A model for the importance of zinc in the dynamics of human sperm chromatin stabilization after ejaculation in relation to sperm DNA vulnerability

Abstract

The focus of this review is the dual functions of the sperm chromatin stabilization and how external factors can interfere with these functions. Zinc depletion after ejaculation allows for rapid and total sperm chromatin decondensation without addition of exogenous disulfide cleaving agents. Zinc depletion without concomitant repulsion of chromatin fibers induces another type of stability that requires exogenous disulfide cleaving agents to allow decondensation. It is essential to extend the present concept, that the sperm chromatin stability is based on disulfide bridges only, to include also the functions of Zn²⁺. It is suggested that the chromatin stability of the ejaculated human spermatozoon is rapidly reversible due to the dual function of Zn²⁺ that stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism: the formation of zinc bridges involving protamine thiols of cysteine and potentially also imidazole groups of histidine. Extraction of zinc from the freshly ejaculated spermatozoon allows two totally different biological results: (1) immediate decondensation if chromatin fibers concomitantly are induced to repel (e.g., through phosphorylation in the ooplasm) and (2) thiols freed from Zn²⁺ are available to form disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (in first ejaculated fraction) represent physiology. Extraction of chromatin zinc can be caused by unphysiological exposure of spermatozoa to the zinc chelating and oxidative seminal vesicular fluid, a situation common to most assisted reproductive techniques (ART) laboratories where the entire ejaculate is collected into a single container in which spermatozoa and secretions are mixed during at least 30 min. Some men in infertile couples have low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to decreased access for the assay to the DNA in superstabilized chromatin.

Keywords:

• assisted reproductive techniques
• ejaculation
• sperm chromatin structure
• sperm DNA damage
• zinc

Introduction

Functional Significance and Effects of the Unique Sperm Chromatin Structure

The purpose of the spermatozoon is to transport a haploid genome unharmed to the egg. To accomplish this transfer of genetic material and chromatin structure of the male gamete is radically different from that of somatic cells. In principle, the sperm chromatin DNA is extremely resistant towards conditions that could harm the DNA, compared to somatic cell DNA. Concomitant with this protected state, the chromatin structure must carry the property to make the DNA available very rapidly in the ooplasm. However, as will be discussed, there is a considerable heterogeneity in sperm DNA protection and delivery among ejaculated human spermatozoa handled in vitro. It is the aim of this review and hypothesis to highlight the importance of zinc for this dual function of the sperm chromatin structure and how extrinsic factors can interfere with the zinc dependent chromatin stabilization.

The transcriptionally inactive DNA in the sperm is packed very densely in an almost crystalline way, due to the exchange of somatic histones with basic protamines. Thereby the DNA is given a very high degree of protection by reducing both the access of a potential source of free radicals (i.e., free water) as well as water soluble compounds that could both contribute to DNA damage. Yet the DNA must be made available rapidly at arrival in the oocyte.

Faulty sperm chromatin packing could be manifested either as a reduced compaction or as a supernormal compaction. A reduced compaction would increase the access to the DNA and thereby enable increased exposure of the DNA to potential damage. In the laboratory tests for DNA damage, a reduced compaction would increase the access to the DNA that might be interpreted as increased DNA damage compared to spermatozoa with less access to the DNA. A supernormal compaction of the sperm chromatin would jeopardize the timing of the rapid delivery of the sperm DNA in the ooplasm. In laboratory tests aimed at revealing damage to the DNA, supernormal compaction would reduce the access of DNA interacting staining that could be interpreted as a reduced level of damage. Therefore, all factors affecting the compaction must be considered to understand normal physiology, pathological outcomes, and to correctly interpret sperm DNA damage tests.

Elements of the sperm chromatin structure

For an understanding of the mechanisms behind normal and disturbed sperm chromatin structure, it is essential to identify the elements that participate in or interact with the possible mechanisms. The change of somatic histones into protamines still leaves 5–10% of human sperm DNA linked to histones. Therefore the histone-linked DNA has a stabilization and structure similar to that in somatic cells and different from that of the DNA linked to protamines.

The Sperm Protamines

Arginine is the predominant component (45–48%) of both protamines (P1 and P2) in human spermatozoa [Gusse et al. 1986] that imparts an abundance of positively charged −NH₃⁺ groups onto the protamines. These groups neutralize the negative charges of the phosphate groups of the DNA backbone, thereby allowing a high degree of compaction of adjacent chromatin fibers [Balhorn 2007].

Similarly the imidazole groups of histidine and the thiols (−SH) of cysteine are likely candidates to interact with zinc in the sperm chromatin due to their efficiency in binding Zn²⁺ [Porath et al. 1975]. Furthermore, imidazole and thiol groups are also possible participants in ion bridges involving Zn²⁺. In classical zinc fingers, a single zinc ion is tetrahedrally coordinated by conserved histidine and cysteine residues, stabilizing the motif. Thiols alone are the basis for the formation of disulfide bridges. In the absence of zinc, the thiol groups could form disulfide bridges between thiols [Bal et al. 2001; Bianchi et al. 1994; 1992; Gatewood et al. 1990; Kvist 1980a].

Serine and threonine residues can be phosphorylated, i.e., bind negatively charged phosphate groups. Thus, serine and threonine provide the basis for negatively charged repulsive forces when these groups are phosphorylated. Furthermore, compaction of adjacent chromatin fibers would be possible when serine and threonine residues are dephosphorylated. Phosphorylation is therefore a candidate to provide an important mechanism to induce a rapid decondensation by repulsion of chromatin fibers while unpacking the DNA in the oocyte.

Sperm decondensation in the oocyte requires gluthathione. If the function of gluthathione is blocked, pretreatment of the oocytes with dithiothreitol (DTT) can make sperm chromatin decondensation possible [Sutovsky and Schatten 1997]. It should be noted, that in addition to disulfide-bridge cleavage, both DTT and glutathione thiols have a high affinity for zinc. In other words, a mechanism for both zinc-chelation and disulfide-bridge cleavage, therefore, seems to be present in the mature ooplasm.

Zinc

During spermiogenesis zinc is incorporated into the sperm nucleus when the compaction of the nucleus starts. An early sign of zinc deficiency is an arrest at spermiogenesis causing a complete lack of elongated spermatozoa [Barney et al. 1968].

The content of zinc associated chromatin in the ejaculated sperm has been calculated to approximately 8 mmol Zn²⁺/kg [Kvist et al. 1985]. Up to one zinc atom for every five sulfur atoms has been detected by X-ray microanalysis of the sperm head. Given the fact that human protamines contain approximately 5 sulfur atoms there appears to be in the order of one zinc ion for every protamine molecule. One protamine molecule provides positively charged −NH₃⁺ (in the guanidinium group of arginine) that neutralize the 20 negatively charged phosphate groups in the 10 base pairs of the DNA, equaling one turn of the DNA-protamine helix [Balhorn 2007]. Thus, it appears that the human sperm chromatin contains one zinc ion for each protamine molecule for each turn of the DNA.

Zn²⁺ has an important structural function in different proteins involved in nucleic acid binding or gene regulation [Berg 1990]. Such zinc-stabilized structures (usually referred to as zinc fingers) secure the tertiary structure and thereby reduce the number of possible conformations of the protein. This gives the protein a conformational stability which is suitable for interaction with other macromolecules like DNA or other proteins [Banecki et al. 1996]. In the classical zinc finger a single zinc ion tetrahedrally synchronizes conserved histidine and cysteine residues. Mostly zinc fingers are arranged of 2 HIS and 2 CYS, but variants in the number of HIS and CYS have been reported. Moreover, by forming stable inter-molecular zinc-bridges Zn²⁺ can also contribute to the quaternary structure. The active enzyme nitric-oxide synthase (NOS) is an example of an inter-molecular zinc-bridge, where one zinc synchronizes two cysteine-residues in each monomer into an active dimer (CYS)₂—Zn²⁺—(CYS)₂ [Raman et al. 1998].

Zinc ions may to a certain degree protect thiols from oxidation into disulfide bridges because zinc ions show no tendency for oxidation or reduction in biological systems. Therefore zinc ions may act as reversible inhibitors of sites requiring free thiols [Chesters 1978; Chvapil 1973]. However, experimentally induced oxidative challenges inactivate the NOS-enzyme by releasing zinc and the thiols become oxidized into disulfide-bridges [Zou et al. 2002].

It is therefore logical to assume that zinc ions can be a functional factor in the DNA-protamine structure by e.g., linking protamines with zinc bridges, where the Zn²⁺ ions bond to link thiol groups of cysteine and possibly imidazole groups of histidine, respectively [Bench et al. 2000; Bianchi et al. 1994; 1992; Gatewood et al. 1990; Kjellberg 1993; Porath et al. 1975; Raman et al. 1998].

The Chromatin of Ejaculated Spermatozoa

The chromatin of human spermatozoa can immediately after ejaculation be induced to decondense rapidly in vitro by the simple removal of zinc by metal chelation with ethylene diamine tetra acetate (EDTA) [Kvist 1980a; 1980b; Roomans et al. 1982] concomitant with exposure to the anionic detergent sodium dodecyl sulfate (SDS; removes membranes and imposes chromatin fiber repulsion) [Björndahl and Kvist 2010; Björndahl and Kvist 1985]. Exposure of human spermatozoa to 6 mM EDTA within one h after ejaculation extracts 86% of the sperm chromatin zinc as revealed by X-ray microanalysis [Roomans et al. 1982]. This indicates that at ejaculation the sperm chromatin has a zinc dependent chromatin stability.

However, storage of spermatozoa in vitro leads to the inherent loss of zinc dependent chromatin stabilization that is superseded by another type of stability [Björndahl and Kvist 2010; Kvist and Björndahl 1985]. This second type of chromatin stability requires disulfide breaking agents to unpackage chromatin. The change of stabilization is enhanced when zinc is removed from spermatozoa in vitro, and can to a large extent be counteracted by storing sperm in an environment with high biological availability of Zn²⁺.

A likely model that zinc forms salt bridges with protamine thiols and potentially also imidazole groups of histidine that both stabilizes chromatin and prevents the development of disulfide bridge dependent chromatin stability, is shown in Figure 1. A salt bridge that comprises zinc, thiols, and imidazole groups is as strong as a covalent disulfide bridge and can serve as a reversible and temporary stabilizer of the sperm chromatin. Removal of zinc from the sperm chromatin within the ooplasm would allow a rapid unraveling of the chromatin fibers. However, removal of zinc without concomitant repulsion of chromatin fibers could increase the oxidation of free thiols into disulfide bridges [Björndahl and Kvist 2010].

Figure 1.  Schematic overview of dual actions by Zn²⁺ stabilizing the structure and at the same time preventing formation of surplus disulfide bridges. Depletion of zinc allows two biologically different outcomes: (1) ‘immediate decondensation’ if repulsive forces are permitted to separate the chromatin fibers; and (2) otherwise unbound thiols close to each other may oxidize into disulfide bridges creating a ‘superstabilized chromatin’.

Support that interaction between zinc and protamine thiols constitutes a basis for secure and rapidly reversible chromatin stability is provided by the decrease during sperm maturation in the epididymis of the amount of chromatin thiols and their reappearance after sperm exposure to DTT. The original interpretation of this result was that thiols form S-S bridges because DTT can break the strong covalent disulphide bridges [Calvin and Bedford 1971]. However, there is an alternative interpretation. Perhaps zinc interacts with thiols making them undetectable. Since DTT also binds zinc, exposure of spermatozoa to DTT could extract zinc and allow free thiols to be detected. Additonal support for this notion is gained from epididymal spermatozoa revealing more thiols if preexposed to acid or EDTA, since both act as zinc chelating agents [Calvin and Bleau 1974; Calvin et al. 1973; Kvist and Eliasson 1978]. Furthermore, decondensation of the chromatin of ejaculated human spermatozoa in liquefied semen can be induced by a very low concentration of DTT (40 µm) if concomitant with EDTA exposure (unpublished data). Additionally, spermatozoa exposed to DTT are deprived of zinc but not magnesium [Kvist and Eliasson 1978].

From this point of view it is possible that sperm chromatin zinc deficiency induced during sperm collection and processing may be one factor jeopardizing the outcome of ART procedures. Moreover, decreased access to the sperm chromatin due to excess formation of disulfide crosslinking can hinder detection of DNA breaks by assays like Comet, TUNEL, and SCSA and give false negative results [From Björk et al. 2009; Pettersson et al. 2009; Tu et al. 2009].

Decondensation induced by zinc withdrawal is global and cannot be explained by decondensation of the 5–10% of the sperm chromatin DNA linked to histones. It is also unlikely that the sulfhydryl-connected zinc (1 zinc for every 5 sulphur) should be confined to the sulphur containing Histone H3 within the 5–10% histone fraction containing some 1–2% of the nuclear sulphur.

Factors influencing sperm chromatin stability at and after ejaculation

During sexual intercourse, spermatozoa are expelled suspended in the prostatic fluid onto the cervical mucus. Prostatic fluid should be regarded as the physiological medium for ejaculated spermatozoa in human. In the clinical setting, the entire ejaculate is collected in a single container. There is extensive contact between this zinc chelating and oxidizing seminal vesicular fluid secretion and the spermatozoa. The laboratory produced ‘semen sample’ is characterized by rapidly changing biochemical properties of the ‘seminal fluid’ [Björndahl and Kvist 2003]. It is often discounted that the ejaculate is not a homogenic and homeostatically regulated fluid like blood plasma; and this is probably due to the misleading term ‘seminal plasma’. The ejaculate is a mixture of various secretions and the composition of the ‘seminal plasma’ varies during ejaculation, liquefaction, and after ejaculation; and it varies between different men and between different ejaculates from the same man. Therefore collection of the entire ejaculate in one single container, according to the golden standard for semen laboratories [World Health Organization 1999], is likely to introduce increased heterogeneity of the sperm chromatin stabilization. Heterogeneity in chromatin stabilization may also be the reason for the variation in uptake of exogenous DNA by mammalian spermatozoa and the variation in susceptibility for enzymatic degradation in sperm DNA.

To comprehend the dynamics in vitro of the sperm chromatin stabilization after ejaculation it is essential to be aware of the sequence of ejaculation and how the sperm chromatin zinc content is influenced by prostatic fluid and seminal vesicular fluid, respectively [Björndahl et al. 1991; Björndahl and Kvist 1990]. Typically, most spermatozoa are expelled in the first 1/3 of the ejaculate together with the slightly acidic, zinc rich prostatic secretion. The prostatic fluid has a high biological availability of zinc which prevents a loss of chromatin zinc, as measured by X-ray microanalysis ([Björndahl and Kvist 1990; Kvist et al. 1985]; Figure 2). The remaining 2/3 of the ejaculate contains mainly seminal vesicular fluid. When the consecutive ejaculate fractions are collected individually (split ejaculate) spermatozoa reveal very different content of sperm chromatin zinc as measured with X-ray microanalysis [Björndahl and Kvist 1990; Kvist et al. 1985]. The amount of zinc in the sperm chromatin is inversely related to the admixture of seminal vesicular fluid to the ejaculate fraction. The admixture of seminal vesicular fluid increases the pH, which causes increased zinc affinity for citrate [Sillén and Martell 1971]. Furthermore, this fluid also adds high molecular weight (HMW) zinc ligands to the mixture ([Arver 1982b]; Figure 3). During liquefaction in vitro, the seminal fluid develops into a zinc chelating medium that depletes spermatozoa of zinc [Arver and Eliasson 1982; Björndahl et al. 1991; Björndahl and Kvist 1990]. A measure of the zinc binding capacity of the seminal plasma is the proportion of zinc bound to HMW ligands of seminal vesicular origin. The proportion of HMW bound zinc was less than 10% among 13 fertile donors, and varied between 2 and 67% among 115 men in infertile couples [Kjellberg 1993]. Thus, seminal vesicular fluid makes the seminal plasma a zinc binding medium, although the total concentration of zinc in seminal plasma appears to be normal. In conclusion, spermatozoa bathed in ‘seminal plasma’ are exposed to conditions that vary between different samples and by duration of exposure, due to variations in the zinc-containing prostatic fluid and the zinc-chelating seminal vesicular fluid and the dynamics in the mixture of these fluids [Arver 1982a; 1982b; Lundquist 1949].

Figure 2.  Schematic representation of the retention of chromatin zinc of spermatozoa expelled suspended in the first ejaculate fractions dominated by prostatic fluid. Zn²⁺ is secreted from the prostate as free zinc and zinc bound to citrate.

Figure 3.  Schematic representation of the zinc chelating properties of the liquefied whole ejaculate. The sperm chromatin can be deprived of Zn²⁺ due to zinc binding action of (1) seminal vesicular proteins and (2) citrate due to increased affinity for zinc at increased pH, both actions caused by the addition of seminal vesicular fluid.

It is of clinical importance that some men in subfertile couples have an abnormal sequence of ejaculation, where the most spermatozoa are expelled suspended in seminal vesicular fluid, leading to extraction of zinc from the sperm chromatin [Björndahl et al. 1991; Björndahl and Kvist 1990]. This is likely to cause changes in the organization of the sperm chromatin resulting in increased vulnerability of the DNA, especially when exposed to oxidative conditions in vitro. Probable causes for this disorder could be ejaculatory duct obstruction [Fisch et al. 2006] or prostatic oedema [Kjellberg 1993] with delayed emptying of prostatic fluid forcing spermatozoa to be expelled primarily with seminal vesicular fluid. Abnormal sequence of events of ejaculation cannot be exposed by routine semen analysis. Examination of a split ejaculate is required for this diagnosis [Björndahl and Kvist 2003].

Relevance of the Sperm Chromatin Structure for Investigations of the Sperm DNA Integrity

Tests like the TUNEL assay and the Comet assay were developed for the loosely packed somatic chromatin. The histone-linked DNA in sperm probably constitutes a DNA fraction that is more accessible to DNA integrity tests. This fraction of DNA contributes to the overall results of these tests but it is highly unlikely to alone account for the results obtained. All sperm DNA integrity tests require procedural steps to decrease the stabilization of the protamine-linked DNA, steps that are not necessary for somatic cell histone-linked DNA. A common methodological problem is that such procedures, whether enzymatic or based on exposure to acids or alkali can also damage DNA. Therefore the commonly used term ‘DNA damage’ or ‘DNA fragmentation’ is not completely correct. At one end of the scale, loosely compacted sperm chromatin would permit easier access to the DNA, and any protocol aimed at breaking down a ‘normal’ stabilization of the sperm chromatin would lead to increased exposure of the DNA to the substances and cause induction of DNA damage. At the other end of the scale supernormal stabilization would decrease access to the DNA. The result would be interpreted as ‘reduced’ DNA damage although it is a matter of reduced access.

It is essential to recognize that the stabilization of the human sperm chromatin is highly variable after ejaculation, depending on the status of zinc content and exposure to the oxidative environment. It is also important to evaluate if a method measures the total content of DNA or only a fraction. If the fraction exposed to the test substances varies, it could also create experimental variations of greater magnitude than the real, biological variations. In the case of the TUNEL assay, it is important to establish a positive control where all spermatozoa react. At present, simple experiments using ‘standard protocols’ only expose 15–50% in positive controls [From Björk et al. 2009]. In the case of the Comet assay, exposure of spermatozoa to cysteine, which can bind zinc as well as cause disulfide bridge disruption, can result in a many times greater head size and tail of the Comet. This could be abolished by removing cysteine after exposure, indicating a possible loss of zinc followed by surplus formation of disulfide bridges before the assay was started [Tu et al. 2009]. Similarly, initial exposure to EDTA decreases the sperm DNA defragmentation index (using Acridine Orange and Flow Cytometry), and after storage even less ‘DNA damage’ was detected [Pettersson et al. 2009].

Conclusion

The nature of the human sperm chromatin and its stabilization is far from completely unraveled. Before it is justified to use the now rapidly emerging sperm DNA damage tests in clinical settings it is important that validation studies investigate if any significant information is obtained with these methods [Castilla et al. 2010]. Furthermore, with the present evidence available on the nature and dynamics of the human sperm chromatin [Barratt et al. 2010] it is essential to continue with the work to standardize methods as well as recognize the importance of zinc and post-ejaculation changes in the status of the sperm chromatin stability.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Abbreviations

ART:	=	assisted reproductive techniques
DTT:	=	dithiothreitol
EDTA:	=	ethylenediaminetetraacetic acid
HMW:	=	high molecular weight
TUNEL:	=	terminal deoxynucleotidyl transferase dUTP nick end labeling.