Essential Role of Histone Replacement and Modifications in Male Fertility

1Spermatogenesis is the process of male gamete production with successive cellular differentiation, which can be subdivided into spermatogonial mitosis, spermatocytic meiosis and spermiogenesis. During spermatogenesis, SSC (spermatogonial stem cells) undergo self-renewal and differentiate into spermatogonia that perform meiosis to generate haploid germ cells and ensure the genetic diversity through meiotic recombination. Then, the haploid germ cells undergo spermiogenesis with a distinct morphological change and chromatin compaction in the sperm nuclei to prevent the paternal genome from mutagenesis and damage. During the nuclear chromatin re-organization in spermiogenesis, the majority of the somatic histones are firstly replaced by testis-specific histone variants, and transition proteins (TPs) are subsequently incorporated in the nuclei of spermatids, protamines (PRMs) further replace TPs in the late spermatids to pack the genome into the highly condensed sperm nucleus. During the histone-to-protamine transition, the histone variants and specific histone modifications play essential roles by modulating the chromatin compaction and higher-order chromatin structure ( Table 1 ). Defects in either the replacement or the modification of histones might result in azoospermia, oligospermia or teratozoospermia, which leads to male infertility ( Table 2 ). The focus of this review is on recent advances in our knowledge of how epigenetic regulators, such as histone variants, histone modification and their related chromatin remodelers, regulate the highly orchestrated chromatin re-organization and facilitate the histone-to-protamine transition during spermiogenesis.

2In eukaryotes, nucleosomes are the packing units of DNA, which contain four types of canonical histones (H2A, H2B, H3, and H4) and the linker histone H1. While canonical histone expression is typically coupled to DNA replication, some non-canonical histones (histone variants) that are distinct form their canonical paralogues in amino acid sequence, are constitutively expressed and have roles in a wide range of processes. Many histones variants are expressed during spermiogenesis and modulate the chromatin structure to facilitate the histone-to-protamine replacement. Here, we summarize the recent advances in our understanding of the role of histone variants during the histone-to-protamine transition.

3Linker histones contribute to form and stabilize the higher-order chromatin structure. In mammals, there are about 11 different subtypes of histone H1. Among these, H1T, H1T2, and HILS1 are testis-specific H1 variants ( Figure 1 ).

4H1T is exclusively detected as early as mid- to late pachytene spermatocytes, and maintains high expression levels in the elongating spermatids ( Figure 1 ). Biochemical and biophysical studies found that, distinct from other somatic H1 variants, H1T binds less tightly to H1-depleted nucleosomes, suggesting it may maintain a relatively open chromatin configuration to facilitate histone replacement during spermiogenesis. Unexpectedly, H1t-null mice are fertile and exhibit no spermatogenesis abnormalities, and the histone-to-protamine transition in H1t-deficient testis is normal. Although the expression of some canonical subtypes, including H1.1, H1.2, and H1.4, is enhanced in H1t-null mice, elevated levels of H1.1 or H1.2 could not be observed in the H1t-deficient spermatids, indicating some other types of H1 variants may play redundant roles in the histone-to-protamine transition.

5H1T2 selectively localizes at the apical pole in the nucleus of round and elongating spermatids but not in mature spermatozoa ( Figure 1 ). Distinct from H1T, H1T2 is critical for spermiogenesis, as homozygous H1t2-mutant males are infertile due to delayed nuclear condensation and aberrant elongation of spermatids. Further analysis shows the protamine levels are substantially reduced in H1t2-null spermatozoa, indicating H1T2 is necessary for the incorporation of protamines, and proper chromitin condensation during the histone-to-protamine transition.

6HILS1 is strongly expressed in the nuclei of elongating and elongated spermatids ( Figure 1 ). HILS1 is the least conserved H1 variant, and a poor condenser of chromatin compared with somatic H1, demonstrating the idea that HILS1 may have a distinct role in the histone-to-protamine transition. In Drosophila, Mst77F encodes a linker histone-like protein that is similar with the mammalian HILS1 protein and expressed in elongating spermatids. The disruption of Mst77F cause male sterile as producing spermatozoa with malformed heads. Although the histone-to-protamine transition occurs independently of Mst77F, the nuclei of spermatid fail to properly condense after the histone-to-protamine replacement in Mst77F mutant male. However, the functional roles of HILS1 in mammalian spermiogenesis need further investigation.

7Multiple testis-specific H2A variants have been identified in mammals, including TH2A, H2AL1, H2AL2, H2AL3 and H2A.B.

8TH2A is present and actively synthesized in early primary spermatocytes and gradually disappears during condensation of spermatid nuclei ( Figure 1 ). TH2A could contribute to the open chromatin structure, as crystal structures of nucleosome core particles (NCPs) with TH2A show the H-bonding interactions between the TH2A/TH2A′ L1 loops are lost and the histone dimer-DNA contacts are dramatically decreased. Although a Th2a-knockout mouse model has yet to be established, mice with knockouts of the testis-specific H2B variants Th2a and Th2b exhibit male infertility with few sperm in the epididymis. In this double-knockout mouse, impaired chromatin incorporation of transition protein 2 (TP2) and elevated H2B could be observed in the mutant testis, suggesting the TH2A and TH2B may regulate the function in chromatin dynamics or the total histone levels to facilitate the histone replacement during spermatogenesis. As the Th2b-null male mice show normal spermatogenesis and fertility, the histone replacement defect in Th2a/Th2b double-knockout male mice is probably caused by the depletion of Th2a or their synergistic effect.

9In late-developing post-meiotic male germ cells, H2AL2 is specifically expressed in condensing spermatids that correlates with the expression of TPs ( Figure 1 ). By comparing H2al2-null mice to wild-type mice, H2AL2 was demonstrated to be required to load TPs onto the nucleosome and for efficient PRMs assembly during the histone-to-protamine transition. Additionally, the nucleosome reconstitution assays revealed that the incorporation of H2A.L.2 can drastically modulate the nucleosome structure to facilitate TPs invading the nucleosomes and further transformation. Thus, H2AL2 could assemble open nucleosomes and allow TPs invading, which further promotes protamine processing and sperm genome compaction.

10H2A.B is spatially and temporally regulated during spermatogenesis and detectable from the pachytene stage to the round spermatids ( Figure 1 ). In vitro studies show that H2A.B is able to destabilize chromatin and has unfolding properties to chromatin, indicating H2A.B might promote chromatin reorganization and further histones displacement by TPs. Male H2a.b-null male mice are subfertile due to the production of abnormal spermatozoa and clogged seminiferous tubules. In H2a.b-null elongating spermatids, H2AL2 could not be detected in pericentric heterochromatin, and the replacement of TP1 by protamines appears to be delayed. These results indicate H2A.B might modulate the dynamics of H2AL2 and TP1 chromatin incorporation and removal to participate in the histone-to-protamine transition.

11The testis-specific histone variant TH2B is one of the earliest histone variants identified in testis. TH2B massively replaces somatic H2B during meiosis and remains the main type of H2B in round and elongating spermatids, suggesting TH2B might be indispensable for meiotic and post-meiotic germ cells. The crystal structure analysis shows the TH2B could not form the water-mediated hydrogen bonds with H4R78, which may affect the stability of the TH2B nucleosome and facilitate histone replacement during spermiogenesis. In a Th2b mutant mouse, which contains modified C-terminus of the TH2B protein and causes a dominant-negative effect, males were infertile and severe abnormalities were seen in the elongating spermatids, which affected subnucleosomal transitional states during histone replacement. In contrast, Th2b-null mice are fertile and show normal spermatogenesis process, indicating a compensatory mechanism that rescues deficiency of TH2B in the histone-to-protamine transition. Indeed, in Th2b-null testis, the expression of somatic H2B was significantly increased and elevated methylation of H4R35, H4R55, H4R67, and H2BR72 could be detected in Th2b-null spermatids. As H4R35, H4R55, H4R67, and H2BR72 participate in the interactions of histone–DNA and histone–histone, and their methylation may impair these intranucleosomal interactions. Thus, the elevated somatic H2B and histone modification in Th2b-null spermatids might rescue the Th2b deficiency in testis.

12In humans, H2BFWT is a testis-specific histone, is synthesized and aggregated in testes, and single nucleotide polymorphisms (SNPs) in this gene is highly associated with male infertility. And spermatid-specific H2B (ssH2B) and H2BL1 have been identified and are strongly enriched in round or elongating spermatids, similar to that of TPs and protamines. However, the functional roles of these H2B variants in the histone-to-protamine transition still need to be further elucidated.

13In addition to the two canonical histones H3.1 and H3.2, three additional H3 variants have been identified and expressed in mammal testes, including H3.3, H3T and H3.5.

14H3.3 differs from canonical H3.1 with five amino acids, is expressed throughout mouse seminiferous tubules, and accumulates in the XY body of spermatocytes. Biochemical and biophysical studies show that H3.3 contributes to an open chromatin configuration and promotes transcription through disrupting the higher-order chromatin structure. H3.3 could be encoded by two gene paralogs in mammal, H3f3a and H3f3b, and the depletion of either H3f3a or H3f3b causes male infertility. The disruption of H3f3a produces abnormal spermatozoa, and the loss of H3f3b leads to growth defects and death at birth, with surviving H3f3b-null males showing complete infertility. In H3f3b-null germ cells, the TP1 is abnormally deposited in elongating spermatids while PRM1 could not be observed in in elongated spermatids and mature spermatozoa, indicating that H3f3b is required for chromatin reorganization and the histone-to-protamine transition. H3T (H3.4) is exclusively expressed in the spermatocyte and diminishes in the elongating spermatids. Biochemical studies clearly indicate that, in the H3T nucleosome, the DNA around the entry-exit regions shows more flexible than that of the H3.1-containing nucleosome, and that the H3T-containing polynucleosome could formed more open configuration than that of H3.1. However, the disruption of H3T leads to sterile males with azoospermia, as spermatocyte and spermatids are absent in the H3t-null testes. Thus, the function of H3T in the later stage of spermatogenesis need further investigated by using spatially and temporally specific knockout mouse models.

15H3.5 is highly expressed in human testis and specifically observed in spermatogonia and spermatocytes. In vitro studies reveal that the H3.5-specific L103 residue, reduces the hydrophobic interaction with histone H4 in the H3.5-containing nucleosome, which corresponds to the H3.3 Phe104 residue. H3.5 is significantly reduced in non-obstructive azoospermia (NOA) patients, whereas the precise roles of H3.5 in spermatogenesis remain largely unknown.

16Covalent conjugation of different post-translational modification of histones has a dramatic effect on the chromatin conformation by affecting the stability of the nucleosome and the histone-DNA interaction. Many types of histone modifications have been identified to facilitate the histone-to-protamine transition, including acetylation, ubiquitination, methylation, and phosphorylation.

17Hyperacetylated histones could facilitate histone eviction, and the acetylation of H2A, H2B, H3, H4 and histone variants have been detected in mammal testis. In Drosophila, inactivation of histone acetyltransferases by anacardic acid prevents the histones degradation and further a protamine incorporation during spermiogenesis, suggesting that histone acetylation is essential for the histone-to-protamine replacement.

18H4 acetylation (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) shows a spatial distribution pattern during spermatogenesis and is indispensable for the histone-to-protamine transition. H4K5ac, H4K8ac and H4K12ac are expressed in spermatogonia and pre-leptotene spermatocytes, disappear in leptotene to pachytene spermatocytes, reappeared in elongating spermatids, and finally disappeared in condensing spermatids ( Figure 1 ). In contrast, H4K16ac could only be detected in elongating spermatids ( Figure 1 ). In vitro analysis shows that H4 acetylation is essential for destabilization and remodeling of nucleosomes, and the incorporation of H4K16ac into nucleosomes prevents the formation of compact chromatin fibers and influence chromatin forming cross-fiber interactions. These findings indicate that H4 acetylation modulates higher order chromatin structure to facilitate the histone-to-protamine transition. EPC1 (Enhancer Of Polycomb Homolog 1) and TIP60 (Tat-interactive protein, 60 kDa), which are two components for the mammalian NuA4 (nucleosome acetyltransferase of H4) complexes ( Figure 2 ), are co-localized to the nuclear periphery near the acrosomes in both round spermatids and elongating spermatids. The depletion of either Epc1 or Tip60 perturbs histone hyperacetylation, especially H4 acetylation, and affects histone replacement during spermiogenesis. Another gene that may play a role in acetylation is SIRT1 (Sirtuin 1), a member of the NAD+-dependent deacetylase. Germ cell-specific Sirt1 knockout mice display reduced male fertility due to decreased spermatozoa number and increased proportion of abnormal spermatozoa. In Sirt1-null elongating and elongated spermatids, acetylation levels of H4K5, H4K8 and H4K12 are decreased and TP2 could not co-localize in the nucleus, leading to a chromatin condensation defect in Sirt1-null spermatozoa. Thus, SIRT1 may modulate other factors to promote H4 acetylation and the histone-to-protamine transition.

19The histone acetylation might be recognized by some chromatin remodelers to confer downstream signaling, and the double bromodomain and extra-terminal domain (BET) proteins have been identified to be critical epigenetic readers binding to acetylated histones and modulating changes in chromatin structure and organization during spermiogenesis. BRDT is a testis-specific BET member protein, which is expressed specifically in spermatocytes and spermatids, and contains two bromodomains that specifically recognize acetylated lysine residues. BRDT binds the hyperacetylated histone H4 tail and co-localizes with acetylated H4 in elongating spermatids. Remodeling assays have shown BRDT regulated the chromatin reorganization dependent acetylation in round spermatids. In mice, the disruption of the first bromodomain in BRDT resulted in male sterility by producing the morphologically abnormal spermatids. In elongating spermatids with BRDT containing a knockout of bromodomain 1 (BD1), TPs and protamines remained in the cytoplasm and histone replacement did not occur, suggesting BRDT is required for the histone-to-protamine transition by mediating the replacement of acetylated histones ( Figure 2 ). Furthermore, BRDT was found to bind with the N-terminus of SMARCE1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1), a member of the SWI/SNF family of ATP-dependent chromatin remodeling complexes, indicating BRDT may cooperate with SMARCE1 to facilitate the histone-to-protamine transition during spermiogenesis ( Figure 2 ).

20Proteasomes catalyze ATP- and polyubiquitin-dependent protein degradation, and they are made up of a 20S catalytic core particle (CP) and regulatory particle (RP). The 20S CP could be activated by cooperation with various RPs, such as PA700/19S, PA28α/β, PA28γ, and PA200. PA200 is highly expressed in the testis, and the disruption of PA200 results in male infertility and severe defects in spermatogenesis. During spermiogenesis, PA200 regulatory could directly recognize acetylated histones through a bromodomain-like module and promote their ubiquitin-independent degradation. In Pa200-null spermatids, results showed that H2B, H3 and elevated H4K16ac could be detected at the end of the elongation stage. Thus, PA200 specifically recognizes acetylated histones and mediates the core histones for acetylation dependent degradation through proteasomes during spermatogenesis ( Figure 2 ).

21Ubiquitin is a 76 amino acid protein that is attached to target proteins to regulate several cellular processes, such as protein degradation, cell signaling, autophagy, DNA damage responses and so on. Ubiquitinated H2A and H2B are enriched in spermatocytes and elongating spermatids. RNF8 is an ubiquitin E3 ligase that participates in DDR (DNA damage repair) by catalyzing the ubiquitination of H2A to promote the recruitment of some DNA damage response factors on the damage sites. The disruption of Rnf8 causes significant late-stage developmental defects in spermatids due to problematic histone-to-protamine replacement, with the canonical histones being detectable in Rnf8-deficient mature spermatozoa. In Rnf8-null mice, both ubiquitinated H2A and H2B are decreased in the testes and H4K16ac is dramatically decreased as well. Further studies showed that ubiquitinated H2A and H2B were essential for the efficient recruitment of the MOF (males absent on the first) acetyltransferase complex, which is highly expressed in elongating spermatids and responsible for H4K16 acetylation in the chromatin. Thus, RNF8 catalyzed histone ubiquitination could modulate H4K16ac by regulating the localization of MOF on the chromatin and facilitate histone removal in the elongating spermatids.

22The RNF8-dependent histone ubiquitination during spermiogenesis could also be modulated by PIWI protein, which is specifically expressed during germline development and enlists piRNAs (Piwi-interacting RNAs) to repress TE (transposable elements) and protect the germ cell genome integrity. In mice, Miwi, Mili, and Miwi2, the Piwi paralogs, have been identified in the testis and are required for male fertility. During spermiogenesis, MIWI binds to RNF8 in the cytoplasm of early spermatids through a piRNAs-independent manner, and APC/C mediated MIWI degradation in late spermatids is essential for nuclear translocation of RNF8, which catalyzes histone ubiquitination and further facilitates histone removal. In both humans and mice, mutations in the conserved destruction box (D-box) of HIWI and MIWI proteins, which lead to their stabilization, cause male infertility due to impaired histone ubiquitination and histone-to-protamine transition. Except MIWI, L3MBTL2 (Lethal 3 malignant brain tumor like 2), a member of the MBT-domain proteins that is implicated in chromatin compaction, could also interact with RNF8. The depletion of L3mbtl2 in germ cells affected male fertility by producing abnormal spermatozoa and the decrease of sperm counts. L3mbtl2 deficiency also caused the reduction of in levels of the RNF8 and histone ubiquitination in elongating spermatids, which further influenced the PRM1 deposition and chromatin condensation during spermiogenesis.

23PHF7 (PHD Finger Protein 7), which contains PHD (plant homeodomain) and RING finger domain, has been identified as a novel H2A ubiquitination E3 ligase in mouse testis. PHF7 is specifically located in the elongating spermatid nuclei, and the disruption of Phf7 led to male mouse infertility as reduction of sperm count and the increased proportion of abnormal spermatozoa. PHF7 could recognize the H3K4me3/me2 through its PHD domain and catalyze H2A ubiquitination by its RING domain. In Phf7-null spermatids, the H2A ubiquitination was dramatically decreased that resulted in the histone retention and protamine replacement defect ( Figure 2 ). Therefore, PHF7 has dual roles during the histone-to-protamine transition that works as an epigenetic reader by recognizing H3K4me3/me2 and as an epigenetic writer through catalyzing H2A ubiquitination to promote histone removal.

24Multiple histone methylation have been identified in elongating spermatids, for instance H3K4me2, H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K79me2, and H3K79me3. Among them, the methylation of H3K4 and plus acetylation might help to achieve a more-open chromatin configuration, whereas H3K9 and H3K27 methylation are known to be associated with a more-repressed chromatin configuration, indicating a balance of “opened” and “closed” chromatin regions during the histone-to-protamine transition. As some histone methyltransferases and demethylases are detectable during spermiogenesis, the histone methylation may be dynamically regulated in testis. Although few mouse models exist that allow precise detection of methylation activity that directly regulates histone replacement during spermiogenesis, some studies have revealed that histone methylation may modulate the histone-to-protamine transition through some other ways. PYGO2 (Pygopus homolog 2) comprises a C-terminal PHD finger, which can recognize the H3K4me3 and is specifically located in the elongating spermatid nuclei. In mice, the reduction of Pygo2 influenced the Tnp, Prm genes expression and caused the abnormal nuclear condensation, which further led to male sterility. Furthermore, PYGO2 associates with a histone acetyltransferase (HAT) activity, and the acetylation of H3 is disrupted in Pygo2 reduced elongating spermatids, indicating PYGO2 may recognize H3K4me3 through its PHD domain and could recruit HAT to facilitate H3 acetylation and further histone-to-protamine transitions. As described before, PHF7 could recognize the H3K4me3/me2 and catalyze H2A ubiquitination to facilitate the histone-to-protamine transitions. The predominant histone methyltransferase SETD2 (SET domain–containing 2) catalyzes the H3K36me3, and knocking out Setd2 in mouse germ cells causes aberrant spermiogenesis, resulting in complete male infertility. Moreover, the disruption of SETD2 causes complete loss of H3K36me3 and impaired activation of Tnp and Prm genes, indicating H3K36me3 may regulate the histone-to-protamine transition by activating Tnp and Prm genes expression. Contrarily, JHDM2A (JmjC-domain-containing histone demethylase 2A) is an H3K9me2/1-specific demethylase. The loss of Jhdm2a in mice exhibits post-meiotic chromatin condensation defects and leads to male infertility. Although global H3K9 methylation has no effect in Jhdm2a-null testis, JHDM2A directly binds to and controls H3K9 methylation at the promoter of Tnp1 and Prm1 genes, which further regulates the sperm genome packaging and chromatin condensation.

25Histone phosphorylation is involved in various cellular processes, and dynamic histone phosphorylation have been observed during spermatogenesis. The phosphorylation of histone H2AX at residue Ser139 (γH2AX) plays important roles in many biological processes, such as meiotic recombination and male sex chromosome inactivation in germ cells. γH2AX is detectable in elongating spermatids, and TSSK6 has been identified to be responsible for the H2AX phosphorylation during spermiogenesis. In mice, targeted deletion of Tssk6 leads to male sterility caused by the impairment in morphology and motility of spermatozoa. In spermatozoa, the loss of TSSK6 blocks γH2AX formation, resulting in elevated H3, H4 and the precursor and intermediate of PRM2. These results indicate that TSSK6 may mediate γH2AX to participate in the histone-to-protamine transition. H4S1 phosphorylation is highly expressed in mouse spermatocyte, round and elongating spermatids. H4S1 phosphorylation has been found to be essential for chromatin compaction and concomitantly histone accessibility, suggesting that H4S1 phosphorylation is required for histone replacement during spermiogenesis. Outside the canonical histones, many phosphorylated residues have been identified, using mass spectrometry analyses, that exist on different testis-specific histone variants, such as H1T, HILS1, TH2A, TH2B. Although many core histones and histone variants phosphorylation have been identified in germ cells, their physiological roles need further investigation.

26A variety of histone lysine modifications have been identified, including butyrylation, crotonylation, malonylation, propionylation, and succinylation. Kcr (Lysine crotonylation) is a newly identified histone modification and is detectable in elongating spermatids, which regulated testis-specific genes activation in post-meiotic germ cells. The CDYL (chromodomain Y-like) protein, which contains a C-terminal CoAP domain that interacts with CoA to achieve its crotonyltransferase activity, may suppress the histone Kcr by converting crotonyl-CoA to β-hydroxybutyryl-CoA. Accordingly, Cdyl-deficient male mice show reduced fertility, decreased epididymal sperm count and sperm cell motility, and dysregulated histone Kcr. In the Cdyl-deficient mouse testes, further analysis showed that the elevated TP1 and PRM2 were localized in a chromatin-free regions, suggesting that histone crotonylation is essential for the histone-to-protamine transition during spermiogenesis.

27Poly-ADP-ribosylation (PARsylation) is a common protein PTM (post-translational modification) observed in higher eukaryotes and involved in many different fundamental cellular functions. All of core histones and the linker histone H1 can be ADP-ribosylated, which could be catalyzed by poly(ADP-ribose) polymerases, such as PARP1 and PARP2, and resolved by PARG (PAR glycohydrolase). The PARP1, PARP2 and PARsylation proteins are specifically detected in elongating spermatids, and the perturbed PARsylation causes reduced male fertility with abnormal retention of core histones, H1T and HILS1 in mature spermatozoa. Thus, PARsylation is essential for the histone-to-protamine replacement, yet the precise PARsylation histone sites need further characterization.

28Between histone eviction and protamine incorporation in the nuclei of spermatids, about ninety percent of the chromatin components consist of TPs, which are arginine- and lysine-rich proteins encoded by Tnp1 and Tnp2. However, the functional roles of each TP are still controversial. TP1 could reduce the melting temperature of DNA and relax the DNA from core particles of nucleosome, whereas TP2 tends to compact the nucleosomal DNA by increasing its melting temperature, indicating TP2 may promote DNA condensation while TP1 facilitates the eviction of the histones. However, a separate study that shown that neither TP1 nor TP2 leads to the conformation changes in supercoiled DNA. These differences might reveal their unique roles during mammal spermiogenesis, as single knockout of either Tnp1 or Tnp2 leads to little morphological alteration of spermatozoa in mouse models. Elevated TP2 and TP1 proteins could be observed in Tnp1-null and Tnp2-null spermatids, respectively. Thus, TP1 and TP2 may compensate for each other in vivo. Indeed, Tnp1 and Tnp2 double-knockout mice show severe abnormal spermiogenesis with a general decrease in sperm motility and abnormal sperm morphology. The chromatin condensation is perturbed in the Tnp1 and Tnp2 double-knockout mice as severe histones retention is detectable, indicating TPs function redundantly yet have unique roles in the histone-to-protamine transition.

29Protamines are basic proteins that replace TPs in late spermatids. Two protamine genes (Prm1 and Prm2) localize on the same chromosome in both humans and mice. Protamines tightly interact with DNA via a central arginine-rich DNA-binding domain. Unlike Tnp genes, the disruption of either Prm1 or Prm2 leads to the male infertility. Protamines have multiple PTM sites, and a total of 11 PTMs have been identified on the protamines of mouse spermatozoa, including acetylation, phosphorylation and methylation. One site of interest is PRM2 S55, which is a candidate phosphorylated substrate residue of CAMK4 (Ca2+/calmodulin-dependent protein kinase IV). Targeted Camk4 knockout male mice are infertile, and the transition protein displacement by PRM2 is perturbed as a specific loss of PRM2 and prolonged retention of TP2 in Camk4-null spermatids. In vitro, PRM2 could be phosphorylated by CAMK4, implicating CAMK4 mediated PRM2 phosphorylation is required for the protamine incorporation during spermiogenesis. Thus, the specific post-translational modifications on protamines may also be essential for the histone-to-protamine transition.

30During the histone-to-protamine transition, many epigenetic regulators work together to facilitate paternal genome re-organization and packaging into the highly condensed nuclei of spermatozoa, through histone variation, specific histone modification and their related chromatin remodelers. Any defects during the histone-to-protamine transition would lead to male infertility. While the morphological changes during spermiogenesis are well characterized, the precise molecular mechanisms underlying the chromatin re-organization, in particular the transition from histones to protamines, are still unclear. It’s difficult to characterize the dynamic processes that occur during histone eviction, transition protein incorporation and protamine insertion. Moreover, 10% of the spermatozoa population in the epididymis has not yet completed the histone-to-protamine transition. These problems may be ascribed to a lack of experimental methods, which could fully recapitulate germ cell development in vitro. Further physiological insights may be gained by developing an in vitro germ-cell culture system that more accurately recapitulates the in vivo histone-to-protamine transition.

31Many histone variants modulate histone replacement by regulating the chromatin structure; therefore, nucleosomes containing these histone variants often maintain a relatively decondensed and open chromatin configuration, facilitating histone replacement during spermiogenesis. The redundant function of histone variants in modulating chromatin configuration ensures that defects in some histone variants have a limited effect on spermatogenesis. Indeed, some mutant histone variants in mouse models are dispensable for male fertility, and mice may show elevated levels of compensatory histones or histone variants. However, the redundant function of histone variants makes it difficult to explore the precise role of each histone variant in histone replacement.

32Although many histone modifications have been identified during the histone-to-protamine transition, many studies are descriptive and correlative. The direct manipulation of histone modification sites to reveal function is still urgently needed. With the development of gene editing tools, for example the CRISPR/Cas9 system, mouse models disrupting these histone modifications may be generated and used to elucidate function and in vivo relevance in the future. The following open-ended questions still need to be answered to provide in-depth investigation in the field.

33In addition to the histone variants and modifications mentioned above, what other novel histone variants and modifications participate in the histone-to-protamine transition? How can we identify them?

34How and where do histone variants replace canonical histones? What signal is needed to initiate replacement?

35As histone variants and modifications are identified that participate in the histone-to-protamine transition, how do we establish an epigenetic modulating network for this process? Which type of histone code is the initiating code?

36Histone hyperacetylation works as a determining event during the histone-to-protamine transition. Is this histone hyperacetylation an initial signal or an indirect consequence of prior events?

37What’s the relationship between these histone variants and modifications? What’s the mechanism underlying the cross talk between them?

38Chromatin assembly is modulated by histone chaperones or other chromatin remodelers. What’s the functional role of histone chaperones during the histone-to-protamine transition? Which histone variants or modifications send a signal to the chaperones?

39(7) How do the transition proteins replace the histone? How do protamines replace the transition proteins? What are the detailed functional roles of transition proteins?

40(8) Are there still post-translational modifications that need to be discovered in order to more accurately describe how histone modification plays a role in spermatozoa maturation?

41These questions and their underlying ideas need further investigation and refining to help us more thoroughly understand the complex molecular relationships and exact regulating mechanisms of the histone-to-protamine transition.

42WT and HG wrote the manuscript and drew the figures; CL and WL proposed the idea and revised the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work and approved it for publication.

43This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA16020701), the National Key R&D Program of China (grant 2016YFA0500901, 2018YFC1004202), the National Natural Science Foundation of China (grants 31771501, 91649202) and the Youth Innovation Promotion Association CAS (2018109).

44The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.