Introduction
In the last years, accumulating research has documented that male germ cell maturation presents windows of vulnerability for epigenetic reprogramming by environmental stressors that can affect fertility and even transmit developmental, metabolic and behavioral traits to offspring (Bale, 2015; Lee and Conine, 2022). This type of non-genetic “Lamarckian” transmission has been established for paternal lifestyle and different forms of chronic stress, drug abuse, dietary change, and social defeat, involving changes in non-coding RNAs cargo, DNA methylation, and histone post-translational modifications (PTMs) (Bale, 2015; Lee and Conine, 2022). However, most of the research in epigenetic inheritance has documented the role of non-coding RNAs and DNA methylation patterns, whereas the mechanisms by which the environment can trigger specific changes on histone PTMs are still largely unexplored.
Spermatogenesis is a finely regulated process where unipotent spermatogonia undergo meiosis and, subsequently, spermiogenesis, to become sperm cells. During the developmental stages of spermatogenesis, male germ cells experience dramatic chromatin reorganization, where approximately 90% (human) to 95% (mouse) of histones are evicted and replaced by protamines to compact the paternal genome (Rajender et al., 2011; Carrell, 2012). Importantly, a small fraction of histones in the sperm genome is retained in specific locations and carries several PTMs that play critical roles in epigenetic regulation of spermatogenesis and early embryonic development (Hammoud et al., 2009; Brykczynska et al., 2010; Yamaguchi et al., 2018; Chioccarelli et al., 2020; Lambrot et al., 2021; Okada, 2022). The epigenetic program of histone PTMs that controls spermatogenesis is regulated by enzymatic families known as writers and erasers of the so-called “histone code,” that modulate chromatin structure and transcription (Kouzarides, 2007). These epigenetic enzymes catalyze the deposition or removal of specific PTMs such as acetylation, methylation, phosphorylation, crotonylation, among others, at specific amino acid residues on the nucleosome’s core histones H2A, H2B, H3, and H4, and also on the linker H1/H5 (Chioccarelli et al., 2020). In male germ cells, histone PTMs not only control active vs. repressed chromatin states but also a range of processes including DNA replication and repair, chromosome maintenance and histone eviction (Luense et al., 2016). So far, the most studied histone PTMs related to the control of germ cells mitosis, meiosis and spermiogenesis are methylated and acetylated lysines (K) on H2A/B, H3 and H4. Histone K acetylation can be dynamically regulated by the opposing action of acetyltransferases (HATs) and deacetylases (HDACs). Acetylation of K residues neutralizes the positive charge on histones, allowing DNA-binding proteins better access to the DNA and resulting in activation of gene expression as well as histone eviction (Miller and Grant, 2013). Unlike acetylation, methylation does not affect histone charge but regulates recognition and interaction with chromatin-binding proteins that control the transcription or respond to DNA damage (Miller and Grant, 2013). Histone K methylation is finely regulated by methyltransferases (KMTs) and demethylases (KDMs) that control the mono-, di-, and/or tri-methylation of specific residues, and this can either activate or repress transcription depending on the residue position, the number of methylations and the presence of other methyl or acetyl groups in the vicinity (Mosammaparast and Shi, 2010; Miller and Grant, 2013).
The role of epigenetic enzymes comes to focus, as they are responsible for the histones’ PTMs writing and erasing balance. Moreover, histone-modifying enzymes have been shown to respond to the organism physiology and metabolism, several disease conditions, and environmental stressors (Bale, 2015; Nebbioso et al., 2018; Gonzalez et al., 2020). Even though epigenetic enzymes are the bridge linking the environment with epigenetic inheritance through retained histones PTMs, an in-depth analysis of their expression patterns throughout the spermatogenic process is still lacking. Moreover, these enzymes provide new targets for therapies for numerous diseases (Prachayasittikul et al., 2017) but the potential effect of these compounds on epigenetic inheritance is generally overlooked. To further elucidate the mechanisms driving the histone PTMs during spermatogenesis, a better understanding of the gene expression profiles of epigenetic modifying enzymes in male germ cells is required. Here, we curated transcriptomic data from spermatogonia to mature sperm populations and analyzed the expression profile and dynamic changes of all the known families of histone K acetylation and methylation writers and erasers. We provide a complete picture of the epigenetic enzymes across spermatogenesis in mice and the developmental windows when the transcription of these enzymes may be more susceptible to environmental disruption. Moreover, we analyzed pre-cleavage zygote translatome, confirming that mRNAs of several histone modifying enzymes carried by sperm are associated with ribosomes for protein synthesis in 1-cell embryo. Not only do we expand the knowledge on epigenetic enzymes with recognized roles in spermatogenesis, but provide evidence of many more whose roles in male germ cell development and zygote have not been described yet.
Discussion
Accumulating evidence has put on focus the existence of windows of vulnerability during spermatogenesis, at which environmental stressors can induce epigenetic reprogramming and the transmission of developmental, metabolic, and behavioral traits to offspring. To further elucidate the mechanisms driving histone PTMs changes during the spermatogenic process, we analyzed the epigenetic enzyme expression programs across germ cell differentiation to mature sperm. Here, we expand the knowledge on enzymes with recognized roles in spermatogenesis and provide evidence of many more whose role in male germ cells development has not been described yet. We show that cell transition across spermatogenesis is characterized by the upregulation of specific histone K methylation and acetylation writers and erasers driving the epigenome changes necessary for spermatogonia differentiation, meiosis entry and spermiogenesis. Moreover, most of these epigenetic enzymes were detected in the mature sperm mRNA pool. Histone modifying enzymes were also detected in the early zygote translatome, suggesting a possible paternal contribution of epigenetic enzymes to the zygote. Our study shows important mechanistic aspects behind transgenerational epigenetics, where epigenetic enzymes not only can respond to environmental stressors and alter the germ cell epigenome but could function as vectors of epigenetic information transmission themselves, by participating at the maternal-to-zygote transition.
The spermatogonia stage seems to be vulnerable for stress-induced epigenetic reprogramming since they reside outside the blood-testis barrier (González et al., 2020). Furthermore, current data support a dynamic stem cell model in which the fate of SGund population is context-dependent and plastic, and several epigenetic mechanisms are implicated in the regulation of spermatogonia maintenance and cell fate (Mäkelä and Hobbs, 2019). Here, we found that the transition to the SGdiff population is characterized by increased expression of writers of activation marks H3K4me and H3K36me and repression marks H3K9me and H4K20me. At the spermatogonial phase, we found high expression of H3K4me epigenetic enzymes, including Kmt2a/b/c writers and Kdm1a/b erasers. In this context, at spermatogonial stage occurs the deposition of monovalent and bivalent H3K4me marking at promoters by Kmt2b (Mll2), preparing germ cells for gene activation at late spermatogenesis and embryonic development (Tomizawa et al., 2018). Accumulating evidence also points to H3K4me2 in the maintenance of transcriptional states during cell development and, its removal by erasers expressed at spermatogonia such as KDM1A (LSD1) is a key step for epigenetic reprogramming and cell fate (Martinez-Gamero et al., 2021). KDM1A was found to mediate spermatogonia commitment and differentiation, as testicular deletion of Kdm1a leads to progressive germ cell loss, downregulation of self-renewal factors and abnormal accumulation of meiotic spermatocytes (Lambrot et al., 2015; Myrick et al., 2017). Interestingly, overexpression of Kdm1a altered the H3K4me2/3 levels and the RNA profile of sperm and embryos transgenerationally (Siklenka et al., 2015; Lismer et al., 2020). Hence, the spermatogonial phase seems to be specially sensitive to alterations in H3K4me balance linked to epigenetic inheritance. SGdiff showed a specific increase of Prdm9, responsible for H3K4me3 and H3K36me3 that mediate the double-strand breaks (DSBs) formation at meiotic hot spots (Getun et al., 2017; Sun et al., 2020). In line with this, it has been shown that meiosis entry of SGdiff is driven by increased levels of H3K36me3 (Godmann et al., 2007; Song et al., 2011; Zuo et al., 2018). Here, we found Setd2 expression until PD stage, and Setd5 specifically upregulated at SGund, suggesting their involvement in the specific H3K36me3 landscape of spermatogonia differentiation. The SGund population also expressed H3K4me/H3K36me eraser Kdm2b, a subunit of non-canonical PRC1.1 complex that was found to protect the polycomb-silenced promoters against ectopic de novo H3K36me methylation (Blackledge and Klose, 2021). Furthermore, KDM2B was recently found to erase H3K79me2/3 and to induce transcriptional repression via SIRT1 chromatin silencing (Kang et al., 2018).
It is established that SGund typically lacks heterochromatin, which forms as they differentiate to the spermatogonia population committed to meiosis (Chiarini-Garcia and Russell, 2002). The SGund showed increased expression of H3K9me erasers Kdm3a/b and Jmjd1c (Kdm3c), that were found to counteract the enzymatic activity of EHMT2 (Tachibana et al., 2007), which is highly expressed in the spermatogonia population. Methylation of H3K9 by EHMT2 blocks gene expression of the stem cell factors OCT4 and NANOG, therefore, demethylation of H3K9 by KDM3 family enzymes may be a key step in the maintenance of self-renewal of spermatogonial stem cells (Chioccarelli et al., 2020; Kuroki et al., 2020). In agreement, we found increased Ehmt1, Suv39h2 and Setdb2 expression at SGdiff stage, consistent with H3K9me2/3 deposition during spermatogonia differentiation. In particular, EHMT1 was found in a complex that binds E2F- and Myc-responsive genes to repress the mitotic program (Ogawa et al., 2002). Moreover, SGdiff population showed decreased Ezh1 and increased Kdm6b compared to SGund, suggesting that spermatogonia differentiation involves H3K27me removal. We also observed that the transition to the SGdiff population is characterized by specific HDACs upregulation. In this sense, it was shown that class IIa HDACs enzymatic activity depends on their recruitment into a complex containing class I HDAC3 and retinoid acid (RA) receptor (RAR), suggesting that these HDACs could mediate chromatin silencing induced by RA, essential for the transition of SGund into SGdiff (Parra, 2015). Moreover, histone acetylation writer Kat2b expressed in SGund functions as a co-activator for RAR and can interact with NCOA1 to promote transcription (Spencer et al., 1997), which is upregulated from SGdiff to LZ stage. SGund also showed peak expression of Kat2a, and its germ-cell-specific knockout results in abnormal chromatin dynamics, leading to increased sperm histone retention and severe reproductive phenotype (Luense et al., 2019). Finally, SGdiff population showed increased Sirt1, described as a key regulator of spermatogonia differentiation since SIRT1-deficient mice show a delay of pre-meiotic differentiation, aberrant expression of spermatogenic genes, abnormal spermatozoa with elevated DNA damage, and reduced fertility (Coussens et al., 2008; Bell et al., 2014). Therefore, the action of certain epigenetic enzymes in pre-meiotic stages seems to enable the post-meiotic transcriptome and chromatin remodeling processes.
Initiation of male meiosis is one of the most important events that coincide with spermatocyte differentiation, and meiotic prophase is accompanied by several alterations of epigenetic and gene expression programs for post-meiotic spermiogenesis (Maezawa et al., 2018). Histone acetylation writers increase during prophase I progression, and H3K9/18/23ac and H4K5/8/12/16/91ac marks have been associated with open chromatin and hot spot cores (Getun et al., 2017; Chioccarelli et al., 2020). Accordingly, MYST enzymes were previously found strongly related to meiosis control, increasing their expression from early pachytene through diplotene stages (Getun et al., 2017). Interestingly, p300/CBP family and KAT2B were found to regulate histone eviction by acetylation of transition nuclear protein (TNP) 2, affecting DNA condensation properties and interaction with histone chaperones (Pradeepa et al., 2009). Among other acetylation writers upregulated during prophase I, MSL3 binds to H3K36me3 marked sites and deposits H4K16ac, controlling meiosis entry and STRA8 (stimulated by retinoic acid 8) functions (McCarthy et al., 2022). On the other hand, we detected several SIRT enzymes throughout prophase I. Deacetylation of H3K9 by SIRT6 modulates telomeric chromatin function (Michishita et al., 2008) while SIRT7 is highly selective toward H3K18ac, and might play an upstream role in DNA repair and telomere maintenance (Wu et al., 2018). We also detected peak expression of Hdac1 and Kdm1a (Lsd1) at the PD stage, that were recently shown to interact with BEND2 and participate in DSB repair, synapsis and transcriptional repression (Ma et al., 2022). HDAC1 was also found to form a complex with DNA methyltransferase DNMT3L to regulate X chromosome compaction (Deplus et al., 2002; Zamudio et al., 2011).
During prophase I, recombination hotspots are mainly marked by H3K4me3 catalyzed by KMT2 family and PRDM9 methyltransferases (Sollier et al., 2004; Buard et al., 2009). Moreover, the levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere, and PRDM9 is capable of placing both marks on the same nucleosomes in vivo (Powers et al., 2016). It was shown that Prdm9 knockout changes the distribution of DSBs across the genome inducing defective synapses and male infertility (Paigen and Petkov, 2018; Bhattacharyya et al., 2019). We also detected peak expression of H3K79me writer Dot1L at PD stage, consistent with previous reports showing increased levels of DOT1L, and H3K79me2/3 from pachytene onwards (Ontoso et al., 2014). The heterochromatic centromeric regions and the sex body are enriched of H3K79me3, while H3K79me2 is present all over the chromatin, but is largely excluded from the sex body despite the accumulation of DOT1L (Ontoso et al., 2014). Repressive histone methylation marks H3K27me3, H3K9me3 and H4K20me3 are present in chromatin regions with reduced recombination or involved in heterochromatin formation. In vitro and in vivo models disrupting the action of H3K9me writers Suv39h1, Setdb1/2 and Ehmt2 detected during prophase I, showed an abnormal distribution of H3K9me3 at pericentromeric heterochromatin, compromised meiotic silencing of unsynapsed chromatin (MSUC), misregulation of meiotic and somatic genes, anomalous synapsis and misssegregation of chromosomes and apoptosis at pachytene stage (Peters et al., 2001; Tachibana et al., 2007; Takada et al., 2011). Interestingly, low protein diet in the father was linked to altered levels of H3K9me2 through EHMT2, that changed tRNAs in sperm and transmitted metabolic phenotypes to the offspring (Yoshida et al., 2020). Also, SETDB1 haploinsufficiency in mice was shown to induce changes in DNA methylation in transposable elements, and to influence coat color in the offspring, further linking epigenetic inheritance with altered epigenetic enzymes levels during spermatogenesis (Daxinger et al., 2016). A recent work by Barral et al., 2022 reported dual regions in mouse embryonic stem cells that rely on the SETDB1 and NSD proteins to generate H3K9me3 and H3K36me3, respectively. They found that SETDB1 removal induces loss of both marks in dual regions, gains signatures of active enhancers, and comes into contact with upregulated genes, providing a mechanistic insight by which genes are controlled by heterochromatin (Barral et al., 2022). Moreover, NSD1-mediated H3K36me2 was found to prevent H3K27me3 deposition by PRC2, modulating PRC2-mediated H3K27me domains demarcation (Blackledge and Klose, 2021). In line with this, we detected the expression of H3K27me writers Ezh1/2 at PreL and LZ stages that were found involved in mammalian X chromosome inactivation (Schuettengruber et al., 2007). Furthermore, Kdm6a is part of the COMPASS complex with Kmt2b/c/d and Ashl2 that activate genes in response to RA by H3K4me deposition and H3K27me removal (Lavery et al., 2020) and moreover, alterations in Kdm6a expression induce defects that persisted transgenerationally (Siklenka et al., 2015; Lesch et al., 2019). Additionally, the loss of H3K27me3 from bivalent signature in the promoter of endonuclease SPO11 induces chromatin activation that favors meiotic entry (Hammoud et al., 2014). We also detected high expression of H2AZK7me1 writer Setd6 at PreL stage, which in ESC was found with H3K27me3 close to differentiation marker genes and removed upon RA signal (Binda et al., 2013), suggesting that SETD6 may have a role at early male meiosis. Finally, we observed expression of the H4K12me writer N6amt1 at LZ stage, a recently described epigenetic mark found at promoters of genes encoding cell cycle regulators (Metzger et al., 2019).
Chromatin organization dramatically changes during mid-to late-spermiogenesis, due to histone eviction and replacing by PRMs to facilitate the condensation and packaging of the paternal genome. In mice, 1%–10% of histones are retained in sperm chromatin and form a heterogeneous mixture of nucleo-histones and nucleo-protamines (Luense et al., 2016). At the spermiogenesis phase, histone acetylation is essential in destabilization and remodeling of nucleosomes. Recently, it has been reported the specific expression of Kat2b and Kat8 at RStid stage, suggesting that these acetyltransferases are responsible for histone hyperacetylation prior to the histone-to-protamine transition (Li et al., 2021). In line with this, we observed that the RStid population showed high transcriptional levels of H4K16ac writers Msl3 and Kat8, and also p300/CBP and Kat2b that keep their expression from spermatocyte stage, due to their crucial role to regulate histone eviction (Pradeepa et al., 2009). Moreover, KAT2B was found in spermatids and involved in H3K9ac, an epigenetic mark detected at unmethylated active genes/enhancers (Hammoud et al., 2014) that could influence gene expression directly after fertilization (Steilmann et al., 2011). Additionally, Hat1 expression was found related to the incorporation of H4K5/12ac and H3.3 variant at DSBs sites and the promotion of DNA repair (Yang et al., 2013). H4K5/8/12 marks have been also observed just before eviction of histones during spermiogenesis (Meistrich et al., 1992; Hecht et al., 2009) and moreover, H4K8/12ac was detected prior to full decondensation of the sperm nucleus, suggesting that these marks are transmitted to the zygote (van der Heijden et al., 2006).
Multiple histone methylation have been also identified in spermatids, pointing to a balance of “opened” and “closed” chromatin regions during the histone-to-protamine transition (Wang et al., 2019). Here, we detected upregulated expression of H3K79me writer Dotl1, H3K9me writer Ehtm2 and H3K27me writer Ezh2 at RStid stage. It was found that DOTL1 is enriched in post-meiotic stages of mouse germ cells and precedes the histone-to-protamine transition (Dottermusch-Heidel et al., 2014). Moreover, Ezh2 and Ehmt2 methyltransferases were detected at spermatid stage, suggesting that spermatids have a potential preference for epigenetic transcriptional repression and heterochromatin formation (Li et al., 2021). During spermiogenesis, there is a reactivation of transcription from MSUC and MSCI sites that is enabled by the deposition of histone crotonylation (Kcr). It was proposed that CDYL, an HKcr eraser, prevents post-meiotic chromatin reactivation by binding to H3K9me3 and H3K27me2/3 marked sites and facilitating H3K9me2 deposition by EHMT2 (Mulligan et al., 2008). Moreover, there is specific H3K27me deposition to establish bivalency at developmental genes (Maezawa et al., 2018), and proteomic studies found substantial H3K27me3/H3K36me2 double marking in RStid and sperm (Luense et al., 2016).
It is established that mature sperm transport a cargo of miRNAs, tsRNAs, lncRNAs, circRNAs, and protein-coding mRNAs, that carry an epigenetic blueprint involved in early embryo development (Guo et al., 2017). Some of these RNAs remain from the last stages of elongated spermatids, and others are acquired along the passage through the epididymis, as sperm absorb epididymosomes released by somatic cells (Trigg et al., 2019). Here, we found that the mature sperm transport a signature of epigenetic enzymes mRNAs similar to the one detected at spermiogenesis phase, and most of these enzymes show positive translation at one cell embryo. After fertilization, the zygote genome is transcriptionally silent, and cellular processes are carried on with inherited RNAs and proteins until the onset of zygotic genome activation (ZGA) around the two to four cells stage (Schulz and Harrison, 2019). Also, at 1-cell stage there is significant removal of some maternal RNAs and specific translation of others, including RNAs specifically delivered by the sperm like the egg-activating factor PLC-zeta (Sone et al., 2005; Yao et al., 2010). The parental pronuclei have asymmetric reprogramming capacities and the reprogramming factors reside predominantly in the male pronucleus. In this context, previous work suggested that KMT5C deposits H4K20me3 to allow the timely and coordinated progression of replication after fertilization (Eid et al., 2016). Also, it was found that EHMT2 activates soon after fertilization and deposit H3K9me2 patterns in the paternal genome (Ma et al., 2015). Here, we detected high sperm values and embryo translation of several enzymes involved in histone acetylation and methylation, including the H4K20me2/3 writer Kmt5a/c, the H3K9me2 writers Ehmt1/2, and the H3K9me3 writers Suv39h2 and Setdb1/2, with established roles in chromatin structure organization. Moreover, some of these enzymes were found increased in the embryo compared to the MII oocyte, as indicative of paternal contribution to their pool levels. Hence, sperm’s epigenetic enzymes contribution to the zygote’s mRNA pool could cooperate to the translation of the machinery necessary for the extensive chromatin remodeling that takes place to allow a new developmental program to start.