Human Sperm Chromatin Stabilization After Ejaculation Biology Essay
The primary focus of this review is to highlight the dual functions of the sperm chromatin stabilization: to preserve a structure that protects the DNA during the transport to the oocyte and then allows a rapid liberation of the DNA in the ooplasm. In vitro, depletion sperm chromatin zinc after ejaculation allows a rapid and total sperm chromatin decondensation without the addition of exogenous disulfide cleaving agents. In contrast, if zinc is depleted without concomitant repulsion of the chromatin fibres another type of stability is induced - a stabilization that requires exogenous disulfide cleaving agents to allow decondensation. It may be that Zn2+ stabilizes the structure and prevents the formation of excess disulfide bridges by formation of zinc bridges involving protamine groups. Extraction of zinc from the freshly ejaculated spermatozoon allows two biologically totally different results: (a) immediate decondensation if chromatin fibers concomitantly are induced to repel (e.g. through phosphorylation in the ooplasm); (b) thiols freed from Zn2+ are available to form disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (normally the first expelled ejaculate fraction) represent the physiological situation. Extraction of chromatin zinc can be caused by unphysiological exposure of spermatozoa to the zinc chelating and oxidative seminal vesicular fluid. In most ART laboratories the entire ejaculate is collected into a single container in which spermatozoa and secretions are forced to mix during an extended period of at least 30 minutes causing an unnatural exposure to the zinc binding and oxidative seminal vesicular fluid. There are men in infertile couples with low content of sperm chromatin zinc most likely 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.
Functional significance and effects of the unique sperm chromatin structure
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The purpose of the spermatozoon is to transport a haploid genome unharmed to the egg. To accomplish this transfer of genetic material the chromatin structure of the male gamete is completely different from that of somatic cells. It is extremely resistant towards conditions that could harm the DNA. Concomitant with this protected state, the chromatin structure must have the property to make the DNA available very rapidly in the ooplasm. It is the aim of this review 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 into 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 contribute to DNA damage. Yet the DNA must be made available rapidly after the arrival in the ooplasm.
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 damage to the DNA, 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 aiming at revealing damage to the DNA, supernormal compaction would reduce the access of the DNA interacting staining and could thus 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 interpret sperm DNA damage test correctly.
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 sperm protamines
Arginine is the predominant amino acid (45-48%) of the protamines (P1 and P2) in human spermatozoa (Gusse, et al., 1986) and brings an abundance of positively charged -NH3+ groups into 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).
Histidine contributes with imidazole groups and cysteine with thiol (-SH) groups. Both groups 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, stabilising the motif.
Thiols alone are the basis for formation disulfide bridges. In the absence of zinc, the thiol groups could form disulfide bridges between thiols (Kvist 1980; Gatewood, et al. 1990; Bal, et al. 2001; Bianchi, et al. 1994; Bianchi, et al. 1992).
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 dithithreitol (DTT) can make sperm chromatin decondensation possible (Sutovsky and Schatten 1997). It should be noted, that beside a disulfide-bridge cleaving action, 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.
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).
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The content of chromatin zinc 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 with 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 (Chvapil 1973; Chesters 1978). 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 for instance linking protamines with zinc bridges, where Zn+2 by ion bonds link thiol groups of cysteine and possibly imidazole groups of histidine, respectively (Porath, et al. 1975; Gatewood, et al. 1990; Bianchi, et al. 1994; Bianchi, et al. 1992; Raman, et al. 1998; Kjellberg 1993; Bench, et al. 2000).
The chromatin of ejaculated spermatozoa
The chromatin of human spermatozoa can immediately after ejaculation be induced to decondense rapidly in vitro by simple removal of zinc by Me2+ chelating EDTA (ethylene diamine tetra acetate) (Kvist 1980; Roomans, et al. 1982) concomitant with exposure to the anionic detergent SDS (sodium dodecyl sulfate; 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 hour 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, once ejaculated in an open container in vitro the inherent, zinc dependent chromatin stabilization is rapidly lost and 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 be reversed and bring about unpacking of the 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 contains both that (1) zinc stabilizes the chromatin and (2) prevents the development of a disulfide bridge dependent chromatin stability, is that zinc forms salt bridges with protamine thiols and potentially also imidazole groups of histidine (Figure 1). A salt bridge that comprises zinc, thiols and imidazole groups is as strong as a covalent disulfide bridge and can therefore serve the purpose of 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 a concomitant repulsion of chromatin fibers could cause that freed thiols oxidize into disulfide bridges (Björndahl and Kvist 2010).
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Additional proof that interaction between zinc and protamine thiols constitute a base for a secure and rapidly reversible chromatin stability is that the detectable amount of chromatin thiols decrease during sperm maturation in the epididymis and reappear after sperm exposure to DTT. The original and sole interpretation of these results was that thiols form S-S bridges because DTT can break the strong covalent disulfide bridges (Calvin and Bedford 1971). However, there is an alternative interpretation involving that zinc interacts with thiols making them undetectable. Furthermore, DTT also binds zinc,. Thus, exposure of spermatozoa to DTT could extract zinc and allow free thiols to be detected. In favor of this is that epididymal spermatozoa reveal more thiols if pre-exposed to acid or EDTA treatment, which both acts 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 very low concentration of DTT (40 mM) 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, SCSA techniques and give false negative results (From Björk, et al. 2009; Pettersson, et al. 2009; Tu, et al. 2009).
Influences on sperm chromatin stability at and after ejaculation
In the normal sexual intercourse spermatozoa are expelled suspended in the prostatic fluid onto the cervical mucus. Thus the prostatic fluid should be regarded as the physiological medium for ejaculated spermatozoa in human. In the clinical setting, the entire ejaculate is collected in one single container. This means that also the seminal vesicular fluid is included and that extensive contact between this zinc chelating and oxidizing secretion and the spermatozoa takes place. Furthermore, the laboratory produced "semen sample" is characterized by rapidly changing biochemical properties of the "seminal fluid" (Björndahl and Kvist 2003). It is commonly disregarded that the ejaculate is not a homogenic and homeostatically regulated fluid like blood plasma; probably due to the misleading term "seminal plasma". The ejaculate fluid is just a mixture of various discharged secretions and the composition of the "seminal plasma" varies during ejaculation, during liquefaction, 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.
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).
Normally, most spermatozoa are expelled in the first 1/3 of the ejaculate together with the slightly acidic, zinc and citrate 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 (Kvist, et al. 1985; Björndahl and Kvist 1990) (Figure 2). The last 2/3 of the ejaculate contains mainly seminal vesicular fluid. When the consecutive ejaculate fractions are collected individually (split ejaculate) spermatozoa from different ejaculate fractions reveal very different content of sperm chromatin zinc as measured with X-ray microanalysis (Björndahl, et al. 1991; Björndahl and Kvist 1990). 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 further reducing the bioavailability of zinc (Sillén and Martell 1971). Furthermore, this fluid also adds high molecular weight zinc ligands (HMW) to the mixture (Arver 1982) (Figure 3). During liquefaction in vitro, the seminal fluid develops into a zinc chelating medium which depletes spermatozoa of zinc (Björndahl, et al. 1991; Björndahl and Kvist 1990; Arver 1982). A measure of the zinc binding capacity of the seminal plasma is the proportion of zinc bound to high molecular weight ligands (HMW) 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 exposed to "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 1982; Lundquist 1949; Arver 1982).
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 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 originally developed for the loosely packed chromatin of somatic cells. Common for all tests for sperm DNA integrity is the requirement for initial procedures to increase the access to the DNA in the tightly condensed sperm chromatin. A common methodological problem is that such procedures, whether enzymatic or based on exposure to denaturing conditions which may include acids or alkali compounds, can also damage DNA. Therefore the commonly used term "DNA damage" or "DNA fragmentation" is not completely correct. A less compacted sperm chromatin would allow 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 may induce DNA damage. At the other end of the scale supernormal stabilization would cause a decreased access to the DNA, and the result is in general interpreted as "reduced" DNA damage although it is a matter of reduced access.
To make interpretations based on scientific evidence 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 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 can be made to react - simple experiments using "standard protocols" exposes only 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 - an increase that could be reversed 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). Furthermore, pre-exposure to EDTA decreases the sperm DNA defragmentation index (using Acridine Orange and Flow Cytometry), and after storage there was a further decrease of "DNA damage" which was prevented by storage with zinc ions (Pettersson, et al. 2009).
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 with validation studies investigating if any significant information is added by the information obtained with these methods (Castilla, et al. 2010). Furthermore, with the evidence on the nature and dynamics of the human sperm chromatin presently available (Barratt 2010) it is essential to continue with the work to standardize methods as well as recognizing the importance of zinc and post-ejaculation changes in the status of the sperm chromatin.
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