Chromosomes in terminally differentiated mammalian spermatozoa are extensively condensed by protamines but a small proportion of histones remain. We examined the primary organization of somatic-type chromatin in lysolecithin-permeabilized human sperm nuclei and report that nucleosomes are closely packed with a periodicity of ∼150 bp. Incubation of nuclei in the presence of exogenous Mg2+ and ATP induced chromatin reorganization leading to an increase in spacing of the nucleosomes to ∼190 bp. This ATP-dependent chromatin rearrangement involved phosphorylation of both protamine and histone H2a. Increase in linker length between nucleosomes correlated with the phosphorylation of H2aX, the major H2a variant in human spermatozoa, predominantly at the C-terminal end. Chromatin reorganization was independent of detectable nuclear dispersion, which is an early chromosomal event in male pronuclear formation during fertilization.
In human spermatozoa, about 85% of the haploid genome is protamine bound (Pro-DNA), whilst the rest is nucleosomal (Nu-DNA; Tanphaichitr et al., 1982). Histones H3 and H4 in sperm nuclei are extensively acetylated (Gatewood et al., 1987, 1990a). De novo methylation of DNA during spermatogenesis differentiates sperm chromatin from that of somatic cells (Groudine and Conkin, 1985). Furthermore, unlike lower vertebrates, some regions of the paternal genome can withstand reprogramming by maternal factors on fertilization and are activated as early as the two-cell stage during mouse and human embryogenesis (Sawicki, 1981; McGrath and Solter, 1984; Surani et al., 1986). These observations led to the speculation that Nu-DNA in sperm represents transcriptionally active genes (Gatewood et al., 1987).
A number of fundamental problems relevant to spermatogenesis, fertilization and early development warrant elucidation of the structural organization of mammalian sperm chromatin. For example, why do protamines fail to dislodge nucleosomes from ∼450 Mb DNA during terminal differentiation? Does spatial distribution of nucleosome-bound chromatin regulate protamine-DNA interactions? Could the existing nucleosomal structure escape chromatin remodelling during male pronuclear formation? Are all the genes having somatic-type chromatin structure activated in early development? In particular, do the reciprocally imprinted neighbouring IGF2 and H19 genes differ in chromatin structure in male germ cells?
Attempts have been made (Groudine and Conkin, 1985; Gatewood et al., 1987) to define chromatin structure in sperm nuclei, but with little success. Limited access of nucleases to super condensed chromatin and an inability to separate the two populations of DNA following digestion of sperm nuclei constitute major problems in these studies. Recently we reported (Banerjee and Hulten, 1994) that micrococcal nuclease digestion of lysolecithin-permeabilized human sperm nuclei preincubated in the presence of ATP and Mg2+, produced mononucleosome-length DNA fragments. We suggested that these DNA fragments would help to elucidate the primary organization of chromatin in human sperm. Here we have tested this possibility and provide experimental evidence that human sperm nuclei unincubated or incubated in the absence of exogenous ATP and Mg2+ contained close-packed nucleosomes with a spacing of 149-152 bp; in contrast, incubation of the nuclei in the presence of exogenous Mg2+ alone or ATP and Mg2+, induced chromatin reorganization resulting in an increase in spacing of the nucleosomes from ∼150 to ∼190 bp. We have also demonstrated that phosphorylation of histone H2a and protamine were necessary for sperm chromatin reorganization. However, physiological spacing of the nucleosomes in rearranged chromatin was dependent upon phosphorylation of H2aX, the predominant H2a variant in human sperm. Since sperm chromatin rearrangement occurred in the absence of sperm nuclear dispersion, we propose that chromatin in sperm nuclei might undergo phosphorylationdependent reorganization prior to decondensation and remodelling during fertilization.
MATERIALS AND METHODS
Preparation of sperm nuclei
Purification of human sperm by sedimentation through Percoll and subsequent treatment with lysolecithin (L-α-lysophosphotidylcholine) were as described previously (Banerjee and Hulten, 1994). The permeabilized sperm were finally resuspended in sperm wash buffer (SWB: 20 mM HEPES-NaOH, pH 7.0, 100 mM potassium glutamate, 250 mM sucrose, 0.5 mM spermidine free base and 0.2 mM spermine tetrahydrochloride) containing 1× protease inhibitor cocktail (leupeptin, chymostain, APMSF, pepstatin A at 10 μg/ml; antipain and aproteinin at 5 μg/ml), 50% glycerol and stored at −70°C.
Incubation of sperm nuclei
Lysolecithin-permeabilized sperm nuclei were incubated at 35°C for 1 hour in 250-300 μl reactions at concentrations of 2-3×103 nuclei per μl in 20 mM HEPES-NaOH, pH 7.9, 150 mM KCl, 0.01 mM EDTA, 1 mM DTT, 5-6 mM Mg2+, 1 mM ATP and 5 mM creatine phosphate (CP).
Micrococcal nuclease digestion of sperm nuclei and separation of Pro-DNA from Nu-DNA
Sperm nuclei with or without preincubation were digested with micrococcal nuclease (MNase) in a reaction buffer containing 3 mM CaCl2 at 35°C for up to 3 minutes. Nuclease activity in aliquots was stopped by adding a mixture of EDTA, EGTA and N-laurylsarcosine at a final concentration of 12.5 mM, 12.5 mM and 62.5 mM, respectively. Alternatively, reactions were terminated by adding EDTA and EGTA, 25 mM each, prior to centrifugation at 13,000 rpm for 10 minutes at 4°C. The chromatin in the pellet and the supernatant were treated with SDS and proteinase K. Following phenol-chloroform extraction, ethanol precipitated DNA was resolved in 1.5% agarose gels, stained with ethidium bromide and photographed as described previously (Banerjee and Cantor, 1990). In some experiments, DNA was alkalitransferred from agarose gel to a nylon membrane (Genescreen+, Dupont) for hybridization to gel-purified radiolabeled mononuclosome-length DNA fragments derived from MNase digested sperm nuclei. Sperm-specific mononucleosome-size DNA was electroeluted from an agarose gel slice and was concentrated by eluting from an Elutip-D (S and S) column and ethanol precipitation.
Phosphorylation of sperm chromosomal proteins and separation by acid urea Triton (AUT) gel electrophoresis
To analyse the phosphorylation of chromosomal proteins, sperm nuclei were incubated as described above in the presence of 125–150 μCi of [γ-32P]ATP (3000 Ci/mM, Amersham) per ml, with or without exogenous ATP and Mg2+. Following incubation, the chromosomal proteins from sperm nuclei were acid extracted (0.5 M HCl), precipitated with trichloroacetic acid (TCA, 25% final), washed with cold acidified acetone and acetone alone (Banerjee et al., 1991). In some experiments, prior to acid extraction, the nuclei were pelleted by centrifugation at 13,000 rpm for 10 minutes, and resuspended in the original volume of the reaction buffer. Chromosomal proteins from HeLa nuclei were extracted with 0.25 M HCl before TCA precipitation. The proteins were resolved in 15% polyacrylamide acid urea Triton (AUT) gels as described previously (Banerjee and Hulten, 1994). Gels stained with Coomassie Blue were destained, photographed, dried and autoradiographed.
N-bromosuccinimide cleavage of H2a/H2aX
Sperm nuclei were incubated for 1 hour in the presence of 5 mM Mg2+ and 500 μCi/ml of [γ-32P]ATP. Chromosomal proteins were acid extracted and resolved in a 15% polyacrylamide AUT gel. The gel was stained with Coomassie Blue in 40% methanol and 10% acetic acid for 5 minutes. The position of H2a/H2aX was marked by incisions made above and below the bands. Subsequently, urea and acid were removed by gentle agitation of the gel in distilled water for 1 hour, followed by treatment with 10 mg/ml of N-bromosuccinimide (NBS) and 5% acetic acid in distilled water for 3 hours at room temperature (Green and Poccia, 1985). NBS was removed from the gel by repeated washes in distilled water, followed by equilibration in SDS-gel buffer for 2 hours. NBS-cleaved peptides in the excised gel slice were resolved in a 15% discontinuous polyacrylamide-SDS gel. Following electrophoresis, the gels were silver stained, photographed, dried and autoradiographed.
Microscopic examination of sperm nuclei
Lysolecithin-permeabilized sperm nuclei, unincubated or incubated in the presence of Mg2+, were mixed with equal volumes (4 μl) of staining buffer (50% glycerol, 5 μg/ml of propidium iodide in reaction buffer) on microscope slides, examined using a fluorescent microscope (Nikon, Japan) equipped with phase rings and photographed as described previously (Banerjee and Hulten, 1994).
Autoradiograms with minimum exposure and the type 55 Polaroid negatives, generated following photography of ethidium bromidestained agarose gels, were scanned using a laser densitometer (LKB 2202 Ultroscan). An ‘intensity fit’ program was used for background correction in each lane, analysis of peak positions and for integrating the area below the peak where necessary. To determine the nucleosome repeat-length in each experiment, two negatives with different background fog were scanned.
Production of nucleosomal DNA fragments following digestion of sperm nuclei with MNase
In order to determine whether the presence of exogenous cofactors is essential for protecting the nucleosome-length DNA fragments, the sperm nuclei were incubated in the presence or absence of ATP, Mg2+ and creatine phosphate (CP) at 35°C. As controls, fresh nuclei or those incubated at 0°C and 35°C were also digested. Results from these experiments (Fig. 1A) demonstrated that MNase digestion of sperm nuclei preincubated in the presence of ATP, Mg2+ and CP resulted in the accumulation of DNA fragments (∼190 bp) which comigrated with those of HeLa cells. Nuclease digestion of fresh sperm nuclei, or nuclei incubated at 0°C and 35°C, produced smaller DNA fragments (147-155 bp), indicating that exogenous ATP and Mg2+ altered the sperm chromatin structure such that Nu-DNA became less accessible to MNase compared to that of controls (Fig. 1A, compare the distribution of high Mr DNA in the fourth panel from left with the rest).
To further investigate the independent role of cofactors in sperm chromatin reorganization, nuclei were digested following incubation, either alone, in the presence of ATP and CP; in Mg2+ alone, or in the presence of ATP, Mg2+ and CP. Results showed (Fig. 1B) that sperm nuclei did contain some endogenous ATP and Mg2+. Since exogenous Mg2+ was sufficient for maximum accumulation of mononucleosome-length DNA fragments, nuclei were subsequently incubated in the presence of Mg2+ alone to maintain an identical specific activity of ATP in all reactions (see below).
ATP hydrolysis is necessary for chromatin reorganization
To examine whether ATP was hydrolysed during chromatin reorganization, the endogenous ATP utilization was competitively inhibited by incubating sperm nuclei in the presence of glucose and hexokinase (HK) prior to MNase digestion. Increasing concentrations of HK substantially reduced the yield of mononucleosomes (Fig. 2A). ATP-dependent reorganization of sperm chromatin could be due to phosphorylation of chromosomal proteins, and nuclei were therefore treated with alkaline phosphatase following incubation with exogenous Mg2+. Phosphatase treatment almost completely abolished the nucleosomal structure in reorganized chromatin (Fig. 2B), indicating that phosphorylation of chromosomal proteins is involved in chromatin rearrangement and maintaining stable nucleosomes.
Nucleosomes in sperm chromatin are closepacked; ATP hydrolysis increases nucleosome spacing to that of human somatic cells
Accumulation of mononucleosome-length DNA fragments following MNase digestion suggested that a spaced nucleosome structure existed in sperm chromatin. However, in repeated experiments, we failed to obtain a somatic-type nucleosome ladder of digested sperm chromatin in ethidium bromide stained agarose gels (Figs 1, 2). This was possibly because the nucleosome ladder (∼15% of the genome) was obscured by randomly cleaved Pro-DNA which represents ∼85% of the nuclear DNA. Attempts to achieve clear nucleosome ladders by blotting these gels and subsequent hybridization to gel-purified radiolabelled mononucleosome-DNA probes were equally unsuccessful (data not shown). However, we observed that short stretches of nucleosome bound chromatin, released during MNase digestion of the nuclei, were separable from the bulk of the high molecular mass chromatin by a brief centrifugation. Accordingly, MNase digested nuclei were centrifuged and the chromatin in the pellet and supernatant were deproteinized (see Fig. 3 for a detailed protocol). Results from these experiments showed that the nucleosomes in sperm nuclei incubated or unincubated in the absence of exogenous Mg2+ were close-packed with an average spacing of ∼150 bp (Fig. 3A); while incubation in the presence of 5 mM Mg2+, resulted in an increased periodicity from ∼150 bp to ∼190 bp (Fig. 3B and see below), confirming that reorganization of sperm chromatin occurred under these conditions. Hybridization of a blot of this gel with 32P-labelled mononucleosome-DNA probes gel-purified from digests similar to those shown in Figs 1, 2, demonstrated that nucleosome ladders in earlier experiments (Figs 1, 2) were indeed obscured by smears of randomly-cleaved Pro-DNA fragments. We concluded that exogenous cofactors (ATP and Mg2+ or Mg2+ alone) were essential, during incubation, to induce sperm chromatin rearrangement.
To quantitate the increase in repeat-length following chromatin reorganization, both Type 55 Polaroid negatives and the autoradiograms were scanned densitometrically (Fig. 4A-C). The average sizes of partially digested fragments were plotted against band (nucleosome) number. The results of such analysis showed that the mean repeat-length of nucleosomes in sperm nuclei with and without incubation were 149.6 bp and 152.2 bp, respectively (Fig. 4D and E), whereas, the periodicity of nucleosomes in rearranged chromatin was 190 bp, which was comparable to that of HeLa cells (Fig. 4F and G).
Involvement of histone H2a and protamine phosphorylation in sperm chromatin reorganization
Disruption of nucleosomal structure (a) by depletion of endogenous ATP by glucose plus HK, and (b) by phosphatase treatment of reorganized chromatin provided an indication that chromosomal proteins might be phosphorylated during incubation of sperm nuclei in the presence of Mg2+. This was tested by incubating the nuclei in the presence of trace amounts of [γ-32P]ATP, both with and without exogenous Mg2+. Acid extracted nuclear proteins were resolved in AUT gels, stained and autoradiographed. Histone H2a (H2aX, H2aZ and ubiquitinated H2a or uH2a) and protamines were the major chromosomal proteins phosphorylated in the presence of exogenous Mg2+ (Fig. 5A). Depletion of endogenous ATP with glucose and an increasing concentration of HK (see Fig. 2A), either substantially reduced (Fig. 5A) or completely inhibited (data not shown) the phosphorylation of these proteins. Protamines, but not histones, were phosphorylated in the absence of exogenous Mg2+ (Fig. 5A). Therefore, phosphorylation of H2a and an increased phosphorylation of protamines were necessary for chromatin reorganization. In agreement with previous reports (Gatewood et al., 1987; Banerjee and Hulten,1994), heteromorphous H2aX constituted the bulk of the histone H2a content in human sperm nuclei, whereas H3 and H4 were extensively acetylated (Fig. 5A).
Heterogeneity in condensation of human sperm chromatin due to highly variable transit periods through the epididymis (Rowley et al., 1970; Bedford et al., 1973), prompted us to examine the phosphorylation of sperm nuclei from numerous individuals. In these experiments, histone H2a and protamine were invariably the major phosphorylated proteins. However, the ratios of protamine to histone phosphorylation in different individuals were not comparable (Fig. 5B, compare with Fig. 5A). This was further established by phosphorylation analysis of five nuclear samples which were prepared at the same time, using the same reagents. Variable levels of endogenous ATP in sperm nuclear preparations might have accounted for this observation. To verify this, nuclei were incubated in the absence and presence of 50 μM exogenous ATP and 5 mM CP. This resulted in a striking increase in the ratio of protamine to histone phosphorylation (Fig. 5C). Variability in the relative phosphorylation of histone H2a and protamine in sperm preparations was therefore most likely to be due to variations in the endogenous ATP pool. The absolute levels of phosphorylation of H2aX and protamines in the presence of exogenous ATP are presumably reflecting the kinetics of the different kinases.
Dephosphorylation of H2aX disrupts nucleosome structure
Nucleosome structure of the reorganized sperm chromatin was shown in Fig. 2B to be disrupted by phosphatase treatment. Chromatin reorganized in the presence of Mg2+ and [γ-32P]ATP was therefore further incubated with or without phosphatase and the phosphorylation of the proteins analysed. Fig. 6A shows that H2aX was more effectively dephosphorylated (64.5%) than protamines (23%). We concluded that phosphorylation of H2aX was necessary to stabilize and alter the spacing of the nucleosomes in sperm chromatin and phosphorylation of protamines alone was not sufficient for chromatin reorganization.
H2aX is mostly phosphorylated at the C-terminal end
All isotypes of eukaryotic H2a (H2a.1, .2, X and Z) have an evolutionarily conserved large hydrophobic core, but the smaller N- and C-termini are less conserved and contain the majority of the phosphorylation sites (Pantazis and Bonner, 1981; Mannironi et al., 1989). In order to determine the relative distribution of 32P incorporated in H2aX, phosphorylated chromosomal proteins were resolved in an AUT gel and treated with N-bromosuccinamide (NBS) which cleaves at three tyrosine residues located very close to each other within the central domain (Fig. 6B). NBS cleavage released large C-terminal and small N-terminal fragments which were separated in SDS-gels (Fig. 6B). The majority of the phosphates were incorporated at the NBS-cleaved C-terminal peptides of H2aX (Fig. 6B). A distinct band migrating above poorly labelled N-terminal fragments might represent larger N-terminal peptides, as observed in mouse testis and cleavage-stage H2a of sea urchin (Green et al., 1991).
Sperm nuclei do not decondense on incubation
Phosphorylation of protamine was suggested to be one of the mechanisms for mammalian sperm chromatin decondensation (Young and Sweeney, 1978). This view was supported by the identification of a protein kinase which phosphorylated protamines in fertilized rabbit embryos (Wiesel and Schulz, 1981). However, phosphorylation of sea urchin sperm-specific H1 and H2b precedes chromatin decondensation (Green and Poccia, 1985) and yet fails (Poccia et al., 1990) to disperse the nuclei. In order to evaluate the effect of chromatin reorganization on morphology, the sperm nuclei were therefore incubated at 35°C in the presence of exogenous Mg2+. Unincubated and incubated nuclei were morphologically indistinguishable (Fig. 7). We concluded that phosphorylation of protamine and H2a were not sufficient to induce chromatin decondensation.
This report describes the primary organization of nucleosomebound genes in human sperm. The nucleosomes display a distinct periodicity despite the presence of histone variants, extensive acetylation of H3 and H4, and the apparent absence of histone H1. Unlike somatic cells, the nucleosomes in sperm nuclei are close-packed. However, sperm chromatin does undergo rearrangement in a process dependent on protamine and histone H2a phosphorylation, resulting in an increase in nucleosome spacing from ∼150 bp to ∼190 bp. This increase in linker-length correlated with the phosphorylation of histone H2aX. De novo phosphorylation of chromosomal proteins and reorganization of sperm chromatin were surprising since maternal factors have been thought to induce such modifications during fertilization (Poccia et al., 1990; Wiesel and Schultz, 1981). Our results show that protamine and histone H2aX phosphorylation are not sufficient for human sperm chromatin decondensation, since the nuclei incubated in the presence of Mg2+ did not disperse.
Phosphorylation of protamine, H2aX and the resulting chromatin rearrangement can be envisaged as a reverse differentiation, in which a reduction in the net positive charge of protamines unlocks chromatin loops, facilitating phosphorylated H2aX-mediated sliding of the nucleosomes. Phosphorylationdephosphorylation of protamine is intimately associated with terminal differentiation of spermatids to spermatozoa (Marushige et al., 1969; Balhorn, 1982). In spermatids, most of the somatic histones are displaced by newly synthesized protamines which are phosphorylated immediately after synthesis at serine and threonine residues (Marushige and Marushige, 1975; Mayer et al., 1981). This phosphorylation is essential for an ordered alignment of protamine along the DNA grooves (Willmitzer et al., 1977a). However, subsequent dephosphorylation of protamine is a prerequisite for increasing its DNA binding constant, leading to complete neutralization of the phosphate-ribose backbone (Willmitzer et al., 1977b). Following dephosphorylation, unbound cysteinyl residues of protamine are cross-linked (disulphide bonds or Zn2+-coordination), generating a supercondensed, crystal-like sperm nucleus (Balhorn, 1982; Gatewood et al., 1990b).
In agreement with previous reports on entirely different systems (Sun et al., 1990; Kleinschmidt et al., 1991), the absence of histone H1 in human sperm (Gatewood et al., 1990a; Banerjee and Hulten, 1994) argues against its universal role in spacing nucleosomes. Coincidentally, ATP-dependent physiological spacing of nucleosomes, assembled on naked DNA in cell-free extracts from Xenopus oocytes (Almouzni and Mechali, 1988) and HeLa cells (Banerjee and Cantor, 1990) has also been shown to correlate with the phosphorylation of H2a (Kleinschmidt et al., 1991; Banerjee et al., 1991). Phosphorylation of the C-termini of H2aX might internally stabilize core particles (Hatch et al., 1983): the N- and C-terminal ends of H2a have distinctly different functions in which N-termini protruding out of the core towards linker DNA facilitate higher order organization of chromatin (Allan et al., 1982; Bohm and Crane-Robinson, 1984). Trypsin sensitive C-termini of H2a (Bohm et al., 1984; Hatch et al., 1983) contacting the interfaces between H2a-H2b dimer and H3-H4 tetramer of the nucleosomes are thought to be accessible to kinase/phosphatase action (Hatch et al., 1983).
Disruption of nucleosome structure by extensive depletion of endogenous ATP (glucose and HK, Figs 2 and 5) or by dephosphorylation of H2aX (Figs 2 and 6), raises the possibility that a low level of phosphorylated histone might be necessary to stabilize sperm nucleosomes. This conjecture is consistent with previous reports on quantitative analysis of sperm histone phosphorylation: In mouse testis tubule cultures (Green et al., 1991), 95% of 32P was incorporated by H2a variants, H2a.1 and H2aX. On the other hand, both unphosphorylated and residually phosphorylated histones were found to be present in terminally differentiated mouse epididymal sperm (Marushige and Marushige, 1975; Mayer et al., 1981). In contrast, sea urchin sperm histones H1 and H2b, are rephosphorylated only on fertilization (Green and Poccia, 1985).
In conclusion, we show that demembranated human spermatozoa contain close-packed nucleosomes and are capable of reorganizing their chromatin in the absence of maternal kinases and fertilization. Physiologically, chromatin rearrangement in terminally differentiated spermatozoa might have more relevance to fertilization and early development than to gametogenesis. Chromatin decondensation and chromatin remodelling are early chromosomal events in male pronuclear formation during fertilization (Green and Poccia, 1985). Sperm chromatin reorganization prior to these events might stabilize the existing nucleosomal structure which can escape remodelling and epigenetic modifications by maternal cytoplasmic components. Such an evasion of maternal reprogramming may have several important consequences. Functionally, transcriptional activation or silencing of genes in Nu-DNA would rely upon paternally imprinted programs. Structurally, nucleosome-bound chromatin domains can directly influence the phasing of the newly assembled nucleosomes during remodelling.
We thank many individuals for providing their germ cells; Roy Tilling for patient help with the densitometry; Fiona McDonald, Uche E. Uche and Yvonne Wallis for reading this manuscript; and Colyn Crane-Robinson for lively discussions and thoughtful comments on the work. We are also thankful to the reviewers, whose suggestions improved the manuscript. This project is supported by the Letten F. Saugstad (LSF) fund and S.B. is a LSF research fellow.