Spermatozoon DNA Structural Peculiarities

Abstract: Spermatozoon DNA Structural Peculiarities: Nuclear Chromatin Organization, Nuclear Chromatin Protection and Nuclear Chromatin Vulnerability. 

The article focuses on researching the peculiarities of spermatozoon DNA and its role in fertilization process. Sperm DNA integrity is essential for the accurate transmission of genetic information. Understanding the essential basis of how DNA is packaged in the spermatozoon has vital implications for male infertility. Sperm nuclei provide much more than half the genetic make–up of the newly fertilized embryo—it presents its DNA in a structural context that is required for the embryo to access the paternal genome in a proper sequence of events. It has a highly compact and complex structure and is capable of decondensation—features that must be present in order for a spermatozoon to be considered fertile. Any form of sperm chromatin abnormalities or DNA damage may result in male infertility. The integrity of sperm DNA is of crucial importance for balanced transmission of genetic information. As a result of integrative studying spermatozoon DNA structural peculiarities: nuclear chromatin organization, nuclear chromatin protection and nuclear chromatin vulnerability, there has been substantiated and explicated the essential peculiarities of chromatin compaction in the sperm nucleus which are vital for fertilization process. Inclusive representation of the nature of chromatin compaction in the sperm nucleus was given.

Introduction

Traditionally, the diagnosis of male infertility is based upon microscopic assessment and analysis of sperm concentration, motility and morphology as basic routine indicators of semen quality. These indicators provide fundamental information about sperm production upon which clinicians base their initial diagnosis. As emphasized in previous ESHRE reports, it is imperative that Semen Analysis be performed to the highest standards. To this end ESHRE’s Special Interest Group in Andrology (SIGA) has been instrumental in providing formal training programmes and external quality control schemes. However, even with appropriate quality assurance, traditional semen parameters provide a limited degree of prognostic and diagnostic information and their predictive power is highest primarily at the lower ranges of the spectrum [Lefièvre et al., 2007; Lewis, 2007]. Sperm production is only part of the fertility indicators. Sperm chromatin and DNA integrity are two vital essentials to ensure that the fertilizing sperm can support normal embryonic development of the zygote.

The nuclear status of sperm cells has shown to be a useful parameter in the assessment of male fertility. It has been shown that the probability of fertilization of oocytes in In–Vitro Fertilization (IVF) is related to the frequency of spermatozoa with chromatin alterations [Hoshi et al., 1996; Lolis et al., 1996], and it has been reported that the vast majority of spermatozoa bound to zona pellucida are characterized by normal chromatin packaging [Hoshi et al., 1996]. It has been postulated that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation and, consequently, in fertilization failure [Sakkas et al., 1996; Samocha–Bone et al., 1998]. Interestingly, sperm chromatin structure features do not seem to be related to conventional parameters of semen quality [Spanò et al., 1984; Evenson et al., 1991; Engh et al., 1992; Sakkas et al., 1995; Bianchi et al., 1996; Lolis et al., 1996; Fosså et al., 1997; Spanò et al., 1998]. In particular, Sperm Chromatin Structure Assay (SCSA) can identify the sperm subpopulations undergoing complete nuclear chromatin maturity [Evenson et al., 1989; Yossefi et al., 1994; Weissenberg et al., 1995; Golan et al., 1996] and can detect sperm samples with suspected impaired fertility [Evenson et al., 1980, 1991; Evenson and Melamed, 1983; Evenson and Jost, 1994; Golan et al., 1996, 1997; Fosså et al., 1997].

Numerous studies have presented the most essential factors and parameters that would be predictive for male fertility potential, including sperm count, motility, acrosome status, cell membrane integrity, morphology and morphometry of whole spermatozoa and/or sperm heads and integrity of nuclear chromatin. These factors and parameters, however, are not able to assess alterations in sperm chromatin organization, such as irregular condensation or DNA damage [Bianchi et al., 1996; Sakkas et al., 1998]. Several studies have shown that male infertility can be caused by sperm DNA damage [Aitken, 1999; Host et al., 2000; Larson et al., 2000; Morris et al., 2002; Tomsu et al., 2002; Benchaib et al., 2003; Carrel et al., 2003; Henkel et al., 2004; Tesarik et al., 2004a]. For this reason, sperm DNA fragmentation should be considered during the assessment of semen quality. Various scientific theories have been proposed to explain sperm DNA damage. In this controversial scenario, some authors think this damage is due to incomplete maturation of the gametes caused by flawed topoisomerase II activity [Bianchi et al., 1993; Manicardi et al., 1995]; others suggest that the alteration in genetic material is the result of an incomplete apoptotic process [Gorczyca et al., 1993; Rodriguez et al., 1997; Sinha Hikim et al., 1997; Richburg, 2000], whereas other authors believe that sperm DNA damage may be the result of excess Reactive Oxygen Species (ROS) production [Aitken et al., 2003]. However, the pathophysiological mechanism leading to sperm DNA damage is understood only incompletely, and no specific Treatment for infertility caused by this condition has yet been proposed. Similarly, it would be difficult to determine inclusively and conclusively the most accurate theory which would transparently represent the whole paradigm of indicators, factors and causes which lead to sperm DNA damage and would present the most effective preventive measures to avoid the negative consequences for the embryo development. Even though the pattern of sperm DNA damage (fragmentation) closely resembles that resulting from apoptosis (also defined as “programmed cell death” or the most accurate definition of apoptosis is programmed cell death) in somatic cells, several studies have questioned the causal relationship between the activation of the classical apoptotic pathway and DNA fragmentation of mature spermatozoa [Sakkas et al., 2002; Henkel et al., 2004; Moustafa et al., 2004; Lachaud et al., 2004]. It is absolutely incontrovertible that the classical cell death signalling pathway, in which caspase activation is followed by phosphatidylserine externalization marking the cell as target for phagocytosis, is active while germ cells remain in a tight association with Sertoli cells, but most Sertoli cell–free germ cells in the diseased testis undergo DNA fragmentation without caspase activation and phosphatidylserine externalization [Tesarik et al., 2004b]. After release from Sertoli cells, spermatids and spermatozoa thus appear to suffer DNA damage independently of the usual cell death signalling pathways. Previous studies have suggested that oxidative stress can be responsible for sperm DNA damage [Fraga et al., 1996; Barroso et al., 2000; Aitken and Krausz, 2001; Agarwal et al., 2003; Moustafa et al., 2004]. The loss of nutritional support by Sertoli cells may aggravate the impact of oxidative stress on sperm cell components [Tesarik et al., 2004b].

If DNA damage detected in ejaculated spermatozoa essentially begins after sperm release from Sertoli cells, it can be hypothesized that the degree of damage increases with time after Sertoli cell release. If this hypothesis is true, sperm populations recovered directly from the testis could be expected to be less affected by this pathological process as compared with ejaculated sperm populations. Moreover, developmental competence of spermatozoa obtained from these two sources was determined by evaluating fertilization rate, the percentage of good morphology embryos, pregnancy rate and implantation rate.

1. Retrospective overview of the various scientific hypotheses, concepts and theories, which outline the spermatozoon DNA organization with special emphasis given to the peculiarities of the organization 

Spermatozoa used for Assisted Reproductive Technology (ART) are, in a vast majority of cases, prepared by Density Gradient Centrifugation (DGC) or by a swim–up preparation in order to favour the isolation of motile and morphologically normal spermatozoa in accordance with strict exclusion criteria. Several studies have shown that, even though various levels of efficiency are reported, both sperm separation methods are quite effective in sorting out spermatozoa with nicked DNA and poorly condensed chromatin as evaluated by a variety of the sperm DNA integrity assays: SCSA [Golan et al., 1996; Larson et al., 1999, 2000; Spanò et al., 1999; Gandini et al., 2004; Zini et al., 2000a, 2000b], terminal transferase–mediated DNA end–labelling (TUNEL) [Younglai et al., 2001; Lachaud et al., 2004; Morrell et al., 2004; Piomboni et al., 2006], Comet assay [Donnelly et al., 2000, 2001; McVicar et al., 2004] and chromomycin A3 (CMA3) [Sakkas et al., 2000; Hammadeh et al., 2001; Tomlinson et al., 2001]. The need to minimize the occurrence of sperm exhibiting minimal DNA damage is an active research issue and new procedures for achieving this goal have recently been described and proposed [Said et al., 2006; Ainsworth et al., 2007]. The packaging of chromatin in its final form into the sperm nucleus is a long and complex process starting in the very early stages of the spermiohistogenesis when histones are replaced firstly by transition proteins and finally by protamines [Meistrich, 1993; Banerjee et al., 1995; Kramer and Krawetz, 1997]. Additional steps occur after ejaculation, with the participation of seminal plasma, in the evolution of chromatin stability, a pathway culminating in a smaller mature sperm cell which requires less energy to support motility and ready to fertilize [Agarwal and Said, 2003]. Packaging of DNA into maximally condensed chromatin is associated with the disulfide cross–linkages established between cysteine molecules of protamines. The progressive sperm chromatin packaging seems to be linked to a complex process of DNA cutting and ligating. Lower packaging quality, found in morphologically normal spermatozoa, may represent one of the major limiting factors in the fertilizing ability, thus stressing the importance of the tertiary and quaternary chromatin structure in the protection of genetic information and possibly in early post–fertilization events.

During recent years, there has been an increased focus on the role of sperm DNA and chromatin integrity in infertility [Evenson et al., 2002; Spanò et al., 2005; Erenpreiss et al., 2006; Evenson and Wixon, 2006a; Aitken and De Iuliis, 2007]. A variety of tests have been described to assess sperm chromatin integrity [Erenpreiss et al., 2006]. One of these tests, the Sperm Chromatin Structure Assay (SCSA) has shown to be a good predictor of fertility, in vivo [Evenson et al., 1999; Spanò et al., 2000; Bungum et al., 2004, 2007] as well as in vitro [Evenson and Jost, 2000; Larrson et al., 2000; Larson–Cook et al., 2003; Saleh et al., 2003; Bungum et al., 2004, 2007; Gandini et al., 2004; Virro et al., 2004; Check et al., 2005; Boe–Hansen et al., 2006; Evenson and Wixon, 2006b]. But further investigations in this field should be done for integrative understanding of the the role of sperm DNA and chromatin integrity in infertility.

2. Inclusive representation of the nature of chromatin compaction in the sperm nucleus. The essential peculiarities of chromatin compaction in the sperm nucleus which are vital for fertilization process: the concepts and conclusions represented in the study “Function of sperm chromatin structural elements in fertilization and development” written by Ward W.S.

The nature of chromatin compaction in the sperm nucleus has been excellently reviewed by Ward (2010). According to this model, the DNA is compacted into doughnut–shaped toroids that contain ∼50 kb of DNA in a semicrystalline state. Interspersed between these toroids are interlinker regions of DNA that form a close association with the nuclear matrix. Within the toroids, extensive creation of intra– and inter–molecular disulphide bridges within and between protamines during epididymal transit generates a chromatin structure that is relatively resistant to damage once the spermatozoa have achieved a state of maturity [Sawyer et al., 2003]. By contrast, the inter–toroid linker regions are histone rich and are, therefore, particularly vulnerable to attack by nucleases [Villani et al., 2010; Ward, 2010] and, presumably, Reactive Oxygen Species (ROS). In normally compacted sperm chromatin, the DNA cleavage would, therefore, preferentially occur at the interlinker regions. However, in defective spermatozoa the chromatin is relatively histone rich [Foresta et al., 1992] creating additional areas of vulnerability that may extend into the toroid regions of the DNA in order to generate the close correlations that have been observed between poor protamination and DNA damage [De Iuliis et al., 2009b; Simon et al., 2011a, b]. Such observations have led to a ‘two–step’ hypothesis of DNA damage in spermatozoa, whereby poor protamination of sperm chromatin during spermiogenesis is held to create a state of vulnerability that is subsequently exploited in attacks on DNA integrity that are largely mediated by Reactive Oxygen Species (ROS) [Aitken and De Iuliis, 2010]. Such a model is clearly consistent with the correlations highlighted above between DNA fragmentation, 8OHdG formation and chromatin protamination. Indeed, these associations are so strong that oxidative damage to sperm DNA can be seen as an indirect deflection of the quality of sperm chromatin remodelling during spermiogenesis [Aitken et al., 2013].

The integrity of sperm DNA is of crucial importance for balanced transmission of genetic information. 

During spermatogenesis, a complex and dynamic process of proliferation and differentiation occurs as spermatogonia are transformed into mature spermatozoa. This unique process involves a series of meioses and mitoses, changes in cytoplasmic architecture, replacement of somatic cell–like histones with transition proteins, and the final addition of protamines, leading to a highly packaged chromatin [Balhorn 1999; Poccia, 1986]. The tissue remodeling that occurs during spermatogenesis is unique in that it is one of the only systems that produces a cell type in which the nucleus is transcriptionally inactive and a large part of the cell is stripped off. It has been known for many years that the chromatin of the mature sperm nucleus can be abnormally packaged [Evenson 1980]. In addition, abnormal chromatin packaging and nuclear DNA damage appear to be linked [Evenson 1980; Manicardi et al., 1995], and there is a strong association between the presence of nuclear DNA damage in the mature spermatozoa of men and poor semen parameters [Sun et al., 1997; Irvine et al., 2000].

Understanding the essential basis of how DNA is packaged in the spermatozoon has vital implications for male infertility. Sperm DNA integrity is vital for the accurate transmission of genetic information. It has a highly compact and complex structure and is capable of decondensation—features that must be present in order for a spermatozoon to be considered fertile. Any form of sperm chromatin abnormalities or DNA damage may result in male infertility. The most vital concepts, presented by Ward W.S. in his study concerning the question of recent advances in the study of sperm chromatin structure and function, have altered the perception of this highly condensed, inert chromatin. As it was shortly resumed by Ward W.S., sperm DNA is packaged very tightly to protect the DNA during the transit that occurs before fertilization. However, this condensation cannot sacrifice chromosomal elements that are essential for the embryo to access the correct sequences of the paternal genome for proper initiation of the embryonic developmental program. The primary levels of the sperm chromatin structure can be divided into three main categories: the large majority of DNA is packaged by protamines, a smaller amount (2–15%) retains histone–bound chromatin and the DNA is attached to the nuclear matrix at roughly 50 kb intervals. Current data suggest that the latter two structural elements are transferred to the paternal pronucleus after fertilization where they have important functional roles. The nuclear matrix organization is essential for DNA replication, and the histone–bound chromatin identifies genes that are important for embryonic development. These data support the emerging view of the sperm genome as providing, in addition to the paternal DNA sequence, a structural framework that includes molecular regulatory factors that are required for proper embryonic development [Ward, 2010].

The paternal genome in spermatozoa is condensed in a manner that is specific to the cell type presumably to protect the DNA during the transit from the male to the oocyte prior to fertilization. The existence of this unique chromatin packaging has important consequences for both the development of improved diagnostics for medical infertility and for the study of higher order DNA structures [Ward, 2010].

Modern andrology’s theories’ paradigms correlate with close scientific focus given to normal and abnormal spermatozoon chromatin structure. Infertility researchers are interested in understanding sperm chromatin structure in order to determine how to best interpret assays for DNA integrity, which affects the outcome of Assisted Reproductive Technologies (ART) [Agarwal and Said, 2003; Evenson and Jost, 2000; Morris et al., 2002; Sakkas et al., 2002; Tomsu et al., 2002; van der Heijden et al., 2008]. Sperm chromatin can be divided into three major structural domains: 

1. The vast majority of sperm DNA is coiled into toroids by protamines [Hud et al., 1995]; 
2. A much smaller percent remains bound to histones [Churikov et al., 2004; Gineitis et al., 2000; Hammoud et al., 2009] and
3. The DNA is attached to the sperm nuclear matrix at MARs (Matrix Attachment Regions) at medium intervals of roughly 50 kb throughout the genome [Martins et al., 2004; Nadel et al., 1995].

Protamine–bound sperm chromatin

The vast majority of sperm chromatin is compacted into toroids that contain roughly 50 kb of DNA [Hud et al., 1993; Hud et al., 1995;  most of the DNA is hidden within the toroid. This component of the sperm DNA exists in semi–crystalline state and is resistant to nuclease digestion. Protamines also contain several cysteines that are thought to confer an increased stability on sperm chromatin by intermolecular disulfide cross–links [Ward, 2010]. Sperm DNA cannot be decondensed in vitro without reducing reagents [Ohsumi et al., 1988], and the disulfide cross–links increase as the sperm cells transit the epididymis after they exit the testis [Ward, 2010].

Another important aspect of this, largest structural domain of sperm chromatin is that its major function is almost certainly only for fertilization, and not for embryonic development. Protamine binding also silences gene expression during spermiogenesis [Martins et al., 2004; Carrell et al., 2007], but its role during fertilization and beyond is probably protective. Three separate lines of evidence support this contention. First, protamines are completely replaced in the first 2–4 hours after fertilization by histones so that the paternal chromatin has the same accessible chromatin as all other somatic cells [Ajduk et al., 2006]. Second, sperm chromatin is resistant to much greater mechanical disruption than somatic cells, supporting the protamines’ role in DNA protection. The structural organization of both histone–bound chromatin and sperm sperm nuclear matrix (MARs) are probably transmitted to the newly formed paternal pronucleus after fertilization and evidence suggests that both are required for proper embryogenesis [Ward, 2010].

Finally, the major unanswered question of protamine–bound chromatin structure concerns the secondary organization. Mudrak et al. (2009) have provided evidence that the protamine toroids are stacked side to side like a package of lifesavers. This model is the most efficient form in which the protamine toroids could be condensed into a highly protective chromatin. Variations of this theme are certainly possible, for example the protamine ‘lifesaver’ chromatin might be compacted so that two adjacent lines of toroids are aligned together with alternating toroids on the same chromatin being packaged in one line [Mudrak et al., 2009]. The condensation of this DNA into the crystalline–like toroids already infers the most important functional characteristics of protamine–bound sperm DNA—that it is unlikely to be active until after decondensation in the oocyte, and that it serves a largely protective function during fertilization [Ward, 2010].

Histone–bound sperm chromatin

Between 2 and 15% of sperm chromatin is bound to histones, rather than protamines [Gineitis et al., 2000; Churikov et al., 2004; Hammoud et al., 2009]. Three important questions regarding sperm histones have recently been addressed. The first question was whether sperm histones are associated with specific sequences within the sperm chromatin, or positioned randomly within the chromatin fiber as the result of incomplete protamine deposition. One group initially focused on the protamine gene locus of sperm that spans 28 kb flanked by two Matrix Attachment Regions (MAR regions) [Wykes and Krawetz, 2003]. This entire region seems to be preferentially associated with histones in spermatozoa, but does contain some protamine–associated DNA, as well. The same group recently surveyed the entire genome, and concluded that histones were interspersed throughout the genome, primarily at gene promoters [Arpanahi et al., 2009]. A separate group performed a similar study and concluded that entire gene families that were important for early development were preferentially associated with histones in spermatozoa [Hammoud et al., 2009]. Consequently, it is resumed that the work from both groups indicates that histones are non–randomly distributed in the sperm genome, and are associated with specific genes [Ward, 2010].

The second, related question was how were these histones distributed—were they interspersed throughout the sperm genome or were they located in discrete regions of the chromatin? The data described above [Arpanahi et al., 2009; Hammoud et al., 2009] suggest that histones are present in two types of distribution—in relatively large tracts of DNA, from 10 to 100 kb, and in smaller tracts of DNA interspersed throughout the genome. This has important implications for the structural organization of sperm chromatin, because if histones were distributed at regular intervals throughout the genome they might be part of a repeating unit of sperm chromatin structure [Ward, 2010].

Finally, a third important question concerning histone–bound DNA in sperm chromatin was whether sperm histones are transmitted to the developing embryo [Ward, 2010]. Shortly after fertilization, the protamines in sperm chromatin are replaced with histones supplied by the oocyte [Ajduk et al., 2006], but in those regions where histones are already present in the sperm DNA this may not be necessary. Van der Heijden et al. (2008) demonstrated that histones with specific modifications in the sperm cell are also present in the paternal pronucleus, suggesting that they were never replaced. The transmission of sperm histones, and the associated chromatin structures, suggest it is possible that the newly fertilized oocytes inherits histone–based chromatin structural organization from the sperm [Ward, 2010].

It was emphasized that the data currently support a model for histone-associated chromatin representing functional genes for both spermiogenesis (possibly representing residual active chromatin that persisted through chromatin condensation) [Martins and Krawetz, 2005; Ostermeier et al., 2005] and for early fertilization [Arpanahi et al., 2009; Hammoud et al., 2009]. Moreover, some of these histone-associated sperm chromatin structures may persist during the structural reorganization of the paternal chromatin when the sperm nucleus decondenses to form the paternal pronucleus [Ward, 2010].

Matrix Attachment Regions

In both somatic [Vogelstein et al., 1980; Gerdes et al., 1994; Dijkwel and Hamlin, 1995; Linnemann et al., 2009] and sperm nuclei [Kalandadze et al., 1990; Moss et al., 1993; Choudhary et al., 1995] chromatin is organized into loop domains that are attached every 20–120 kb in length to a proteinaceous structure termed the nuclear matrix [Ward, 2010]. This organizes the chromatin into functional loops of DNA that help regulate DNA replication [Vogelstein et al., 1980; Gerdes et al., 1994; Dijkwel and Hamlin, 1995] and gene transcription [Cockerill and Garrard, 1986; Nelson et al., 1986; Choudhary et al., 1995; Ostermeier et al., 2003]. This loop domain structure is present throughout the entire sperm chromatin even though the tertiary structure of most of the DNA is very different in spermatozoa. Ward W.S. explicated in his study the hypothesis that each protamine toroid contains a single DNA loop domain [Ward, 2010]. Between each protamine toroid, is a nuclease sensitive segment of chromatin the scientists term the toroid linker, which is also the site of attachment of DNA to the nuclear matrix, or Matrix Attachment Regions (MAR) [Ward, 2010]. The nuclease sensitivity suggests that these protamine linker regions are bound by histones, and this is consistent with the wide distribution of histones throughout the genome [Arpanahi et al., 2009]. Thus, the organization of sperm DNA into protamine toroids and by the nuclear matrix is directly linked [Ward, 2010].

What is the most essential to note is that a functional role for the sperm nuclear matrix in the function of the paternal genome during early embryogenesis. Spermatozoa with structurally disrupted sperm nuclear matrices do not support embryonic development after Intracytoplasmic Sperm Injection (ICSI) unlike those with intact matrices. To test the role of the sperm nuclear matrix organization directly, it is experimentally possible to remove the other two types of sperm chromatin organization—protamine condensation and histone–bound nucleosomes—by Treatment with high salt and reducing reagent. This Treatment leaves only the sperm nuclear matrix with associated loop domains attached and the resulting nuclei are called sperm nuclear halos [Nadel et al., 1995; Kramer and Krawetz, 1996]. When sperm halos were injected into oocytes, pronuclei formation was normal and DNA replication proceeded [Shaman et al., 2007]. This was true even when up to 50% of the DNA that was not attached to the matrix was removed by restriction endonuclease Treatment. DNA, alone, injected into oocytes did not form pronuclei nor did the DNA replicate. However, when the sperm nuclear matrix (MARs) are reversibly cleaved by Topo 2b, the paternal DNA is degraded at the time of the initiation of DNA synthesis 5.5 hours after Intracytoplasmic Sperm Injection (ICSI) [Yamauchi et al., 2007]. Oocytes injected with intact sperm halos did not develop to the blastocyst stage (unpublished data) suggesting that while the organization of DNA into loop domains by the sperm nuclear matrix is required for DNA replication, it is not sufficient for development [Ward, 2010].

Sperm chromosomes

An overview of sperm chromatin structure must include an accurate representation and transparent discussion of the least understood element, the higher order structure of the chromosomes. There are many models for mitotic chromosomes and, while important differences exist, there is general agreement on the basic folding pattern of the DNA [Pienta and Coffey, 1984; Boy de la Tour and Laemmli, 1988]. The higher order structure of chromosomes in interphase somatic cells is much less well understood, partially because it is much less uniform. However, it is clear that the DNA is bound to histones in nucleosomes that are in various stages of condensation or openness, depending on their function [Grewal and Elgin, 2007; Kloc and Martienssen, 2008]. The scientific group under the management of Zalenskaya I.A. have elegantly demonstrated that sperm chromatin of all species tested are folded into hairpin–like structures with the centromeres positioned near the center of the sperm cell with the telomeres of each chromosome paired and arrayed around the periphery of the sperm nucleus [Zalenskaya et al., 2000; Churikov et al., 2004; Solov’eva et al., 2004]. They also demonstrated that individual chromosomes are partitioned into territories that do not overlap [Mudrak et al., 2005; Zalensky and Zalenskaya, 2007]. Beyond these facts, models for the higher order structure of sperm chromatin are lacking. Understanding how sperm chromosomes are folded would provide unique insights into the structure of somatic cell chromosomes [Ward, 2010].

Ultimately, Ward W.S. explicated the conclusion that, of the three types of sperm chromatin structure discussed, two are inherited by the embryo and are probably required for proper development. This has two important implications. For infertility research, it suggests that methods to manipulate spermatozoa for Assisted Reproductive Technology (ART) should be developed that maintain the integrity of the sperm chromatin structure, as well as taking into consideration the integrity of the paternal DNA. Inheritance of sperm chromatin structural elements by the embryo: DNA in round spermatids is packaged by histones (top) but during spermiogenesis, most of these are replaced by protamines (middle, red). After fertilization, the protamines are removed, and histones supplied by the oocyte replace them (bottom, light green). However, some histones that were retained in the spermatozoon (middle, dark green) are probably retained in the newly formed paternal pronucleus after fertilization. Sperm nuclear Matrix Attachment Regions (MARs) are probably retained in the paternal pronucleus as well [Ward, 2010].

The second implication relates to the use of sperm chromatin as a model for eukaryotic DNA packaging. Sperm chromatin presents an interesting model for eukaryotic chromatin, and for studying the function of the nuclear matrix, in particular, because most of the DNA is condensed into chromatin that is difficult to decondense. Only what is thought to be the most active part of any cell’s chromatin, the attachment to the nuclear matrix, required for DNA replication, and important genes for development, is left associated with histones. Thus, the sperm cell has already fractionated the chromatin, naturally, into inactive and active chromatin by condensing most of the DNA with protamines [Ward, 2010].

As discussed above, the data suggest that the structural organization of the histone–bound chromatin and the sperm nuclear matrix (MARs) of the sperm cell are inherited by the paternal pronucleus after fertilization. Both of these structural elements of sperm chromatin are associated with different functions in the embryo [Ward, 2010]. In the case of sperm nuclear matrix (MARs), it is clear that the embryo cannot develop past the first cell cycle without proper organization by the nuclear matrix [Shaman et al., 2007], and it is likely that the histone–bound sequences are just as important for later embryonic development [Arpanahi et al., 2009; Hammoud et al., 2009]. The data, represented by Ward W.S. supports an emerging view that sperm nuclei provide much more than half the genetic make–up of the newly fertilized embryo—it represents its DNA in a structural context that is required for the embryo to access the paternal genome in a proper sequence of events [Ward, 2010].

3. Evaluation of the integrity of sperm DNA dimension: the concepts and conclusions represented in the study “Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications—a position report” written by Barratt L.R.C., Aitken R.J., Björndahl L., Carrell T.D., de Boer P., Kvist U., Lewis E.M.S., Perreault D.S., Perry J.M., Ramos L., Robaire B., Ward S. and Zini A.

Evaluation of the integrity of sperm DNA include, but are not limited to, the TUNEL assay, the Sperm Chromatin Structure Assay (SCSA), the Comet Assay and the Sperm Chromatin Dispersion Assay. These assays are already in clinical use in some infertility centres for two primary reasons. The first is to attempt to predict future Assisted Reproductive Technology (ART) outcome or explain previous failure (failures). The second is to be able to detect sperm with DNA that is sufficiently damaged as to result in transmission of genetic defects to the embryo and subsequent offspring. To optimize Assisted Reproductive Technology (ART), especially Intracytoplasmic Sperm Injection (ICSI), clinicians would like to be able to choose sperm with intact DNA. As conducted at present, these DNA integrity assays provide an assessment of the distribution of cells in a given ejaculate. However, these assays also destroy the cells and so cannot be used to identify or select an individual intact sperm for use in Intracytoplasmic Sperm Injection (ICSI). Before they become routine clinical assays, the experts need to comprehend what these assays are telling them about the integrity of the male genome. This will come from a thorough knowledge of the sperm chromatin structure and how it is packaged [Barratt et al., 2010].

Most DNA in sperm is bound to protamines, as somatic histones are replaced during spermiogenesis. However, biochemical analyses of sperm proteins indicate retention of some histones resulting in a nuclear protein composition that is about 90% protamine and 10% histone. The scientists have a fairly clear understanding of how the protamines package sperm DNA [Barratt et al., 2010]. The protamine–bound DNA is coiled into tightly compacted toroids that contain about 50 kb of DNA [Conwell et al., 2003], although the histone–bound DNA is believed to be organized into nucleosomal chromatin [Zalenskaya et al., 2000; Wykes and Krawetz, 2003; Hammoud et al., 2009]. Sperm DNA is so well protected that, unlike somatic cell chromatin, it is resistant to nucleases and sonication [Tateno et al., 2000]. The precise packaging of the histone portion of sperm chromatin is not well understood. The scientists are only now beginning to appreciate how this histone bound DNA is distributed throughout the chromatin, but they still do not know the size of the histone bound DNA segments [Barratt et al., 2010]. Recent evidence suggests that histone bound DNA in sperm cells is associated with gene families that are important for cell differentiation and early embryo patterning [Hammoud et al., 2009].

How are the current assays expected to interact with sperm DNA that is associated with protamine versus histone fractions? Reagents used in the TUNEL assay, for example, would not be expected to be able to access the DNA packaged and stabilized by protamines into toroids. This is because it uses the action of the enzyme Terminal deoxynucleotidyl Transferase (TdT) and if protamine bound chromatin is resistant to nucleases, it would be expected to be resistant to other enzymes as well. However, improperly stabilized protamine and histone bound DNA would be expected to be accessible to most of the assays currently in use for sperm DNA assessment. TdT can access DNA breaks in nucleosomal DNA, so the TUNEL assay would be expected to reveal this type of chromatin structure [Barratt et al., 2010]. The SCSA partially denatures sperm with acid which would preferentially extract histones rather than protamines [Ballachey et al., 1987; Larson–Cook et al., 2003]. Therefore, the SCSA would also be expected to identify single stranded DNA in histone bound sperm chromatin [Barratt et al., 2010]. However, SCSA would also measure single–stranded DNA in the protamine bound DNA if the stabilization of the protamine–DNA complex have been compromised [Dias et al., 2006] Finally, most protocols for the comet assay involve high salt extraction in the presence of a reducing reagent which removes both protamines and histones [Tomsu et al., 2002; McVicar et al., 2004]. The Comet assay therefore probably detects chromatin breaks in both types of chromatin with equal efficiency. A more detailed discussion of these points can be found elsewhere. Consequently, there are three integrative questions were formulated by the scientific group under the management of Christopher L.R. for inclusive understanding sperm DNA’s organization, sperm DNA’s protection and sperm DNA’s vulnerability: 

1. How can the experts and clinicians specifically assess the DNA damage in histone bound versus protamine bound DNA in sperm chromatin 
2. How meaningful is this distinction for clinical prognosis and
3. How can we assess the damage to the whole genome as opposed to specific fractions? [Barratt et al., 2010].

At the present time the experts have a limited understanding of how the sperm chromatin is formed during spermiogenesis. It was hypothesized, investigated and proved that protamines largely replace the histones during sperm nuclear condensation and that during this process the sperm DNA is unwound by topoisomerases and other proteins through the normal and necessary induction of strand breaks [Boissonneault, 2002; Kwan et al., 2003]. It is clear that when this packaging is not complete, DNA single and double strand breaks appear in the fully mature sperm [Marcon and Boissonneault, 2004]. Furthermore, the protamine deposition can also be incomplete, resulting in ratios of histone to protamine and of protamines 1 to protamines 2 that differ from normal. Both types of defects in spermiogenesis are associated with subfertility or infertility [Aoki et al., 2006a, b; Oliva, 2006]. Yet, the scientists do not know how topoisomerase cleaves sperm DNA during spermiogenesis or how the protamines interact to form sperm chromatin toroids. A host of additional aspects of sperm DNA packaging are also emerging as potential indicators of prognosis, including chromosome position within the sperm nucleus [Zalenskaya and Zalensky, 2004], the presence of mature mRNAs [Ostermeier et al., 2002, 2004] and newly described pi and microRNAs in sperm chromatin [Li et al., 2001; Martins and Krawetz, 2005; Carrell, 2008]. Currently, the experts can explain that they know a lot about how sperm chromatin packaging differs from somatic cell packaging, but it is absolutely incontrovertible to note with a special emphasis that they do not understand the specifics of how that packaging occurs [Barratt et al., 2010]. This will be important for a real understanding of what current sperm DNA assays are measuring.

Changes in sperm chromatin during sperm transport from the testis to the oocyte: the role of zinc

Zinc is incorporated into the sperm nucleus during spermiogenesis. The chromatin contains around 8 mmol Zn2+/kg which equates to one zinc ion for every 10 base pairs of the DNA, equalling one turn of the DNA–protamine helix [Kvist et al., 1985]. However, the role of zinc in sperm chromatin structure and function is only partly understood. The chromatin of more than 90% of spermatozoa can be experimentally decondensed in vitro by exposure of freshly ejaculated sperm to the anionic detergent SDS together with the divalent cation chelating EDTA [Björndahl and Kvist, 1985; Kvist et al., 1988]. This observation suggests a potential rapid mechanism for decondensation of the sperm chromatin that relies on Zn2+ depletion and interruption of macromolecules. Thus, at ejaculation, sperm exhibit zinc–dependent chromatin stability. However, upon in vitro culture, sperm become more resistant to decondensation and subsequently require disulfide bond reduction to enable unpackaging of the chromatin. This change is enhanced when zinc is withdrawn from sperm in vitro, and can, to a large extent, be counteracted by storing sperm in a buffer containing Zn2+. Although the role of zinc—to bind thiols—is simple, the consequences of this organization are complex based upon the following observations [Barratt et al., 2010]. 

1. Zinc primarily contributes to rapidly reversible chromatin stability.
2. If zinc is lost, it will not contribute to a sufficient stabilization of the chromatin, leaving the DNA more accessible and therefore more vulnerable to factors that might degrade it (endogenous enzymes or exogenous chemicals). 
3. Extraction of zinc can elicit chromatin decondensation if repulsion of macromolecules (DNA–protein filaments) is induced simultaneously, e.g. by phosphorylation or in experimental studies by the action of detergents like SDS [Kvist et al., 1987]. 
4. If macromolecules are not repelling the chromatin threads, the lack of zinc can allow the formation of disulphide bridges (S–S dependent chromatin stability) [Barratt et al., 2010].

What happens to the sperm chromatin during ejaculation, liquefaction and after liquefaction during further processing for ART? The zinc content of the sperm head and the type of chromatin stability are influenced by the composition of the surrounding ‘seminal fluid’ which comprises a mixture of various secretions that vary during ejaculation, liquefaction and after ejaculation. Normally spermatozoa are expelled in the first ejaculatory expulsions suspended in the zinc–rich prostatic fluid and the zinc–chelating seminal vesicular fluid is expulsed in later fractions [Barratt et al., 2010]. Upon mixture during and after liquefaction, spermatozoa are thus exposed to a series of differing environments that can have a marked influence on chromatin stability [Björndahl and Kvist, 2003]. Of clinical importance is that in some men the emptying of prostatic fluid is delayed and sperm are expelled in primarily zinc–chelating vesicular fluid, leading to extraction of zinc from the sperm chromatin [Barratt et al., 2010].

Mechanisms of DNA damage in male germ cells and spermatozoa

One of the potential mechanisms often cited as a cause of DNA damage in the male germ line is abortive apoptosis. The general idea behind this assertion is that as male germ cells metamorphose into highly differentiated spermatozoa, they progressively loose their capacity to undergo programmed cell death in the form of apoptosis. Since these cells are transcriptionally and translationally silent, it could not be otherwise [Barratt et al., 2010]. Thus, instead of engaging in a complete apoptotic response leading to cell death, differentiating haploid germ cells are thought to undergo a restricted form of this process leading to DNA fragmentation in the nucleus whereas retaining the capacity to differentiate into mature functional spermatozoa that may still be capable of fertilization [Sakkas et al., 2004]. Clearly, haploid germ cells are capable of activating a process that resembles apoptosis in some respects because both caspase activation and phosphatidylserine exteriorization have been observed in spermatozoa [Weng et al., 2002]. It is possible that by expressing apoptotic markers on their surface, senescent spermatozoa ensure that their ultimate phagocytosis in the female tract will be silent and not associated with a full–blown inflammatory response [Kurosaka et al., 2003]. This default senescence pathway may resemble the intrinsic apoptotic cascade in many respects but in one important detail, it is very different. In an archetypal somatic cell, stimulation of the intrinsic apoptotic pathway leads to the sudden appearance of endonucleases that are either released from the mitochondria (such as endonuclease G) or activated in the cytosol (caspase–activated deoxyribonuclease) and then they move into the nucleus to cleave the intra–nucleosomal DNA. However, in spermatozoa the physical separation of the mitochondria and cytoplasmic space from the sperm nucleus means that such mechanisms cannot be operative. As a result, the claim that “apoptosis” is a significant cause of DNA damage in spermatozoa might not be true for cells entering this process as mature gametes [Barratt et al., 2010]. However, sperm mitochondria represent a major source of Reactive Oxygen Species (ROS) in these cells [Koppers et al., 2008], that can become activated during the intrinsic apoptotic pathway. Additionally, what is essential to mention is that some DNA damage in the male germ line is the result of a programmed senescence pathway, characterized by the activation of mitochondrial Reactive Oxygen Species (ROS) formation, oxidative DNA base damage and unresolved DNA strand breakage [Aitken and De Iuliis, 2009]. If this is the case, then the most essential question is not what induces spermatozoa to undergo this restricted form of apoptosis—because this is their default condition. The vital question is what prevents these cells from entering this pathway. The answer to this lies in defining pro–survival factors that will prevent spermatozoa from initiating apoptosis [Barratt et al., 2010].

Conclusion

Sperm DNA integrity is very essential for the accurate transmission of genetic information. Understanding the essential basis of how DNA is packaged in the spermatozoon has vital implications for male infertility. Sperm DNA integrity is vital for the accurate transmission of genetic information. It has a highly compact and complex structure and is capable of decondensation—features that must be present in order for a spermatozoon to be considered fertile. Any form of sperm chromatin abnormalities or DNA damage may result in male infertility.

Consequently, what is vital to emphasize is that packaging of DNA into maximally condensed chromatin is associated with the disulfide cross–linkages established between cysteine molecules of protamines. The progressive sperm chromatin packaging seems to be linked to a complex process of DNA cutting and ligating. Lower packaging quality, found in morphologically normal spermatozoa, may represent one of the major limiting factors in the fertilizing ability, thus stressing the importance of the tertiary and quaternary chromatin structure in the protection of genetic information and possibly in early post–fertilization events [Spanò et al., 1999].

In support of this conclusion, it was reported that in–vivo fecundity decreases progressively when >30% of the spermatozoa are identified as having DNA damage. Several methods are used to assess sperm chromatin/DNA, which is considered an independent measure of sperm quality that may yield better diagnostic and prognostic approaches than standard sperm parameters (concentration, motility and morphology). The clinical significance of this assessment lies in its association not only with natural conception rates, but also with Assisted Reproduction success rates. Therefore, screening for sperm DNA damage may provide useful information in cases of male idiopathic infertility and in those men pursuing assisted reproduction. Treatment should include methods for prevention of sperm DNA damage [Agarwal and Said, 2003].

Significant and fundamental questions remain to be answered as part of a detailed understanding of the basic structure of chromatin and its repackaging during spermatogenesis, sperm maturation, ejaculation and during unpackaging in the oocyte. Although we do know how sperm chromatin packaging differs from that of somatic cell chromatin, we do not understand the specifics of how that packaging occurs. Furthermore, the scientists are at the beginning of their understanding and uncertain where in the lifecycle of the cell the DNA damage originates, and uncertain of the causes (e.g. oxidative in nature) or the nature of the damage (e.g. single and/or double strand breaks and/or DNA cross linking). Other basic questions remain unanswered: does the origin and nature of the damage suggest less/more severe consequences? How does the oocyte recognize and repair the damage? Is there a threshold of repair? Is there a degree of damage beyond the oocyte’s ability to repair? [Barratt et al., 2010]. Additionally, exciting areas are now emerging such as the presence of histones, mature mRNAs and newly described microRNAs in sperm chromatin. All the above explicates the importance of developing additional hypotheses and theories for further investigation and to better study the essence of spermatozoon DNA structural peculiarities: Nuclear Chromatin Organization, its Protection and its Vulnerability. Comprehensive research is absolutely required to address the key questions for further formulating the most inclusive strategies for prevention of any variations of DNA abnormalities of spermatozoon or for efficient sperm chromatin remodeling, which is possible to implement in every day practice of Fertility Clinics’ laboratories. 

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