁcation. In that model, mice overexpress a human version Mest and Peg3. Interestingly, a deregulation of the methylation of histone H3 lysine 4 (H3K4) demethylase KDM1A during status of these DMRs were reported elsewhere to be linked spermatogenesis. Spermatozoa of these mice showed reduced with the occurrence of chronic diseases and metabolic disorders H3K4 dimethylation within CpG islands of genes implicated in in the oﬀspring (Murphy and Jirtle, 2003; Jirtle and Skinner, development. In their progeny were observed severely impaired 2007). embryonic development and reduced survival rates across three A great number of studies have demonstrated that generations (Siklenka et al., 2015). However, KDM1A was not environmental exposures play an important role in the found expressed in the germline of subsequent generations, accumulation of epigenetic patterns at speciﬁc loci, which the transgenerational defects were therefore unlikely to result further aﬀects normal development and even causes various from transmitted modiﬁcations of sperm histone PTM. Since no diseases via tightly regulated gene expression at the epigenetic changes in DNA methylation were observed at CpG islands, the level. In addition to the modiﬁcations of epigenome by authors hypothesized that the transgenerational inheritance was nutrition, other environmental factors can alter epigenome mediated by sperm-borne RNA (Siklenka et al., 2015). such as toxins, unhealthy lifestyles, etc. (Schaefer and Nadeau, Several studies investigated speciﬁcally the role of small 2015). For example, in mice, male exposure to pesticides non-coding RNAs (sncRNAs) in transgenerational inheritance Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance because they are long known to be present in the germ cell lineage represented miRNAs, namely miR19, into fertilized eggs resulted and their involvement in the regulation of DNA methylation in the appearance of similar phenotypes in the developed and histone modiﬁcations is well documented (Holoch and embryos (Grandjean et al., 2015). Moazed, 2015). Those studies found that sperm-derived ncRNAs, Changes in sperm ncRNA content following an including miRNA and small tRNA derived from tRNAs, have environmental stress is not limited to metabolic stresses the potential to inﬂuence embryonic development (see above) since Gapp et al. (2014) reported that a traumatic stress such as and lead to transgenerational inheritance (Holoch and Moazed, maternal separation in early life was associated with behavioral 2015; Rodgers et al., 2015; Chen et al., 2016a; Sharma et al., and metabolic conditions in the progeny (Gapp et al., 2014). 2016). For example, a nutritional stress in rodents such as a Here too, sperm miRNAs were shown to be involved since high fat diet can modify the sperm miRNA content which micro-injection of sperm miRNAs collected from traumatized constitutes potential epigenetic signals. These signals will drive males into fertilized oocytes led to similar phenotypes. oﬀspring health and will initiate the transmission of metabolic Altogether, these studies showed that the sperm epigenome in abnormalities in future generations (Fullston et al., 2013). In particular its DNA methylation proﬁle or/and its ncRNA content 2016, two independent studies showed that a high-fat or a low- has the potential to alter the health of the next generations. protein diet given to male mice was associated with increased levels of fragmented transfer RNAs species (stRNA) in sperm CONCLUSIONS AND FUTURE that were subsequently associated with metabolic disease in their PROSPECTS oﬀspring (Carone et al., 2010; Chen et al., 2016a; Sharma et al., 2016). To attest that sperm stRNA generated by the diets were The sperm epigenome, established during spermatogenesis in the at the origin of the inherited oﬀspring phenotypes, both research testis, is highly specialized and unique. The studies mentioned groups microinjected these diet-induced small tRNA into control in the present review demonstrate that sperm is more than oocytes fertilized with sperm issued from males not subjected to a vehicle transferring its haploid genome to the oocyte. DNA the regimens. Chen et al. reported that a subset of stRNA, exhibits modiﬁcations, chromatin proteins and associated marks as well changes in expression proﬁles and RNA modiﬁcations in male as sperm-derived RNA (in particular sncRNA) constitute a mouse fed with HFD, compared with mice fed a normal diet speciﬁc epigenetic landscape, shown, in case of disruption, (ND) male mouse. Injection of sperm stRNAs from HFD male to result in male infertility, abnormal embryo development mouse into normal zygotes generated metabolic disorders in the and/or transgenerational inheritance. Future studies will have F1 oﬀspring and altered gene expression of metabolic pathways to elucidate and clarify the role and underlying mechanisms of in early embryos and islets of F1 oﬀspring (Chen et al., 2016a) sperm epigenetics in those processes, in particular its impact on Moreover, the bioinformatics analyses show that sperm stRNA the oﬀspring health. If one takes into account the additional which are diﬀerentially expressed between HFD and ND match eﬀects due to variations in the oocyte epigenome, predicting preferentially to gene promoters rather than coding regions the risk of diseases in the oﬀspring associated with germ cell which are associated with metabolic genes. Interestingly, Sharma epigenome alterations will certainly be a rather complex issue. et al, showed that other diet stresses such as protein restriction in male mice aﬀects small RNA levels in mature sperm with AUTHOR CONTRIBUTIONS increased level of 5 fragments of glycine tRNA. Furthermore, they showed that these stRNA are gained during the epididymal AC, JC, JH-B, and AK: drafted the manuscript; JD: critically transit trough epididymosomes: vesicles that fuse with sperm reviewed the manuscript. during epididymal transit. The study showed that stRNA which are aﬀected by low protein diet can regulate expression of ACKNOWLEDGMENTS transcripts driven by endogenous retroelements (MERVL) in the embryos (Carone et al., 2010; Sharma et al., 2016). Due to space constraints, we could not include all the relevant These 2016 studies conﬁrmed an earlier report showing that citations on the subject matter, for which we apologize. State male mice fed a western diet (high fat and high sugar diet) grants from the University of Clermont Auvergne-(UCA), exhibited an altered sperm miRNA proﬁle associated with an INSERM, CNRS and former Région Auvergne support the increase in metabolic disorders in their oﬀspring (Grandjean activities of the research team headed by JD. 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Paternally induced transgenerational inheritance of susceptibility Conﬂict of Interest Statement: The authors declare that the research was to diabetes in mammals. Proc. Natl. Acad. Sci. U.S.A. 111, 1873–1878. conducted in the absence of any commercial or ﬁnancial relationships that could doi: 10.1073/pnas.1321195111 be construed as a potential conﬂict of interest. Wendt, K. D., and Shilatifard, A. (2006). Packing for the germy: the role of histone H4 Ser1 phosphorylation in chromatin compaction and germ cell development. Copyright © 2018 Champroux, Cocquet, Henry-Berger, Drevet and Kocer. This is an Genes Dev. 20, 2487–2491. doi: 10.1101/gad.1477706 open-access article distributed under the terms of the Creative Commons Attribution Wossidlo, M., Arand, J., Sebastiano, V., Lepikhov, K., Boiani, M., Reinhardt, R., License (CC BY). The use, distribution or reproduction in other forums is permitted, et al. (2010). Dynamic link of DNA demethylation, DNA strand breaks and provided the original author(s) and the copyright owner are credited and that the repair in mouse zygotes. EMBO J. 29, 1877–1888. doi: 10.1038/emboj.2010.80 original publication in this journal is cited, in accordance with accepted academic Wu, F., Caron, C., De Robertis, C., Khochbin, S., and Rousseaux, S. practice. No use, distribution or reproduction is permitted which does not comply (2008). Testis-speciﬁc histone variants H2AL1/2 rapidly disappear from with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 19 May 2018 | Volume 6 | Article 50 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Cell and Developmental Biology Pubmed Central http://www.deepdyve.com/lp/pubmed-central/a-decade-of-exploring-the-mammalian-sperm-epigenome-paternal-uZvzf7LnHC

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Copyright: Copyright © 2018 Champroux, Cocquet, Henry-Berger, Drevet and Kocer.
ISSN: 2296-634X
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DOI: 10.3389/fcell.2018.00050
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Abstract

REVIEW published: 15 May 2018 doi: 10.3389/fcell.2018.00050 A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance 1 2 1 1 Alexandre Champroux , Julie Cocquet *, Joëlle Henry-Berger , Joël R. Drevet and Ayhan Kocer * GReD, Laboratoire “Génétique, Reproduction and Développement,” UMR Centre National de la Recherche Scientiﬁque 6293, INSERM U1103, Université Clermont Auvergne, Clermont-Ferrand, France, INSERM U1016, Institut Cochin, Centre National de la Recherche Scientiﬁque UMR8104, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Edited by: The past decade has seen a tremendous increase in interest and progress in the ﬁeld of Reinhard Stöger, sperm epigenetics. Studies have shown that chromatin regulation during male germline University of Nottingham, United Kingdom development is multiple and complex, and that the spermatozoon possesses a unique Reviewed by: epigenome. Its DNA methylation proﬁle, DNA-associated proteins, nucleo-protamine Johannes Beckers, distribution pattern and non-coding RNA set up a unique epigenetic landscape which Helmholtz Zentrum München - Deutsches Forschungszentrum für is delivered, along with its haploid genome, to the oocyte upon fertilization, and therefore Gesundheit und Umwelt, Germany can contribute to embryogenesis and to the offspring health. An emerging body of Chris Murgatroyd, compelling data demonstrates that environmental exposures and paternal lifestyle can Manchester Metropolitan University, United Kingdom change the sperm epigenome and, consequently, may affect both the embryonic *Correspondence: developmental program and the health of future generations. This short review will Julie Cocquet attempt to provide an overview of what is currently known about sperm epigenome and julie.cocquet@inserm.fr Ayhan Kocer the existence of transgenerational epigenetic inheritance of paternally acquired traits that ayhan.kocer@uca.fr may contribute to the offspring phenotype. Specialty section: Keywords: sperm, paternal epigenome, chromatin, histone post-translational modiﬁcations, protamines, embryo This article was submitted to development, DNA methylation, sperm non-coding RNA Epigenomics and Epigenetics, a section of the journal Frontiers in Cell and Developmental INTRODUCTION Biology The principal function of spermatozoa is to deliver the haploid paternal genome to an oocyte Received: 19 December 2017 Accepted: 18 April 2018 during fertilization. Spermatozoa are highly specialized cells generated in the testis through a Published: 15 May 2018 diﬀerentiation process called spermatogenesis. They are equipped with speciﬁc structures to achieve fertilization, such as a ﬂagellum which confers mobility and a very compact nucleus which Citation: Champroux A, Cocquet J, ensures protection of the paternal genome. Their chromatin organization diﬀers signiﬁcantly from Henry-Berger J, Drevet JR and that of somatic cells: while, in the latter, the chromatin is packed into nucleosomes containing Kocer A (2018) A Decade of Exploring 146 bp of DNA wrapped around an octamer of basic proteins named histones (Kornberg, the Mammalian Sperm Epigenome: 1974), in mammalian sperm cells, the chromatin is organized in a very compact structure Paternal Epigenetic and described as a “doughnut loop” or “toroid” in which the DNA is wrapped around even more Transgenerational Inheritance. basic, smaller proteins, called protamines. As a result, the sperm nucleus is about seven times Front. Cell Dev. Biol. 6:50. doi: 10.3389/fcell.2018.00050 smaller than that of an interphase somatic cell (Ward and Coﬀey, 1991). During the last step of Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance spermatogenesis, histones are progressively replaced by DNA METHYLATION AND BEYOND protamines through a series of complex chromatin remodeling DNA methylation is the most studied epigenetic modiﬁcation events which have been the focus of recent studies (see for in the literature, probably owing to its robustness compared to instance, Gaucher et al., 2012; Montellier et al., 2013; Barral et al., histone post-translational modiﬁcation (PTM) or RNA. 2017; Liu et al., 2017). The key steps of histone-to-protamine DNA methylation is an important regulator of gene expression transitions are (i) opening of the chromatin, owing to the demonstrated to be globally involved in gene regulation, and, combination of post translational modiﬁcations of histones, more speciﬁcally, in transposon silencing, genomic imprinting, in particular histone hyperacetylation, and incorporation of maintenance of genome integrity or X chromosome inactivation histone variants, (ii) eviction of most histones while transition (for reviews, see Bird, 2002; Hackett et al., 2012; Messerschmidt proteins and some late expressed histone variants are transiently et al., 2014). It consists in the addition of a methyl group of incorporated, (iii) removal of transition proteins and of most S-adenosyl-1-methionine to carbon ﬁve of cytosine resulting remaining histones, while protamines are incorporated (For in the formation of 5-methylcytosine (5mC). In mammals, reviews on this topic, see Rathke et al., 2014; Hoghoughi et al., cytosine methylation occurs mostly in a context of CpG 2017). The majority of histones are replaced by protamines dinucleotides. CpG islands (CGIs), which possess high CpG during spermatogenesis but some do persist in mature sperm density (i.e., ∼1 kb-long regions with greater than 50% CpG), (Figure 1; Tanphaichitr et al., 1978; Gatewood et al., 1990; Oliva are predominantly unmethylated. Other CpGs (in a non- and Dixon, 1991; Wykes and Krawetz, 2003). First seen as CGI context) are usually methylated (Deaton and Bird, 2011; histone remnants of an ineﬃcient replacement process, it is now Messerschmidt et al., 2014). Overall, DNA methylation at rather clear to the scientiﬁc community that sperm persisting promoter regions is associated with gene repression (except for histones contribute to the paternal information brought to promoters with low CpG content, for review see Messerschmidt the oocyte and the developing embryo. There is nevertheless et al., 2014). DNA methylation is carried out by DNA an ongoing debate regarding the genomic location of those methyltransferases (DNMTs) that are divided in two classes: persistent histones (Hammoud et al., 2009; Brykczynska et al., DNMT3A, DNMT3B, and DNMT3L (Bestor T., 1988; Bestor 2010; Carone et al., 2014; Samans et al., 2014; Kocer et al., T. H., 1988) are responsible for de novo methylation, while 2015; Royo et al., 2016). Interestingly, it has also been recently DNMT1 is involved in methylation maintenance (for review, demonstrated that spermatozoa do not only transmit their DNA see Chen and Li, 2004). A fourth de novo DNMT has been and chromatin to the embryo. Along with the haploid genome recently identiﬁed in rodents (Barau et al., 2016; Jain et al., and various epigenetic marks carried by the DNA and associated 2017). chromatin proteins, spermatozoa bring along a complex array of In gametes, DNMTs also establish a diﬀerential DNA RNAs (both coding and non-coding). Recent reports have shown methylation on ICR (Imprinting Control Regions) a that some of these RNAs may aﬀect the developing embryo phenomenon known as paternal or maternal imprinting and/or the progeny (Chen et al., 2016a; Sharma et al., 2016). depending on whether it takes place in male or female germ cells Therefore, from now on, the issues of male fertility, successful (Kaneda et al., 2004; Kato et al., 2007). These ICR are maintained reproduction, optimal embryonic development, oﬀspring’s health in the embryo meaning that somatic cells present, at a few and that of the subsequent generations cannot be solely attributed loci, paternal or maternal mono-allelic methylation patterns to sperm DNA integrity. The picture is now more complex and which results in a maternal or paternal mono-allelic expression, beside an optimal paternal genetic code one has to consider respectively (Hanna and Kelsey, 2014). This phenomenon only the highly dynamic and environmentally susceptible epigenetic concerns a minor part of the genome (to date there are fewer than information carried by the paternal nucleus. 30 ICR in the mouse genome) but has an important signiﬁcation In the present review, we focus on sperm-speciﬁc chromatin in term of development and health. Most of them are maternal features and epigenome. Subsequently, with the literature ICR and only 3 of them are established in male gametes. available to date we illustrate the impact that the sperm Transposon silencing is also mediated by DNMTs and epigenome may have on early embryo development and the established in the male gametes with a high eﬃciency. consequences of environmental eﬀects on paternal epigenome All these distinct sex-dependent DNA methylation proﬁles inheritance. are essential for male gametogenesis as demonstrated by reports that loss of function of DNMT3A and 3B leads to THE MAMMALIAN SPERM EPIGENOME an arrest in meiosis, an overexpression of repeated elements such as LINEs (long interspersed nuclear elements) and Epigenetics (from “epi” in Greek which means above/upon/near) IAP (intracisternal A particles) and, a loss in spermatocytes is the study of heritable changes in gene expression that are via apoptosis; while loss of function of DNMT3L leads not caused by modiﬁcations in the primary DNA sequence to male sterility because de novo methylation is absent in (Goldberg et al., 2007). Epigenetic information is cell speciﬁc, germ cells (Walsh et al., 1998; Bourc’his and Bestor, 2004; dynamic and responsive to environmental inﬂuences. Epigenetic Kaneda et al., 2004; Kato et al., 2007). In human, a recent regulation can occur via at least three main processes in study shows that single-nucleotide polymorphisms (SNPs) mammals: (i) DNA methylation and associated modiﬁcations, in diﬀerent DNMT genes induce idiopathic male infertility (ii) the histone/chromatin code which consists mainly in histone associated with abnormal semen parameters (Tang et al., variants and their post-translational modiﬁcations, and (iii) 2017). Coding and non-coding RNA. Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance FIGURE 1 | The sperm epigenome. During spermatogenesis, most histones are replaced by protamines (PRM). However, few histones remain in chromosomal domains called ≪ solenoid ≫ as well as in the short DNA segments connecting two adjacent toroids which are also attached to the sperm nuclear matrix (Matrix Attachment Regions = MAR, red bar). The sperm DNA is thus mainly packaged into toroids (protamine rich regions) and, for a minor proportion, into solenoids (histone rich regions) overall allowing a great condensation of the chromatin (A). This condensed state of the sperm nucleus is further enhanced during epididymal maturation via intra- and inter-protamine disulﬁde bonds (B). The sperm nucleus also harbors epigenetic marks at various levels: for example, on the sperm DNA there are complex methylation proﬁles with regions rich in 5-methylcytosine (5 mC) as well as in 5-hydroxymethylcytosine (5hmC). At the level of the sperm chromatin, persisting histones are concerned by a vast array of post-translational modiﬁcations (PTM; A). Finally, a third epigenetic information is associated with the sperm cell it is represented by a complex pool of RNA (mRNA and several classes of non-coding RNA). This RNA-mediated epigenetic information is acquired both during spermatogenesis and the post-testicular maturation processes (i.e., during epididymal transit; black arrow) with non-coding RNA being transferred from the epididymal epithelium toward sperm cell via lipid-rich exosomes named, epididymosomes (B). C, cytosine; G, guanine; SH, thiol group; S-S, disulﬁde bridge. Overall, depending on the cellular type and development During the formation and migration of PGC at E12.5 to E13.5 period, the genome is more or less methylated with two major days in mouse, DNA methylation is erased up to a point that reprogramming time points during mammalian development: in solely 10% of total CpG remain methylated. This is required primordial germ cells (PGC) and pre-implantation embryos. because PGC are derived from embryo cells that have already Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance acquired a somatic fate (Hajkova et al., 2002; Guibert et al., 2012; IAP (Intracisternal A-particle) is resistant to this genome-wide Seisenberger et al., 2012). The erasure of CpG methylation (5mC) reprogramming with potential for transgenerational inheritance is a key component of PGC speciﬁcation, but the dynamics (see below). and underlying mechanisms of this process remained unclear Numerous studies have correlated abnormal sperm DNA (Hackett et al., 2012). Recent studies have shown that conversion modiﬁcation patterns with male infertility (for review, see of 5mC to 5hmC (5-hydroxymethylcytosine) is instrumental to Cui et al., 2016), starting with defects in DNA methylation the DNA demethylation process and involves enzymes belonging on ICR that are associated with failures in spermatogenesis to the ten-eleven translocation (Tet) family (Tahiliani et al., (Hartmann et al., 2006; Denomme et al., 2017). In clinic, patients 2009; Gan et al., 2013; Hackett et al., 2013). Further down, showing overall low DNA methylation or a non-methylated 5hmC can itself be oxidized by Tet into 5fC (standing for 5- Igf2/H19 locus present a reduced sperm quality, a decrease formylcytosine) and 5caC (standing for 5-carboxycytosine; He in sperm count and mobility when compared to fertile men et al., 2011; Ito et al., 2011). In mammals, the Tet family contains (Niemitz and Feinberg, 2004; Boissonnas et al., 2010; Zama and three members (Tet 1–3) that are expressed diﬀerently according Uzumcu, 2010). Other abnormal proﬁles in DNA methylation to tissue and stage of development (Tahiliani et al., 2009). Tet 1 on imprinting gene regions such as Mest (mesoderm speciﬁc and 2 are present in embryonic stem (ES) cells and PGC (Hackett transcript) or on spermatogenesis implicated genes such as for et al., 2012) while Tet3 is expressed in the oocyte, spermatozoon example Dazl (deleted in azoospermia like) were shown to be and in the preimplantation embryo. In mouse, PGC, at around associated with oligozoospermia, i.e. low sperm concentration E15.5, DNA methylation is rapidly re-acquired, and the germ (Marques et al., 2008; Poplinski et al., 2010). More recently, it cell-speciﬁc proﬁle is fully established after birth in pachytene was also reported that modiﬁcation in 5hmC pattern in sperm spermatocytes through the action of DNMT 3A, 3B, 3L, and 3C is associated with male infertility (Wang et al., 2015; Eﬁmova (Guibert et al., 2012; Seisenberger et al., 2012; Barau et al., 2016). et al., 2017). In these reports, infertile males were shown to At the end of gametogenesis, spermatozoa are extensively contain higher rate of 5 hmC than fertile males and infertility methylated with ∼90% of methylated CpG, a situation that correlated with defects in sperm morphology and a high sperm is diﬀerent from that of the oocyte where only 40% at CpG DNA fragmentation rate (Eﬁmova et al., 2017). Several studies are methylated (Popp et al., 2010). DNA modiﬁcations in have also reported that a link exists between assisted reproductive spermatozoa are mostly in transposons and intergenic regions, technologies (ART) and loss of imprinting resulting in an while gene bodies and CGI are sparsely methylated (Smith et al., increased incidence of genomic imprinting disorders in children 2012). In many species, the sperm DNA methylation pattern conceived via ART (for review, see Ventura-Juncá et al., 2015). appears to be diﬀerent from that of somatic cells but similar These include Beckwith-Wiedemann syndrome (Chang et al., to that of ES cells (Weber and Schübeler, 2007; Farthing et al., 2005), Angelman syndrome, Silver-Russel syndrome (Chopra 2008). For instance promoters of transcription and signaling et al., 2010) and retinoblastoma (Marees et al., 2009). factors controlling early development such as Sox-, Fox-, Hox Nevertheless, a study shows that mice produced by or Gata- family are equally hypomethylated in ES cells and intracytoplasmic sperm injection (ICSI) present altered spermatozoa (Hammoud et al., 2014). Interestingly, these same DNA methylation on imprinted genes (de Waal et al., 2012) regions apparently contain persisting histones (Hammoud et al., but oﬀspring of ICSI-derived males exhibited normal epigenetic 2009; Brykczynska et al., 2010; Erkek et al., 2013) suggesting a role proﬁles in their somatic tissues, suggesting correction of the of the sperm epigenome in the regulation of gene expression after observed altered epigenome by germ-line speciﬁc epigenetic fertilization. reprogramming (de Waal et al., 2012). These results suggested In the oocyte, zygote and early embryo, the DNA methylation that ART procedures can lead to epigenome alterations that pattern is the opposite, i.e. methylation restricted to CGI and are normally corrected in the germ line through epigenetic gene bodies (Smith et al., 2012). Other cytosine modiﬁcations, reprogramming and thus not propagated to subsequent such as 5hmC, 5fC, and 5caC are minor compared to 5mC generations. but despite representing only a small fraction of modiﬁed CpG, their location could be biologically meaningful. In their study of 5hmC at diﬀerent stages of male germ cell diﬀerentiation, Gan SPERM CHROMATIN et al. observed that 5hmC represents less than 2.5% of 5mC in male germ cells but is very dynamic as it changes in genomic The eukaryotic genome is compacted into the nucleus via a regions related to the regulation of gene expression (such as multi-layer structure, the chromatin. The association of a histone transposons and piRNA clusters) and correlates positively with octamer with ∼146 bp of DNA forms the basal chromatin genes expressed during spermatogenesis, with high level of 5hmC structure of all somatic cells, the nucleosome. The organization found in coding exons of highly expressed genes (Gan et al., of the sperm chromatin is unique compared to that of other cells, 2013). as its basal unit is the nucleoprotamine. During the last phase In mouse, a few days after fertilization, DNA methylation of spermatogenesis takes place an extensive remodeling of male is erased, with substantial diﬀerences in timing between the germ chromatin that results in the replacement of most histones paternal and maternal pronuclei (Messerschmidt et al., 2014). by smaller and more basic proteins than histones, the protamines. However, a small portion of DNA methylation, principally This leads to a chromatin structure known as “doughnut loop” or imprinted regions and a sub-category of transposons called “toroid” containing 50–100 Kb of DNA (depending on the species Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance analyzed). This structure is ∼6–10 times more condensed than Gene expression and chromatin organization in somatic cells a classical somatic nucleosome (Ward and Coﬀey, 1989, 1991; is also inﬂuenced by nucleosome remodeling, i.e. incorporation Balhorn, 2007). In mammals, including human, two protamines, of histone variants that have diﬀerent properties and functions Prm1 and Prm2 exist. The ratio between the two Prm is 1:1 compared to that of canonical histones leading to subtle changes in human sperm but shows a wide range of variability between in chromatin organization. The incorporation of histone variants species (Corzett et al., 2002). However, the protamine ratio depends on cellular types and cell cycle (for review, see Talbert is tightly controlled and an aberrant ratio is associated with and Henikoﬀ, 2017). male infertility (Balhorn et al., 1988; Aoki et al., 2006). In Histone PTMs and histone variants also play a major role order to study the structure and composition of the mammalian in the reorganization of sperm chromatin. First, because they sperm chromatin, two main approaches have been used: either are involved in the extensive chromatin remodeling process that DNA digestion by endonucleases, such as DNase I or Mnase takes place in elongating spermatids and leads to the replacement (Pittoggi et al., 1999; Zalenskaya et al., 2000; Hammoud et al., of histones by protamines. Secondly, because a small portion 2009; Brykczynska et al., 2010; Erkek et al., 2013), or high of histones (∼1–10% depending on species) is maintained in salt treatments to disrupt proteins-DNA associations coupled spermatozoa, and those persisting histones bear PTMs and to digestion of free DNA typically by using EcoRI and BamHI include non-canonical histones (so called histone “variants”). It is endonucleases (Wykes and Krawetz, 2003; Arpanahi et al., 2009). worth noting that a majority of histone variants are encountered Taken together, these studies showed that the sperm DNA is at one point during spermatogenesis; they are involved in mainly associated with protamines and that only few regions establishing a male germ cell-speciﬁc gene expression program remain associated with histones both canonical and variant ones and/or in histone-to-protamine chromatin remodeling process (Hammoud et al., 2009; Brykczynska et al., 2010; Erkek et al., (see Table 1). Consequently, deﬁciency in histone variants is 2013). From these studies, it was found that about 2% histones often associated with defective spermatogenesis (see Table 1). persist in the sperm nucleus of mouse, hamster and bull, while in Prior to histone eviction, in spermatids, histone variants human around 5–10% of histones remain (Balhorn et al., 1977; are incorporated and many histone residues, modiﬁed. This Gatewood et al., 1990; Bench et al., 1996; Tovich and Oko, 2003; leads to nucleosome destabilization and chromatin opening (for Hammoud et al., 2009; Erkek et al., 2013). Persisting histones reviews, see Rathke et al., 2014; Hoghoughi et al., 2017). Histones were reported to be organized into solenoid structures within are replaced by a transitory structure composed of transition the protamine embedded chromatin and also, associated with proteins (TPs) that regulate protamine processing and assembly linker DNA segments connecting adjacent toroids, a chromatin (Goudarzi et al., 2014). Interestingly, one particular spermatid- structure formed by protamines (Sotolongo et al., 2003; Ward, expressed histone variant H2A.L2 is incorporated along with 2010; Noblanc et al., 2013) as schematized in Figure 1. transition proteins after most histones have been removed, Chromatin organization in somatic cells depends on the and facilitate nucleoprotamine assembly (Barral et al., 2017). composition of nucleosomes in histone variants and histone In addition, many modiﬁcations of histone residues have been PTMs. To date, the most extensively studied histone PTMs reported to occur prior to histone removal; the most striking and are lysine acetylation (which often correlates with nucleosome described in the literature being histone H4 hyperacetylation (see destabilization and transcriptional activity) and methylation, Goudarzi et al., 2014). It is the molecular signal recognized by which depending on the residue can impact on gene expression the testis-speciﬁc bromodomain protein BRDT which controls either by activation or repression. For example, histone H3 the replacement of histones by transition proteins (Gaucher trimethylated at lysine 27 (termed H3K27me3) deposited by et al., 2012). Other chromatin remodeling proteins and enzymes the histone-lysine N-methyltransferase enzyme EZH2, leads to have been described to be involved in histone to protamine chromatin compaction and thus to inhibition of transcription. transition. This is the case of CHD5 (Chromodomain helicase This mark is notably found in the constitutive heterochromatin. DNA binding protein 5; Li et al., 2014; Zhuang et al., 2014), In contrast, H3K4me3 (i.e., trimethylation at lysine 4 of histone CDYL (Chromodomain Y-like transcription corepressor) which 3) is associated with actively transcribed genes (Barski et al., 2007; acts as a crotonyl-coA-hydratase (Liu et al., 2017), the histone for review, see Bernstein et al., 2007). The recent development acetyl transferase TIP60 (Dong et al., 2017), the ubiquitin ligase of mass spectrometry-based analyses of histone PTM has RNF8 (Lu et al., 2010) or PIWI (Gou et al., 2017). In human, low immensely complicated the picture with 20 diﬀerent types of germ cell expression of CHD5 and BRDT is associated with male modiﬁcations identiﬁed to date resulting in several hundred infertility (Steilmann et al., 2010; Li et al., 2014; Zhuang et al., diﬀerent histone PTMs (for review, see Huang et al., 2015; 2014). Andrews et al., 2016). Also, a recent study identiﬁed 11 PTMs Along with histone H4 acetylation at multiple sites, on protamines both in mouse and human sperm including other PTMs occur on histones prior to their turn-over, phosphorylation and acetylation (Brunner et al., 2014). The such as methylation, phosphorylation, crotonylation and authors suggested that PTMs of protamines are involved in the ubiquitinylation of histone H3 and H2B residues (Wendt and process of deposition of protamines in sperm and their eviction Shilatifard, 2006; Govin et al., 2010; Lu et al., 2010; Tan et al., after fertilization. These concurred with earlier observations that 2011; Brunner et al., 2014; Dottermusch-Heidel et al., 2014a,b; phosphorylation of Prm was necessary for its deposition on DNA Pentakota et al., 2014; Rathke et al., 2014; Mishra et al., 2015; during spermatogenesis (Dadoune, 2003). Hada et al., 2017; Hoghoughi et al., 2017). Some of them are Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance TABLE 1 | Variant histones in mammalian sperm nucleus. Acronym Description Phenotype of knockout mice References H1t Testis-speciﬁc variant of histone H1 No data Drabent et al., 2003 H1t2 H1 histone family, member N, testis-speciﬁc Male infertility due to sperm elongation and Martianov et al., 2005; Tanaka (H1fnt) nucleus condensation defects et al., 2005 γH2A.X Speciﬁcally present at DNA breaks during Male infertility due to failure of sex-body Celeste et al., 2002; sperm chromatin remodeling formation and defects in pairing meiotic sex Fernandez-Capetillo et al., 2003; chromosome in spermatocytes Blanco-Rodríguez, 2009; Turinetto and Giachino, 2015 H2A.Z Involved in transcription, DNA repair, chromatin Early embryo lethality Suto et al., 2000; Meneghini et al., cohesion, centromeres structures and 2003; Greaves et al., 2007; Xu eu/heterochromatin boudaries maintainance et al., 2012; Sharma et al., 2013 MacroH2A Or Associated to heterochromatin and interaction Reduced litter Pehrson and Fried, 1992; Pehrson mH2A with histones deacetylases, silencing of sex Perinatal death in the absence of two et al., 2014 chromosomes during spermatogenesis MacroH2A isoforms TH2A Testis-speciﬁc variant of histone H2A No data When both genes Trostle-Weige et al., 1982 are knocked-out: male infertility due to male infertility due to sperm TH2B Testis-speciﬁc variant of histone H2B No phenotype nucleus Montellier et al., 2013; Shinagawa condensation Canonical H2B et al., 2015 compensation defects H2A.L1 & 2 Found in condensed spermatids on Male infertility associated with sperm chromatin Govin et al., 2007; Barral et al., pericentromeric heterochromatin decondensation 2017 H3.3 Variant of histone H3 Male infertility due to sperm chromatin Szenker et al., 2011; Yuen et al., decondensation associated lack of Prm protein 2014 H3t Testis-speciﬁc variant of histone H3 Male infertility associated with loss of Govin et al., 2004; Ueda et al., spermatocytes 2017 H3.5 Testis-speciﬁc variant of histone H3 in human No data Schenk et al., 2011 CENP-A Centromeres-speciﬁc variant of histone H3 Embryos die at preimplantation stage Palmer et al., 1991 Beside the names (acronyms and extended names) of the histone variants found in the mouse sperm nucleus, the table reports the effects recorded when the corresponding gene was knocked-out. Prm, Protamine. transmitted to the embryo via the persisting histones; their role compartments) that are enriched in persisting histones (Ke et al., and eﬀect remain to be determined. 2017), as measured by Micrococcal nuclease (Mnase) digestion Finally, chromatin organization and gene regulation also assays (see below). depend on a higher order (3D) chromatin structure, in which A burning question related to the sperm epigenome is the megabase-size genomic regions form topologically associated location, function and contribution to the embryo of the small domains (TADs; Dixon et al., 2012; Sexton et al., 2012). portion of histones that persist in spermatozoa. To address Those domains of higher order chromatin structure are stable this question, several groups have undertaken studies consisting during cell diﬀerentiation and conserved through evolution in Mnase digestion of sperm chromatin followed by high (Dixon et al., 2015). They can be partitioned into diﬀerent throughput sequencing (Hammoud et al., 2009; Brykczynska compartments (A/B compartments; Lieberman-Aiden et al., et al., 2010; Erkek et al., 2013; Carone et al., 2014; Samans et al., 2009) which present good correlation with active and repressive 2014), but the answer is not clear to date. While it is clear that chromatin regions, as deﬁned by the composition in histone the positioning of sperm remaining histones is not random, PTMs. Quite surprisingly in view of the diﬀerent architectures there is an ongoing debate about the location in the sperm of sperm and somatic cell chromatin, somatic TADs appear to genome of these histones (Arpanahi et al., 2009; Hammoud be partially conserved in spermatozoa. High resolution maps et al., 2009, 2011; Brykczynska et al., 2010; Erkek et al., 2013; nevertheless showed an elevated number of additional long- Noblanc et al., 2013; Carone et al., 2014; Samans et al., 2014; range intra-chromosomal interactions (>2 Mb) many of which Royo et al., 2016). Some studies have shown that persisting occurring between diﬀerent TADs (inter-TADs), as well as inter histones are enriched at promoters of genes involved in early chromosomal contacts (Battulin et al., 2015; Jung et al., 2017; embryonic development (e.g., transcription factors, HOX genes, Ke et al., 2017). Interestingly, the organization in A/B TADs signaling proteins, etc.), microRNAs clusters, genes subjected compartments in sperm correlates with the presence of persisting to genomic imprinting and binding sites of the chromatin histones. B compartment are “repressive” domains (genes in insulator protein CCCTC-binding factor (CTCF; Arpanahi et al., B compartments showing lower expression than those in A 2009; Hammoud et al., 2009). Besides, nucleosomes were also Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance found to be preferentially retained at sparse methylated DNA enzymes of the RNA interference machinery including DROSHA sequences enriched in CpG (Hammoud et al., 2011; Erkek et al., and DICER (Subramanyam and Blelloch, 2011; Yang and Lai, 2013). When focusing on histone PTMs, those groups observed 2011) and participate to the RNA-induced silencing complex enrichment of H3K4me2/3 and H3K27me3 bivalent marks at (RISC) involved in gene silencing (for review, see Martinez promoters of developmental genes, while H3K4me2/3 alone was et al., 2002; Holoch and Moazed, 2015). Piwi-interacting RNA found at the promoter of genes with a role in spermatogenesis (termed piRNA) are another signiﬁcant part of the sncRNA and H3K27me3 alone marks promoters of genes repressed during found in spermatozoa. They are DICER-independent and are gametogenesis and early embryo development (Hammoud et al., 26–31 nucleotides long. As their name implies, piRNA interact 2009; Brykczynska et al., 2010). However, histone variant TH2B with PIWI family proteins MIWI, MIWI2 and MILI (Malone was found in gene promoters involved in sperm maturation, and Hannon, 2009). piRNA are germ cell speciﬁc and comprise fertilization and capacitation while H2A.Z was detected at peri- two subgroups according to their expression stage arbitrarily centromeric regions (Greaves et al., 2006; Hammoud et al., 2009). separated into pre-pachytene piRNA and pachytene piRNA (for These data suggest that the organization of the paternal genome review Luo et al., 2016). These piRNA are derived from repeated could inﬂuence early embryonic development (also see below). sequences and act on the silencing and DNA methylation Other reports have, however, found that persisting histones are of transposable elements (Deng and Lin, 2002; Kuramochi- predominantly associated with intergenic sequences outside of Miyagawa et al., 2004, 2008; Aravin et al., 2007; Carmell et al., gene regulatory regions (Carone et al., 2014; Samans et al., 2007; Luo et al., 2016). 2014; Kocer et al., 2015). Carone et al. (2014) reported that Recent studies have discovered that small tRNA, a novel nucleosomes are mainly distributed in gene-poor regions in class of RNA, are in fact more abundant than miRNA as they the mouse sperm, and that a subcategory of nucleosomes is constitute the majority of small ncRNA in sperm (Peng et al., retained at CTCF binding sites (Carone et al., 2014). Samans 2012; Chen et al., 2016a; Sharma et al., 2016). The sperm et al., showed nucleosome enrichment within distal intergenic small tRNA (stRNA) are mainly fragments of the 5 end of regions and introns as well as with centromere repeats and tRNA, and range in size from 29 to 34 nt. Although details of retrotransposons including LINE1 and SINEs, (Samans et al., their biogenesis remain unknown, they could have a potential 2014). But the computational approach used in this study was role in transgenerational eﬀects. This will be discussed below. contested as it appeared to induce a bias toward repetitive Otherwise, sperm tRNA harbor numerous RNA modiﬁcations elements (Royo et al., 2016). These rather conﬂicting data may or RNA-editing that contribute to their stability such as 5- be also partly explained by the diﬀerent protocols that were used methylcytidine and N -methylguanosine and has been recently in order to recover histone-bound sperm DNA domains (Kocer summarized by Chen et al., (Chen et al., 2016b). Beside stRNA, et al., 2015). In ﬁne, to date, there is no strong consensus as 28s rRNA-derived small RNAs were also recently shown enriched to which speciﬁc DNA sequences in sperm are associated with in mature sperm and possibly associated with inﬂammatory nucleosomes. situations (Chu et al., 2017). Recent studies provide evidences that beside the spermatogenetic importance of sncRNA, there is a post- SPERM RNA testicular transfer of sncRNA to sperm during post-testicular Another category of epigenetic regulators of gene expression is sperm maturation. It was shown that epididymosomes, a the vast and heterogeneous family of non-coding RNAs (ncRNA). heterogeneous population of small membrane bound vesicles They can be grouped according to their size in long or small non- that are released from the epididymal epithelium (for review, see coding RNA. In this review, we will focus on small non-coding Sullivan and Saez, 2013) bring to transiting sperm cells a load RNA (sncRNA) as they are the most described in male germ of sncRNA (Reilly et al., 2016; Sharma et al., 2016). In line with cells. In spermatozoa, sncRNA could be the basis of epigenetic these works, Dixon’s group published recently that the content information transmitted to the embryo as they enter the oocyte of sncRNA spermatozoa changed during the post-testicular upon fertilization. At the end of spermatogenesis, most of the maturation which takes place in the epididymis. The authors cytoplasm and RNA content of spermatozoa are ejected. Only a show that microRNAs (miRNAs) are highly represented in the limited number of RNA molecules remain, and they have been spermatozoa of the proximal epididymis decreased during the the focus of several studies. In addition to mRNA fragments, epididymal transit, inversely the piRNA are enriched in the several small non-coding RNA (sncRNA) were shown to be mature spermatozoa collected from cauda epididymis. These present in sperm (Kramer and Krawetz, 1997; Wykes et al., 1997, results demonstrate the complexity and dynamic nature of 2000; Ostermeier et al., 2004; Miller et al., 2005; Rassoulzadegan sncRNA proﬁle of spermatozoa (Pantano et al., 2015; Hutcheon et al., 2006; Krawetz et al., 2011; Yuan et al., 2016). Initially, the et al., 2017). To add to this complexity, a recent study, in which main classes of sncRNA found in sperm were miRNA, endo- the miRNA proﬁle of spermatozoa was investigated at the single siRNA and piRNA, the most abundant being piRNA (associated cell level, suggests that spermatozoa from the same individual with repeated sequences Krawetz et al., 2011; Pantano et al., 2015; have diﬀerent miRNA contents (Yu et al., 2017). Future studies Hutcheon et al., 2017). MicroRNA (miRNA) and endogenous are needed to shed light on the role of these post-testicular small interfering RNA (endo-siRNA) are sncRNA of about 20– transferred sncRNA especially in the way they might inﬂuence 24 nucleotides. They are synthetized in the nucleus and they embryo development, therefore contributing to another level of maturate in the cytoplasm compartment. They are catalyzed by paternal inheritance (Figure 1). Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance CONTRIBUTION OF THE SPERM genome (Probst et al., 2010; van de Werken et al., 2014). The pathway involved in this process is completely diﬀerent EPIGENOME TO THE EMBRYO between human and mouse embryos. In the human embryo, Paternal Pronucleus constitutive heterochromatin (cHC) of paternal pronucleus is As described above, the paternal nucleus is tightly compacted established by the H3K9/HP1 maternal chromatin modiﬁers due to protamines. Upon fertilization, it is remodeled via the which recognize the canonical cHC (H3K9me3 and H4K20me3) replacement of protamines with maternally derived histones carried by paternal genome. Whereas in the mouse embryo, (McLay and Clarke, 2003). The decondensed paternal DNA the sperm paternal heterochromatin is devoid of canonical then expand to approximately three times the size of the heterochromatin marks and is mainly established by PRC1/2 mature sperm nucleus, resulting in the formation of the paternal protein complexes (van der Heijden et al., 2006; Probst et al., pronucleus, compatible with DNA replication and the fusion 2010; Casanova et al., 2013; van de Werken et al., 2014). of the maternal and paternal pronuclei. Its timing has been Paternally-derived modiﬁed histones also seem to play a placed by many studies within the ﬁrst hours after fertilization critical role in establishing a totipotent embryo. For a correct depending on the species concerned (Nonchev and Tsanev, development, the paternal pronucleus is hyperacetylated shortly 1990). In studies on human sperm, protamine removal was after fertilization with acetylation of lysines 5 and 16 of H4 and observed to be completed in the hour following intracytoplasmic lysines 9, 14, 18, and 27 of histone H3 (Adenot et al., 1997; sperm injection (Jones et al., 2011). In studies in which porcine Santenard et al., 2010). sperm was used for in vitro fertilization (IVF), it was shown that Beside remaining histones and protamines, the sperm nucleus 80% of the protamines were removed within 3 h post-fertilization contain Matrix Attachment Region (MAR) that allow the (Shimada et al., 2000). On a mechanistic side, sperm nucleus formation of the male pronucleus and the replication of the protamine removal was shown to rely on an antioxidant activity zygote DNA following fertilization (Linnemann et al., 2009). mediated by maternal glutathione allowing for the reduction of Indeed, intracytoplasmic injection of sperm-DNA alone without disulﬁde bonds between protamines thus facilitating the paternal MAR into oocyte or co-injected with an isolated sperm nuclear chromatin decondensation (Perreault et al., 1988; Figure 2). matrix does not allow the formation of the male pronucleus Considering the fact that the sperm nucleus brings along or its replication. While injection of sperm nuclear matrix only persisting histones (canonical and variants) and associated associated with short DNA fragments at the base of the toroids PTMs, the question about the contribution of those persisting (so DNA fragments associated with sperm MAR) allows the histones to the embryo was asked (i.e., are they replaced by formation of the male pronucleus and DNA replication, despite maternal histones or not?). Of course, in view of the ongoing sperm DNA degradation ranging from 20 to 50% (Shaman et al., debate regarding the genomic location of sperm histones, their 2007a,b). contribution and functional role in the embryo cannot be fully understood. Most data regarding the location of sperm histones Paternal DNA Demethylation/Methylation after fecundation are based on immunodetection. It was reported Before the fusion of the two pronuclei, a demethylation step that some sperm persisting histones variants are removed after takes place. The removal of the majority of 5mC in both fertilization. For example, H2AL1/2 rapidly disappear after genomes is crucial to establish the pluripotency of the inner fertilization in the mouse (Wu et al., 2008): First detected in cell mass of the blastocyst. For both the maternal and paternal the centromeres of spermatids, these variants remain enriched pronuclei, DNA demethylation is mediated by (i) an active DNA in heterochromatin domains until displaced from paternal DNA demethylation process dependent on Tet enzymes, including shortly after fertilization (Wu et al., 2008). In contrast, histone Tet3, and the successive formation of 5 hmC, 5 caC, and 5 fC H3 replication-dependent variants H3.1 and H3.2 (Tagami et al., (Tahiliani et al., 2009). and (ii) a passive replication-dependent 2004) were detected in the male pronucleus after fertilization demethylation process (Guo et al., 2014; Shen et al., 2014; Wang and prior to DNA synthesis, though in a much lower abundance et al., 2014). Most of the DNA inherited from the father appears than in maternal chromatin (van der Heijden et al., 2005, to be demethylated before DNA replication (Mayer et al., 2000; 2008; Hajkova et al., 2010). These sperm-derived proteins are Oswald et al., 2000; Santos et al., 2002). On the contrary, the detected until the zygotic S phase initiates, at which point maternal genome undergoes slower, mainly passive, replication- they become indistinguishable from their newly incorporated dependent DNA demethylation. Only a small but signiﬁcant maternal counterparts (van der Heijden et al., 2008). H3.3 plays portion of its DNA appears to be actively demethylated by a an essential role during zygotic S-phase in the transcription of Tet3-dependent process (Guo et al., 2014; Shen et al., 2014; peri-centromeric domains that trigger their silencing following Wang et al., 2014). Tet3 is predominantly located in the male the ﬁrst cell cycle by acquisition of H3K27 methylation in pronucleus and results in an important accumulation of the the male pronucleus (Santenard et al., 2010). Four hours after 5 hmC mark in the male pronucleus (Gu et al., 2011; Iqbal fertilization, the newly formed male pronucleus appears to et al., 2011; Amouroux et al., 2016). In zygotes deﬁcient for carry only nucleosomes containing H3.3, while the female Tet3, 5mC level remains constant on the paternal genome pronucleus exists in a relatively zero-H3.3 state (Santenard et al., leading to a delay in the activation of paternal allele of genes 2010; Akiyama et al., 2011). Moreover, after fertilization, the essential for embryonic development, such as Nanog and Oct4. embryo has to rearrange paternal pronucleus heterochromatin Interestingly, it is the maternally derived Tet3 enzyme which is and euchromatin regions to form a functional embryonic responsible for the conversion of 5mC to 5 hmC in the paternal Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance FIGURE 2 | Dynamics of epigenome reprogramming of the male pronucleus. After fertilization, in one cell embryo, the sperm nucleus undergoes many remodeling events. First, protamines are replaced by maternally-derived histones (A). In addition, in some areas of the sperm nucleus where persisting histones remain, some will be replaced by canonical or other variants. Furthermore, the majority of 5-methylcytosine residues (5 mC) will be erased by two processes: a TET-TDG-BER repair process that will introduce non-methylated cytosines (C) or the AID/APOBEC/BER pathway that will change mC in thymine (T) then will repair the T-G mismatches in non-methylated cytosines (B). Some regions will be protected from DNA demethylation including repeats elements, transposons, and imprinting control regions (ICR) thanks to the presence of the Stella protein and H3K9me2 histone modiﬁcation (C). AID/APOBEC, cytidine deaminase; APE, Apurinic/Apyrimidic endonuclease; BER, Base excision repair; C, cytosine; G, guanine; GSH, Glutathione; 5m, 5-methylcytosine; 5hm, 5-hydroxymethylcytosine; 5f, 5-formylcytosine; 5caC, 5-carboxycytosine; Lig, Ligase; MAR, matrix attachment region; Pol, Polymerase; Prm, Protamine; PTM, post-translational modiﬁcations; S-S, disulﬁde bridge, TDG, Thymine DNA glycosylase; TET, Ten eleven translocation; XRCC1, X-ray repair cross-complementing protein 1. genome (Gu et al., 2011). A combined deﬁciency of Tet1 and The diﬀerent methylated-cytosine derivatives (5hmC, 5fC, Tet3 results in an increase in the 5mC mark and a loss of and 5caC) are ultimately transformed into non-methylated 5hmC at the eight-cell stage. In addition, these embryos show a cytosine by the DNA repair pathway TDG-BER (standing decrease in the expression of genes involved in the biosynthesis for thymidine DNA glycosylase-base excision repair) that is of cholesterol; the few embryos that survived showed signs of a independent of replication (Kohli and Zhang, 2013; Figure 2). holoprosencephaly associated with neurological disorders (Kang TDG enzymes recognize only the 5fC and 5caC forms whereas et al., 2015). 5hmC undergoes a deamination in 5hmU by the intervention of Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance cytosine deaminase AID/APOBEC (Zhang et al., 2012; Xue et al., embryo, as injection of a miR-34c inhibitor into zygotes was 2016). An abasic site is thus generated bringing in the players of shown to induce an arrest in embryo cell division (Liu et al., the BER repair pathways including APE1 and XRCC1 (Weber 2012). This results from the inhibiting role of miR-34c on Bcl2 et al., 2016). XRCC1 is located on the male pronucleus but absent (or B-cell leukemia/lymphoma 2) gene expression and on its from the female pronucleus (Hajkova et al., 2010; Wossidlo anti-proliferative function. et al., 2010). In addition, the DNA methyltransferase DNMT1 is It is worth noting that several studies have shown a link excluded from the nucleus of preimplantation embryos (Howell between DNA methylation and non-coding RNA. Dnmt3A and et al., 2001; Hirasawa et al., 2008), thus inducing the absence of 3B mRNA expression was shown to be negatively regulated re-methylation of the DNA immediately after fertilization. by miR-29b in mouse early embryos and alterations of miR- Importantly, some regions of the paternal sperm nucleus will 29b activity was shown to change the DNA methylation level escape demethylation after fertilization, that is the case of some in mouse preimplantation embryos leading to developmental categories of transposable elements (IAP) and imprinted genes arrest at the morula stage (Zhang et al., 2015). In another (Lane et al., 2003). Recently, studies have shown that a few study, when oocytes were fertilized using sperm derived from single copy genes (which are not imprinted) also escape the mice knocked-out for DICER or DROSHA, thus showing demethylation wave (Hackett et al., 2013; Tang et al., 2015). defective miRNAs/endo-siRNAs biogenesis, a deregulation in the To explain the absence of active DNA demethylation in expression of the embryo preimplantation genes was seen (Yuan speciﬁc regions of the male pronucleus a mechanism has been et al., 2016). Interestingly, the phenotype of these embryos could evoked. It involves the Stella protein (also known as PGC7 be saved by injecting a pool of sperm RNA from WT mice (Yuan or DPPA3) initially identiﬁed in the PGC. It is assumed that et al., 2016). Stella protects from DNA demethylation, at particular paternal Altogether these studies show the importance of all the facets imprinted loci (such as Rasgfr1) by preventing the binding of sperm epigenome not only for sperm quality and fertilization of the Tet3 protein and maintaining the presence of the abilities but also for subsequent embryo development. In histone methylation mark H3K9me2 (Nakamura et al., 2007, addition, recent studies suggest that the sperm epigenome could 2012; Bian and Yu, 2014). Interestingly, the persisting histone go beyond that and have an impact on several generations. repressive marks are associated with imprinting repression such as H3K27me3, H3K9me3, and H4K20me3 (McEwen and Ferguson-Smith, 2010). The loss of function of the Stella protein leads to an arrest in embryo development associated with a TRANSGENERATIONAL EFFECTS OF THE loss of 5mC both in male and female pronuclei (Nakamura PATERNAL EPIGENOME et al., 2007). Two other proteins have been identiﬁed to protect ICR from demethylation: Zfp57, a zinc ﬁnger protein Recent studies in animal models together with epidemiological of the KRAB family and Trim28. The interaction of these two data have suggested that epigenetic factors are responsible proteins, which speciﬁcally target ICR, induces the recruitment for the transmission of pathologies across generations. of repressor complexes such as NuRD (Nucleosome Remodesling This phenomenon was called epigenetic inheritance or Deacetylase), Setdb1 (a histone methyltransferase) and DNMTs transgenerational epigenetic inheritance. Epigenetic inheritance (Quenneville et al., 2011; Zuo et al., 2012). The loss of Trim28 involves the transmission of non-DNA base sequence is embryonic lethal due in part to defects in the expression information to the oﬀspring over multiple generations via of imprinted genes (Messerschmidt et al., 2012) while the loss the germline. Epigenetic inheritance can occur when parents of Setdb1 leads to a de-repression of retrotransposons and an (F0 generation) are exposed to multiple environmental insults increase in DNA double strand breaks (Kim et al., 2016). including nutritional stress, psychological stress, toxins and drugs exposure (Figure 3). Beside environmental origins, epigenetic Paternally Derived RNA inheritance might come from parents carrying mutations Since the discovery of sperm mRNA and non-coding RNA in epigenetic regulators (such as enzymes involved in the transmission to the embryo after fertilization, studies were establishment of DNA methylation or chromatin modiﬁcations, conducted to determine the impact(s) and role(s) of these etc.; Sharma et al., 2016). Both paternal and maternal gametes RNAs in embryonic development (Kramer and Krawetz, 1997; have been shown to transmit epigenetic information to the next Ostermeier et al., 2004; Miller et al., 2005; Jodar et al., 2013). generation (i.e., intergenerational transmission; Huypens et al., Regarding the sperm mRNA cargo, it was estimated that ∼18,000 2016). If epigenetic inheritance eﬀects due to the mother lifestyle mRNA are delivered to the embryo (Ostermeier et al., 2004). or/and environmental exposures particularly during oogenesis The same group identiﬁed six of these sperm mRNA (mRNA and during pregnancy (Gluckman et al., 2008; Jimenez-Chillaron for clusterin, AKAP4, Prm2, Cdh13, Foxg1b, and Wnt5a) and et al., 2009; Radford et al., 2014) have been widely studied and are suspected them to participate to paternal pronucleus formation now well established, only recently has it become apparent that and to control the events of early embryo development. paternal epigenetics eﬀects may also greatly inﬂuence oﬀspring Regarding sperm-borne non-coding small RNA, studies have health (Schaefer and Nadeau, 2015). This part of the review aims shown their impact in preimplantation embryo development. at summarizing recent progresses made on paternal epigenetic A striking example is that of miR-34c, a male germ line- inheritance and how environmental factors and paternal lifestyle speciﬁc miRNA that is transmitted to the embryo. Mir-34c was can alter the paternal epigenetic information, in particular its demonstrated to have a key role in the ﬁrst division of the mouse DNA methylation/modiﬁcation. Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance FIGURE 3 | Paternal epigenetic transmission to subsequent generations. Environmental exposures of male mice (F0) can modify the sperm epigenome. Epigenetic information about these exposures can be transmitted to the next F1 generation and also to the F2 generation or more F3. The offspring of subsequent generations can be subject to transgenerational phenotypes. Paternal Experiences Transmitted to the diet] could result in the appearance of metabolic disorders in the oﬀspring that were directly linked to changes in sperm Future Generation via Sperm Epigenome epigenetics (Carone et al., 2010; Ng et al., 2010; Fullston Examples of Sperm Epigenome Modiﬁcations and et al., 2013; Mejos et al., 2013; Wei J. et al., 2014; Schaefer Consequences on the Offspring and Nadeau, 2015; de Castro Barbosa et al., 2016; Schuster Several research groups using rodent models have recently et al., 2016). The detected epigenetic alterations in the oﬀspring shown that feeding male mice with unbalanced diets [either a low protein diet, a high-fat diet (HFD) or a Folate-deﬁcient concerned changes in DNA methylation at regulatory regions of Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance genes involved in metabolic processes such as glucose–insulin may increase the level of reactive oxygen species in testes, homeostasis, cholesterol biosynthesis (Carone et al., 2010; Ng which has a severe impact on DNA methylation proﬁles in et al., 2010; Fullston et al., 2013). the sperm and this germline epigenetic information can be For example, Ng et al. (2010) reported that feeding male mice transmitted from generation to generations (Anway et al., with a HFD promoted in the female oﬀspring a state of impaired 2006; Guerrero-Bosagna et al., 2012; Skinner et al., 2015). glucose-insulin homeostasis. This was evidenced by alterations Moreover, the authors demonstrate that epigenetic mechanisms in the female oﬀspring transcriptome of retroperitoneal adipose can inﬂuence and promote the occurrence of a number of DNA and pancreatic islet tissues. Changes in the methylation status sequence mutations. Indeed, by analyzing the diﬀerential DNA of the female oﬀspring Il13ra2 gene that is part of the Jak–Stat methylation regions (epimutations) and genetic mutations (copy signaling pathway was also detected in that model (Ng et al., number variation: CNV), they demonstrate an increase in copy 2010, 2014). In another study, it was reported that male mice number variants associated with altered DNA methylation in consuming a low-protein diet produced oﬀspring with increased the F3 generation (Skinner et al., 2015). Besides alterations in level of cytosine methylation in trans-acting regulatory sequences DNA methylation, exposure to environmental factors can lead of key genes involved in lipid and cholesterol biosynthetic to changes in sperm non-coding RNA and histone retention in pathways such as Pparα (Carone et al., 2010). These changes sperm of DDT-exposed male mice (Skinner et al., 2018). were correlated with the down-regulation of the expression Since changes in the methylation status of the paternal of the corresponding genes in the oﬀspring (Carone et al., genome were not always observed, other causative factors of 2010). Interestingly, in that particular model, the authors did paternal epigenetic inheritance have been suspected and looked not show any signiﬁcant change in cytosine methylation at for. Changes in chromatin packaging via sperm nuclear protein classical imprinted loci in the oﬀspring. In addition, the global modiﬁcations and non-coding RNA content were brought cytosine methylation proﬁle of the paternal sperm was not forward and explored. As discussed above, sperm chromatin is signiﬁcantly diﬀerent between male mice that were fed or mainly made of protamines which will be expelled shortly after not fed the diet. This suggested that paternal sperm DNA fertilization; the so-called persisting histones, despite its limited methylation modiﬁcations were probably not at the origin of amount, has therefore the most potential for a contribution the phenotypical trait recorded in the oﬀspring in that particular to the next generation(s). Any modiﬁcation of these persisting model. sperm histones may also be considered as part of the epigenetic In diﬀerent models, it was however shown that a diet-induced information received by the future embryo. Very few studies have stress indeed provokes changes in the DNA methylation proﬁle of succeeded to establish a relation between sperm histone changes the father’s sperm cells (Fullston et al., 2013). In particular, Wei and transgenerational defects. In one particular study, Vassoler Y. et al. (2014) reported that a situation of diet-induced paternal et al. (2013) showed that the administration of cocaine to male prediabetes modiﬁes the sperm methylation status of several rats resulted in an increased level of brain-derived neurotrophic genes such as the phosphatidylinositol (PI) 3-kinase subunits factor protein (BDNF) in the prefrontal cortex of their sons. This Pik3ca and Pik3r1 of the insulin pathway. It was proposed that was shown to be associated with an increased level of acetylated these altered sperm methylation proﬁles were at the origin of histone H3 within the Bdnf promoter in the medial prefrontal the metabolic disorders recorded in the next generations (Wei Y. cortex (Vassoler et al., 2013). This epigenetic modiﬁcation was et al., 2014). also found in the promoter of the Bdnf gene in the paternal In humans, the association between a paternal condition sperm. Although interesting, these results are not suﬃcient to and epigenetic changes in their children has been described in ascertain a transgenerational eﬀect related to sperm histone many instances. For example, Soubry et al. explored putative modiﬁcation because they did not show eﬀect across several correlations between paternal obesity and children epigenetic generations. proﬁles (Soubry et al., 2013, 2015). They reported that children of A more convincing association was shown via the use obese fathers showed low methylation at diﬀerentially methylated of a mutant mice for a protein involved in chromatin regions (so-called DMRs) of several imprinted genes such as modiﬁcation. In that model, mice overexpress a human version Mest and Peg3. Interestingly, a deregulation of the methylation of histone H3 lysine 4 (H3K4) demethylase KDM1A during status of these DMRs were reported elsewhere to be linked spermatogenesis. Spermatozoa of these mice showed reduced with the occurrence of chronic diseases and metabolic disorders H3K4 dimethylation within CpG islands of genes implicated in in the oﬀspring (Murphy and Jirtle, 2003; Jirtle and Skinner, development. In their progeny were observed severely impaired 2007). embryonic development and reduced survival rates across three A great number of studies have demonstrated that generations (Siklenka et al., 2015). However, KDM1A was not environmental exposures play an important role in the found expressed in the germline of subsequent generations, accumulation of epigenetic patterns at speciﬁc loci, which the transgenerational defects were therefore unlikely to result further aﬀects normal development and even causes various from transmitted modiﬁcations of sperm histone PTM. Since no diseases via tightly regulated gene expression at the epigenetic changes in DNA methylation were observed at CpG islands, the level. In addition to the modiﬁcations of epigenome by authors hypothesized that the transgenerational inheritance was nutrition, other environmental factors can alter epigenome mediated by sperm-borne RNA (Siklenka et al., 2015). such as toxins, unhealthy lifestyles, etc. (Schaefer and Nadeau, Several studies investigated speciﬁcally the role of small 2015). For example, in mice, male exposure to pesticides non-coding RNAs (sncRNAs) in transgenerational inheritance Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 May 2018 | Volume 6 | Article 50 Champroux et al. Paternal Epigenetic and Transgenerational Inheritance because they are long known to be present in the germ cell lineage represented miRNAs, namely miR19, into fertilized eggs resulted and their involvement in the regulation of DNA methylation in the appearance of similar phenotypes in the developed and histone modiﬁcations is well documented (Holoch and embryos (Grandjean et al., 2015). Moazed, 2015). Those studies found that sperm-derived ncRNAs, Changes in sperm ncRNA content following an including miRNA and small tRNA derived from tRNAs, have environmental stress is not limited to metabolic stresses the potential to inﬂuence embryonic development (see above) since Gapp et al. (2014) reported that a traumatic stress such as and lead to transgenerational inheritance (Holoch and Moazed, maternal separation in early life was associated with behavioral 2015; Rodgers et al., 2015; Chen et al., 2016a; Sharma et al., and metabolic conditions in the progeny (Gapp et al., 2014). 2016). For example, a nutritional stress in rodents such as a Here too, sperm miRNAs were shown to be involved since high fat diet can modify the sperm miRNA content which micro-injection of sperm miRNAs collected from traumatized constitutes potential epigenetic signals. These signals will drive males into fertilized oocytes led to similar phenotypes. oﬀspring health and will initiate the transmission of metabolic Altogether, these studies showed that the sperm epigenome in abnormalities in future generations (Fullston et al., 2013). In particular its DNA methylation proﬁle or/and its ncRNA content 2016, two independent studies showed that a high-fat or a low- has the potential to alter the health of the next generations. protein diet given to male mice was associated with increased levels of fragmented transfer RNAs species (stRNA) in sperm CONCLUSIONS AND FUTURE that were subsequently associated with metabolic disease in their PROSPECTS oﬀspring (Carone et al., 2010; Chen et al., 2016a; Sharma et al., 2016). To attest that sperm stRNA generated by the diets were The sperm epigenome, established during spermatogenesis in the at the origin of the inherited oﬀspring phenotypes, both research testis, is highly specialized and unique. The studies mentioned groups microinjected these diet-induced small tRNA into control in the present review demonstrate that sperm is more than oocytes fertilized with sperm issued from males not subjected to a vehicle transferring its haploid genome to the oocyte. DNA the regimens. Chen et al. reported that a subset of stRNA, exhibits modiﬁcations, chromatin proteins and associated marks as well changes in expression proﬁles and RNA modiﬁcations in male as sperm-derived RNA (in particular sncRNA) constitute a mouse fed with HFD, compared with mice fed a normal diet speciﬁc epigenetic landscape, shown, in case of disruption, (ND) male mouse. Injection of sperm stRNAs from HFD male to result in male infertility, abnormal embryo development mouse into normal zygotes generated metabolic disorders in the and/or transgenerational inheritance. Future studies will have F1 oﬀspring and altered gene expression of metabolic pathways to elucidate and clarify the role and underlying mechanisms of in early embryos and islets of F1 oﬀspring (Chen et al., 2016a) sperm epigenetics in those processes, in particular its impact on Moreover, the bioinformatics analyses show that sperm stRNA the oﬀspring health. If one takes into account the additional which are diﬀerentially expressed between HFD and ND match eﬀects due to variations in the oocyte epigenome, predicting preferentially to gene promoters rather than coding regions the risk of diseases in the oﬀspring associated with germ cell which are associated with metabolic genes. Interestingly, Sharma epigenome alterations will certainly be a rather complex issue. et al, showed that other diet stresses such as protein restriction in male mice aﬀects small RNA levels in mature sperm with AUTHOR CONTRIBUTIONS increased level of 5 fragments of glycine tRNA. Furthermore, they showed that these stRNA are gained during the epididymal AC, JC, JH-B, and AK: drafted the manuscript; JD: critically transit trough epididymosomes: vesicles that fuse with sperm reviewed the manuscript. during epididymal transit. The study showed that stRNA which are aﬀected by low protein diet can regulate expression of ACKNOWLEDGMENTS transcripts driven by endogenous retroelements (MERVL) in the embryos (Carone et al., 2010; Sharma et al., 2016). Due to space constraints, we could not include all the relevant These 2016 studies conﬁrmed an earlier report showing that citations on the subject matter, for which we apologize. State male mice fed a western diet (high fat and high sugar diet) grants from the University of Clermont Auvergne-(UCA), exhibited an altered sperm miRNA proﬁle associated with an INSERM, CNRS and former Région Auvergne support the increase in metabolic disorders in their oﬀspring (Grandjean activities of the research team headed by JD. 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Dynamic link of DNA demethylation, DNA strand breaks and provided the original author(s) and the copyright owner are credited and that the repair in mouse zygotes. EMBO J. 29, 1877–1888. doi: 10.1038/emboj.2010.80 original publication in this journal is cited, in accordance with accepted academic Wu, F., Caron, C., De Robertis, C., Khochbin, S., and Rousseaux, S. practice. No use, distribution or reproduction is permitted which does not comply (2008). Testis-speciﬁc histone variants H2AL1/2 rapidly disappear from with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 19 May 2018 | Volume 6 | Article 50

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Frontiers in Cell and Developmental Biology – Pubmed Central

Published: May 15, 2018

References

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DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells
Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism
Genomic patterns of DNA methylation: targets and function of an epigenetic mark
Perinatal exposure to bisphenol A exacerbates nonalcoholic steatohepatitis-like phenotype in male rat offspring fed on a high-fat diet
Paternally induced transgenerational inheritance of susceptibility to diabetes in mammals
Packing for the germy: the role of histone H4 Ser1 phosphorylation in chromatin compaction and germ cell development
Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes
Testis-specific histone variants H2AL1/2 rapidly disappear from paternal heterochromatin after fertilization
The structural organization of sperm chromatin
Mammalian spermatozoal mRNAs: tools for the functional analysis of male gametes
Haploid transcripts persist in mature human spermatozoa
Histone H2A.Z controls a critical chromatin remodeling step required for DNA double-strand break repair
Uracil-DNA glycosylase UNG promotes Tet-mediated DNA demethylation
HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis
Alternative miRNA biogenesis pathways and the interpretation of core miRNA pathway mutants
Microfluidics-based digital quantitative PCR for single-cell small RNA quantification
Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development
Histone H3.3 regulates dynamic chromatin states during spermatogenesis
Chromatin structure of telomere domain in human sperm
Epigenetic effects of endocrine-disrupting chemicals on female reproduction: an ovarian perspective
Expression and potential role of microRNA-29b in mouse early embryo development
Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA
CHD5 is required for spermiogenesis and chromatin condensation
Zinc Finger Protein ZFP57 Requires Its Co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain

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A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance

A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

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A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance

A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance

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