Keywords

Key Points

  • Chromatin packaging is an integral part of spermatogenesis, and sperm DNA is packed into almost crystalline status that is at least six times more condensed in comparison to mitotic chromosomes.

  • During spermiogenesis, somatic histones in the haploid spermatid are replaced by testis-specific histones, which in turn are replaced by transition proteins and finally by protamines, leading to dense chromatin compaction in sperm.

  • Sperm histones undergo several posttranslational modifications, predominantly methylation and acetylation, to repress the transcriptional activity in sperm.

  • Compaction or proper packaging of chromatin is essential for shutting down the transcription activity in sperm and also for protecting its DNA from damage during its transit from testis through epididymis into the female reproductive tract.

  • Defects in chromatin packaging affect the morphology of sperm and its transcriptional activity and are associated with infertility or the outcome of ARTs.

  • Significantly higher histone-protamine ratios are observed in sperm from infertile men; a direct correlation exists between sperm protamine levels, DNA integrity, and sperm quality.

1 Introduction

WHO estimates as reported in 2012 indicate that about 50 million couples worldwide suffer from infertility (Mascarenhas et al. 2012). Male infertility accounts for almost 50% of the infertility. Asthenozoospermia, oligoasthenozoospermia, oligozoospermia, teratozoospermia, globozoospermia, azoospermia, and aspermia are the observed manifestations in male infertility. There is a considerable population of infertile individuals where none of these manifestations are observed and thus are referred to as idiopathic. Although research world over has been overwhelming with respect to female infertility, with respect to male infertility, it is limited probably because of the general perception that all problems of male infertility can be bypassed using assisted reproductive technologies (ARTs) such as IVF and ICSI. Increasing evidence is now available on the problems associated with ICSI (Wennerholm et al. 2000; Belva et al. 2007; Bonduelle et al. 2004; Morris et al. 2002). One of the biggest drawbacks of ICSI is that the genetic quality of sperm is overlooked leading to embryonic loss despite successful fertilization following ICSI. Genetic quality of sperm is determined by the integrity of its DNA and its compaction during spermiogenesis. A positive correlation has been observed between chromatin condensation and successful pregnancy in IUI and ICSI couples (Ioannou et al. 2016; Irez et al. 2015; Morris et al. 2002). In order to understand the impact of chromatin compaction on male fertility, it is imperative to understand the process of chromatin condensation.

2 DNA Packaging and Chromatin Compaction During Spermatogenesis

Spermatogenesis is a well-synchronized and tightly regulated process by which haploid male germ cells are formed. In the third and final stage of spermatogenesis, i.e., spermiogenesis, the haploid round spermatids undergo extensive morphological changes and nuclear remodeling to give rise to structurally distinct cell, the spermatozoa. Mature spermatozoon contains nucleus carrying haploid male genome, which is sixfold more compact as compared to any somatic cell of the body. Two major nucleoproteins involved in DNA compaction are nucleosomes and protamines. Nucleosome is the histone octamer composed of dimer of each histone H2A, H2B, H3, and H4. Histone H1 binds to the DNA in between two nucleosomes and is thought to be involved in higher-order chromatin structure formation. Each histone of nucleosome core particle (NCP) is divided into two parts: structured region made up of core and C-terminal region of histone and N-terminal unstructured tail which protrudes out of the nucleosome and interacts with DNA. Approximately 146 bp DNA is wrapped around each nucleosome. Posttranslational modifications (PTMs) like acetylation, phosphorylation, and ubiquitination occurring especially on N-terminus can influence chromatin structure either directly by adding negative or positive charge and altering histone-DNA interaction or indirectly by recruiting modification-specific chromatin remodeling factor (Pivot-Pajot et al. 2003).

Protamines are arginine-rich, small, basic, major nucleoproteins in sperm. They are synthesized in late-stage spermatid. Around 80–85% sperm DNA is compact due to protamination. In case of mammals, protamines are of two types protamine 1 (P1) and protamine 2 (P2). The presence of P1 in association with sperm DNA can be observed in nearly all vertebrates, whereas P2 is present only in primates, many rodents, and a subset of other placental mammals (Balhorn 2007). The number of protamine genes and copies present per haploid genome varies from species to species. Mammals have single-copy genes of P1 and P2, located on chromosome 16 (Reeves et al. 1989). P1and P2 are products of gene Prm1 and Prm2, respectively. The precursor protein of Prm2 undergoes proteolytic processing at its N-terminus to give rise to p2, p3, and p4. P2 family proteins, p2, p3, and p4, differ in 3–4 residues at N-terminus. The arginine-rich DNA-anchoring domains by which protamines bind with the negatively charged DNA and the multiple serine and threonine residues that can be used as phosphorylation sites form the structural elements of protamines (Balhorn 2007). The cysteine residues allow disulfide bond formation and thus link two adjacent protamines, which leads to further compaction of DNA.

During the process of spermiogenesis, nucleohistone to nucleoprotamine transition occurs. This transition is not direct but a gradual process, comprising of well-defined events, which involves first replacement of somatic histone by testis-specific histone variants and subsequently by transition proteins and then protamines.

At the round spermatid stage, the DNA compaction is the same as that of any somatic cell of the body. After the completion of second meiotic division, there is a surge of transcription observed characterized by two features not observed in somatic cells, namely, (a) the use of specialized transcriptional machinery and (b) the expression of large numbers of spermatogenic-specific genes which includes transcription of proteins like transition proteins, protamine, etc. required for spermiogenesis. At the same time, hyperacetylation of somatic histone H4 is observed. In vitro studies have indicated the role of hyperacetylated histones in nucleosome disassembly and replacement of histone by protamines (Oliva et al. 1987; Awe and Renkawitz-Pohl 2010). It has also been shown that bromodomain-containing protein (BRDT) binds with the hyperacetylated H4 and initiates nuclear remodeling (Pivot-Pajot et al. 2003; Moriniere et al. 2009).

Hyperacetylated histones then get replaced by testis-specific histone variants. Excepting histone H4, testis variants have been reported till date for core histones H2A, H2B, H3, and linker histone H1. During spermiogenesis, testis-specific histones get replaced by transition proteins (TP). Mammals, including mouse, rat, human, ram, and boar, predominantly have two types of transition proteins, viz., transition protein 1 (TP1) and transition protein 2 (TP2) (Akama et al. 1996; Chevaillier et al. 1998; Steger et al. 1998). Both TP1 and TP2 are encoded by single-copy genes, Tnp1 and Tnp2, respectively (Rathke et al. 2014). TP1 is a 6200 Da protein with about 20% arginine and 20% lysine, distributed uniformly, and no cysteine (Kistler et al. 1975). TP2 is a 13,000 Da protein with about 10% arginine, 10% lysine, and 5% cysteine (Grimes et al. 1975). It has a highly basic C-terminal domain and an N-terminal domain that forms zinc fingers (Meetei et al. 2000). TP1 is abundantly expressed (Heidaran et al. 1988), and its sequence is highly conserved in various mammals as compared to TP2 (Kremling et al. 1989). The role of TPs is not extensively studied. TP1−/− and TP2−/− knockout mice have been shown to be less fertile than normal mice and show abnormal chromatin condensation (Zhao et al. 2001). TP1 and TP2 double-knockout mice are sterile, and spermatogenesis is severely impaired suggesting their important role in spermiogenesis (Zhao et al. 2004).

Transition proteins remain associated with DNA for a short period of time and rapidly get replaced by protamines. Immediately after their synthesis, protamines get phosphorylated. Phosphorylation is thought to be essential for their nuclear transport as protamines can bind to their nuclear receptor and get transported only when phosphorylated (Mylonis et al. 2004). After binding of protamine to DNA, dephosphorylation takes place, and the disulfide bond formed between protamine further compacts the DNA. Chromodomain helicase DNA-binding protein 5 (Chd5) has a key role in the DNA compaction. It is involved in H4 hyperacetylation, histone variant expression, and removal and replacement of the histones with nucleoprotamines, and Chd5 deficiency in mice leads to defective sperm chromatin compaction and infertility (Li et al. 2014). Low expression of Chd5 has also been observed in the testis of infertile men by the same group.

3 Testis-Specific Histones

Replacement of histone by protamine is not 100%, and about 5–15% histones are still retained in mature human spermatozoa (Tanphaichitr et al. 1978; Gatewood et al. 1987; Zalensky et al. 2002). Retained histones have been found to be specifically enriched in the regulatory region of genes that are important for the earliest development stages postfertilization (Hammoud et al. 2009). Later it was shown by MNase sequencing that infertile males have random distribution of retained histones in spermatozoa (Hammoud et al. 2011). Testis-specific histone variants are thought to have specific biological function during spermiogenesis, as demonstrated by knockout studies with different variants. Table 17.1 summarizes the testis-specific histone variants known to date and their localization and influences on fertility.

Table 17.1 Testis-specific histone variants and their localization and influences on fertility
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Among the retained histones, TH2B is the major testis-specific histone variant present in mature sperm. TH2B differs from H2B mainly at its N-terminus. N-terminus of H2B has been shown to be associated with chromosome condensation in meiotic cell (de la Barre et al. 2001). N-terminus of TH2B has additional three potential phosphorylation sites (Ser12, Thr23, and Thr34) and repositioning of two others (ser5 and ser60), which are not seen in H2B, resulting in a different phosphorylation map of the N-terminal tail for TH2B (Pradeepa and Rao 2007).

Presence of TH2A/TH2B in nucleosome has been shown to induce a more open chromatin structure (Padavattan et al. 2015). This open chromatin structure facilitates the removal of histones and their replacement by protamines, thus enabling further compaction of DNA (Montellier et al. 2013). We have earlier reported reduced TH2B in asthenozoospermic individuals (Parte et al. 2012). However, TH2B knockout male mice have been shown to be fertile as the absence of TH2B is compensated by overexpression of somatic H2B variants and modifications on other histones. But Th2b+/tag mice show arrest at condensing spermatid stage leading to lack of sperm in epididymis and consequently infertility (Montellier et al. 2013). However, double knockout for TH2A/TH2B causes defect in spermatogenesis in males. Histone replacement during spermiogenesis is also affected. The mice showed reduced testis and epididymis weight and are sterile. Secondary spermatocytes at interkinesis (the interphase between meiosis I and II) are more abundant in the mutant testis than in the wild type, suggesting extended interkinesis in mutant mice (Shinagawa et al. 2015). Interestingly, it is the TH2A and TH2B from oocyte that is involved in activation of paternal genome postfertilization (Shinagawa et al. 2014). The dynamic changes in chromatin structure during spermiogenesis, epididymal maturation, and up to early embryonic development are summarized in Fig. 17.1.

Fig. 17.1
figure 1

Chromatin dynamics in the sperm during its journey from a round spermatid to the formation of an embryo. Circles with no pattern represent somatic histones, circles with pattern indicate respective testis-specific histones, while circles with diagonal lines indicate the respective maternally expressed testis-specific histones. TSHV testis-specific histone variant, TP transition protein

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4 Histone Modifications in Sperm and Their Influence on Sperm Fertilizing Ability/Embryonic Development

Several posttranslational modifications (PTMs) have been observed in mouse and human sperm (Fig. 17.2). In mouse sperm, 26 PTMs have been reported in specific residues of core histones and linker histone and 11 PTMs on PRM1 and PRM2 (Brunner et al. 2014). Comprehensive assessment of the histone modifications in normal human sperm revealed 102 modifications (Schon et al. 2015). Modifications are observed on the linker histone H1, the canonical histones, as well as their variants. While modifications on H4 are conserved, those on H3 vary between individuals. The modifications are not altered on cryopreservation of the sperm. Some PTMs of histones are uniquely distributed in human sperm, and this distribution varies among individuals and also between the sperm of a single individual (Krejci et al. 2015). Variations among individuals have been observed in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1 in the sperm-head fractions. Levels of acetylated (ac) histones H4 are relatively stable. Lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1 are seen in sperm with P2 deficiency. H3K9me2 and levels of P2 show a strong correlation. While the localization of H3 lysine 4 methylation (H3K4me) or H3 lysine 27 methylation (H3K27me) is highly similar in the gametes of infertile men compared with fertile men, a reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes has been noted. Also, the methylation status of certain developmental promoters and imprinted loci are altered in a subset of infertile men (Hammoud et al. 2011). Recently, histone PTMs and their relative abundance in distinct stages of mouse spermatogenesis and in human spermatozoa have been identified (Luense et al. 2016). They observed a strong conservation of histone PTMs for histone H3 and H4 between mouse and human sperm; however, H1, H2A, and H2B showed very little conservation (Luense et al. 2016).

Fig. 17.2
figure 2

Posttranslational modifications (PTMs) reported on the core histones in mouse and human spermatozoa. Colors red, blue, green, and yellow represent histones H2A, H2B, H3, and H4, respectively. The extension outside the circle represents the N-terminal region, and that inside it represents the C-terminal region. The segments on the circle represent core region of the respective histone. The PTMs reported on histones in spermatozoa of mouse, human, or both are as shown in the legend to the figure

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In sperm, genes relevant to spermatogenesis are marked by H3K4me2, and the genes involved in developmental regulation are marked by H3K27me3 (Brykczynska et al. 2010). While H3K4me2 is an activating mark, H3K27me3 has been shown to be a repressor of genes. This means that prior to fertilization, the genes involved in early embryonic development are repressed by H3K27me3, while those involved in spermatogenesis are maintained in an active state by H3K4me2. Reduction in H3K4me2 induced by human KDM1A histone lysine 4 demethylase transgene overexpression during mouse spermatogenesis has been shown to severely impair development and survival of the offspring, a defect which is also seen in two subsequent generations (Siklenka et al. 2015). H3K4me2 was reduced at the CpG islands of genes involved in development. While this region is majorly marked by H3K27me3, work of Brykczynska has shown that some of the developmentally regulated genes are also marked by H3K4me2. H3K4me3 has also been demonstrated to be important for spermatogenesis; loss of H3K4 methyltransferase MII2 reduces H3K4me3 consequently rendering the male mice sterile (Glaser et al. 2009). H3K9me2/1-specific demethylase JHDM2A also known as JMJD1A is essential for spermatogenesis, and its loss causes infertility in male mice due to incomplete chromatin condensation (Okada et al. 2007).

Stage-specific modifications have been identified for TH2B with acetylated TH2B being most abundant in spermatogonia (28.9%) compared to spermatocytes (8.3) and spermatids (11.2%). At the C-terminus, phosphorylation at K116 and methylation at K117 were observed in combination in TH2B isolated from these stages (Lu et al. 2009). However, its functional relevance is not known. Various PTMs like acetylation, methylation, and phosphorylation have been identified on TH2B from tetraploid spermatocyte and haploid spermatid. LC–MS/MS analysis of TH2B from spermatocytes identified six acetylation, three monomethylation, and one phosphorylation site, while that of TH2B from round spermatids identified four acetylation and two monomethylation sites. In silico analysis showed altered histone-histone as well as histone-DNA interactions in TH2B-bearing nucleosome. Also acetylation on N-terminal tail of TH2B has been shown to weaken its interactions with the DNA (Pentakota et al. 2014). Its physiological relevance remains to be determined.

5 Protamines and Male Infertility

Protamines and histones are the two major nuclear proteins in many vertebrate species including mice, rat, human, etc. These proteins play major roles during chromatin condensation at spermiogenesis. Several reports indicate that P1 and P2 are expressed in nearly the same amount in fertile human sperm and alteration in P1/P2 ratio is associated with male infertility (Aoki et al. 2005a, 2006; Zhang et al. 2006; Hammoud et al. 2009).

The first documented report highlighting the importance of protamines revealed the absence of protamines in the spermatozoa of infertile (oligozoospermic) patients (Silvestroni et al. 1976). This was followed by a study on 7 infertile and 17 fertile individuals where increased P1/P2 ratio in six of the seven patients was observed (Balhorn et al. 1988). Thereafter, a good number of studies have indicated that fertile men express P1 and P2 in same amount, while alteration in this ratio correlates with male infertility; infertile men show either decreased or increased P1/P2 ratio (Balhorn 2007; Balhorn et al. 1988; Belokopytova et al. 1993; de Yebra et al. 1993; Mengual et al. 2003; Aoki et al. 2005a). Balhorn’s group has shown that the percentage of protamines is different in the patients with abnormal seminal parameters compared to patients with normal parameters. Also within the heterogeneous population of spermatozoa, round-headed spermatozoa from patients contain less protamines and more histones and intermediate proteins than normal spermatozoa. Further, protamine levels vary between individual sperm of infertile males and correlate with viability and DNA integrity (Aoki et al. 2006). Interestingly, studies employing Percoll separation for fractionating sperm have shown that P1/P2 ratio and total protamine from different Percoll fractions within the same sample were not significantly different. However, there were significant differences in P1/P2 ratios in the oligozoospermic and asthenozoospermic groups as compared to normozoospermic indicating that P1/P2 and amount of protamine retention were independent of morphology and motility of sperm cells (Mengual et al. 2003).

This alteration in P1/P2 ratio might be due to alteration in expression of either of two protamines or both. Several studies have indicated lower P2 and increased P2 precursor in infertile men, indicating abnormality in the processing of precursor protein (de Yebra et al. 1993, 1998; Carrell and Liu 2001; Torregrosa et al. 2006). Aoki et al. observed a P1/P2 ratio around 1 in fertile donors; in infertile group, the P1/P2 ratios were either less than 0.8, between 0.8 and 1.2, or greater than 1.2 in 13.6%, 46.7%, and 39.7% of the patients, respectively. P1 and P2 were both under-expressed in patients with a normal P1/P2 ratio. In patients with a high P1/P2 ratio, P1 was normally expressed and P2 was under-expressed. They also reported that patients with abnormal P1/P2 ratios displayed significantly reduced semen quality and sperm penetration ability (Aoki et al. 2005a).

Several studies have also reported the presence of protamine transcript in sperm. Significantly aberrant protamine mRNA ratio was found in infertile individuals, and it correlates with DNA fragmentation and IVF success (Steger et al. 2001; Rogenhofer et al. 2013; Ni et al. 2014a). Significantly higher PRM1 and PRM2 mRNA copy numbers have been observed in normozoospermic versus teratozoospermic samples (Savadi-Shiraz et al. 2015). In contrast, transition protein 2 (TNP2) transcript abundance was significantly higher in teratozoospermic samples and positively correlated with sperm-head defects.

6 Protamines, DNA Compaction, and Integrity

Protamines are essential for sperm-specific packing of DNA. Compaction of DNA shuts off transcription as the DNA is no more amenable to the transcription factors and RNA polymerase. It also protects the DNA from any damage thus maintaining its integrity. This ensures that postfertilization the paternal genome is delivered in a form that allows developing embryo to accurately express genetic information. DNA compaction during chromatin condensation changes a transcriptionally active chromatin into a transcriptionally silent chromatin, and all the genes that are required for spermatogenesis and sperm function are transcribed prior to this transition, i.e., until the round spermatid stage. Live imaging studies in Drosophila have shown that histone-to-protamine transition starts 50–60 h after completion of meiosis and lasts for 5–6 h (Awe and Renkawitz-Pohl 2010). In mice although there is no direct evidence such as live imaging, indirect evidences suggest that this transition starts approximately 156 h after completion of meiosis and it lasts for 120–126 h, i.e., from step 10 to step 15 of spermiogenesis (reviewed by Rathke et al. 2014). This period is characterized by DNA breaks and repair which allows relief from the torsion stress and facilitates removal of the histones and replacement by transition proteins and subsequently protamines (Marcon and Boissonneault 2004). Thereafter, selective translation of the stored mRNA takes place as per the requirements of the sperm.

It is well established that about 5–15% histones are retained in normal human sperm. Elegant studies by Hammoud et al. have shown gene clusters important for embryonic development to be associated with the retained histones in sperm of fertile men (Hammoud et al. 2009). This implies that improper packaging due to higher histone retention as seen in sperm chromatin of infertile men may expose many more gene clusters. A subsequent study by the same group showed that in infertile men, histones retention was random genome-wide, unlike fertile men where the histone retention was seen only at specific gene clusters (Hammoud et al. 2011). The epigenetic marks H3K4me or H3K27me were also reduced on the retained histones in the infertile men. They speculate that these changes may be responsible for the poor reproductive outcome post ICSI/IVF in infertile men.

Any defects in chromatin packaging wreaks havoc with the sperm ability to fertilize or sire a viable offspring either by allowing the untimely transcription of certain genes, allowing certain modifications of histones that may switch the transcription on or off, or increasing the vulnerability of the DNA to drug-induced damage. Observations from the chromodomain helicase DNA-binding protein 5 (CHD5) KO mice are a testimony to the effect of improper condensation on sperm morphology and fertility of the male offspring (Zhuang et al. 2014). H4 hyperacetylation, which is vital for histone replacement during spermiogenesis, is reduced in these mice, and the sperm show deformed nuclei and abnormal head morphology. However, in these mice transcription of important genes, controlling spermatogenesis was not affected. Several groups have shown very lucidly the correlation between protamine compaction, DNA integrity, and sperm quality (Franken et al. 1999; García-Peiró et al. 2011; Manochantr et al. 2012; Utsuno et al. 2014). Chromatin packaging as studied by CMA3 and acidic aniline blue staining negatively correlates with normal sperm morphology (Franken et al. 1999). Utsuno et al. observed abnormal protamination in significantly higher number of spermatozoa with abnormal head morphology compared to those with normal head morphology. DNA fragmentation was also higher in the protamine-deficient spermatozoa. Studies on DNA damage in men undergoing IVF treatment revealed a positive association between DNA damage and abnormal sperm morphology and motility and negative correlation with sperm concentration (Morris et al. 2002). Protamine 2-deficient mice sperm demonstrate a direct correlation between PRM2 haploinsufficiency and frequency of DNA damage as seen from comet assays and ultrastructural analysis (Cho et al. 2003). In studies with human sperm, a positive correlation has been shown between protamine deficiency and sperm DNA damage (De Iuliis et al. 2009; Nili et al. 2009; Tarozzi et al. 2009; Razavi et al. 2010; Manochantr et al. 2012; Utsuno et al. 2014). Several studies have correlated altered P1/P2 ratio with susceptibility to DNA damage (Aoki et al. 2005b, 2006).

7 DNA Integrity and ART Outcomes

DNA integrity also influences sperm penetration and fertilizing ability, IVF and embryo quality, and development in ICSI outcome (Khara et al. 1997; Carrell et al. 1999; Carrell and Liu 2001; Nasr-Esfahani et al. 2004; de Mateo et al. 2009). DNA fragmentation and CMA3 positivity indicative of protamine deficiency negatively correlate with the fertilization rate in ICSI patients; DNA methylation negatively correlated with DNA fragmentation (Tavalaee et al. 2009). However, Tarozzi et al. observed a close relationship between sperm protamination and fertilization and pregnancy only in IVF; in ICSI there was a correlation between DNA fragmentation and pregnancy (Tarozzi et al. 2009). In men enrolled for ICSI, a positive association was seen between sperm damage and impairment of postfertilization embryo cleavage (Morris et al. 2002). In another study of individuals referred for ICSI, CMA3 positivity showed a significant negative correlation with fertilization rate post ICSI (Iranpour 2014). An isolated study using cleavage-stage frozen-thawed embryos from cycles of IVF and ICSI has however observed no significant difference in the biochemical pregnancy, clinical pregnancy, and miscarriage rates between sperm showing DFI <30% and those >30% (Ni et al. 2014b). The group did find some association between DFI and blastocyst formation in the ICSI group. A recent study investigating the influence of sperm DNA fragmentation on the pregnancy outcome and pregnancy loss after ART in couples going for either autologous ICSI, ICSI using donor eggs, or IUI observed that while the pregnancy rates were not significantly different, pregnancy losses correlated positively with the DNA fragmentation which was measured as DNA fragmentation index (DFI). The study indicates that sperm samples showing DFI >27% are associated with an increased risk of early pregnancy loss (Rilcheva Violeta et al. 2016). A similar observation has been reported earlier (Jin et al. 2015). Additionally, this group observed that when the DFI exceeded 27.3%, the live birth and implantation rates were significantly reduced in women with reduced ovarian reserve vis-a-vis women with normal ovarian reserve.

Conclusions and Future Directions

DNA integrity and its proper compaction in sperm are vital to its fertilizing ability as well as for early embryonic development in the preimplantation stage. Poor DNA compaction in sperm severely hampers its fertilizing ability and further development that accounts for fertility loss in natural conception or poor success of IVF/ICSI procedures. While literature is replete with evidences on histone retention and protamine deficiency in infertile cases, our knowledge on impact of several histone modifications on the fertility of male is limited and needs attention. Further research in this direction may identify sperm chromatin tests that may predict the success of ARTs. At the same time, further studies are needed to understand the significance of the retained histones in sperm maturation and their contribution toward the fertilizing ability of sperm.