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J. Biol. Chem., Vol. 278, Issue 32, 29471-29477, August 8, 2003

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The Structural Organization of Sperm Chromatin^*

Susan M. Wykes   and Stephen A. Krawetz  ¶ || **

From the ^¶Department of Obstetrics and Gynecology, the Center for Molecular Medicine and Genetics, and the ^||Institute for Scientific Computing, Wayne State University School of Medicine, Detroit, Michigan 48201

Received for publication, April 30, 2003 , and in revised form, May 22, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The packaging of the male haploid genome within the differentiating spermatid nucleus is facilitated by small basic nuclear proteins called protamines. Although the majority of the DNA in human sperm chromatin is bound by these proteins, a small percentage retains a nucleosomal-like component. These histone-enriched regions may possess enhanced nuclease sensitivity and have been postulated to designate certain genes involved in early embryogenesis. We have shown previously that the chromatin domain containing the two human protamines PRM1 and PRM2 and the transition protein TNP2 forms a DNase I-sensitive conformation in pachytene spermatocytes, a requisite event prior to the haploid expression of its members in round spermatids (Kramer, J. A, McCarrey, J., Djakiew, D., and Krawetz, S. A. (1998) Development 125, 4749–4755). Interestingly, this configuration persists in mature spermatozoa subsequent to the transcriptional silencing of the locus. It was therefore postulated that the retained, enhanced DNase I-sensitive conformation of the PRM1PRM2TNP2 domain in human sperm may be preferentially histone-enriched. To address this tenet, we examined the chromatin structure of the human PRM1 PRM2 TNP2 domain using a PCR-based assay. The results show that this retained, enhanced DNase I sensitive domain reflects an enrichment of histones at discrete regions across the locus. In addition, a similar examination of other genes and repetitive sequences suggests the non-random distribution of histones and protamines within the sperm nucleus. A discussion of these results and their functional significance is presented.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
In mammals, the haploid phase of spermatogenesis, termed spermiogenesis, marks the initial expression of the nuclear packaging proteins, the transition proteins, and the protamines (1). These proteins facilitate the molecular remodeling and species-specific compaction of the male genome within the differentiating spermatid nucleus (2). There are two types of protamines, i.e. protamine-1, which is found in nearly all mammals (3), and protamine-2, which is confined to relatively few species that include human (4), mouse (5), and stallion (6). In addition to the protamines, several mammalian species retain a nucleohistone component within their sperm chromatin, including human (7), mouse (8), and boar (9). Proton-induced x-ray emission spectroscopy has been used to examine the relative amounts of protamine in mammalian sperm. The results showed that, although the total amount of protamine remained constant, the relative proportion of each protamine was variable. For example, the relative amount of protamine-2 in human, mouse, and hamster constitutes 67, 34, and 43% of the total protamine, respectively (10). This interspecies variability has raised the issue of whether the evolution of a second protamine in mammals is functionally redundant. To address this, the expression of the Prm1 or Prm2 genes was disrupted in mouse embryonic stem cells. The resulting haplo-insufficient chimeras were infertile, displaying nuclear condensation abnormalities. This showed that both protamines were essential for normal sperm development (11).

The relationship between function and nuclear organization has been well established in somatic cells (12); however, the functional significance of sperm nuclear architecture remains to be clearly defined. Chromosomes within the sperm nucleus are arranged in a hairpin-like structure with the centromeres confined to the interior of the nucleus and the telomeres at the periphery (13). It has been suggested that the close proximity of telomeres to the egg's environment renders it one of the first structures to participate in the initial events of male pronuclear formation, immediately subsequent to fertilization (14).

Similar to the somatic nucleus, the DNA in sperm chromatin is organized into looped domains attached at their bases to the nuclear matrix (15). Studies examining the global configuration of the sperm nucleus have shown that spermatozoa function abnormally with a disrupted nuclear matrix and do not produce viable offspring (16). These results suggest that the spatial organization of the male-haploid genome provides important epigenetic information critical for both sperm function and early development (17).

The DNA in human sperm chromatin is partitioned into both a nucleohistone and a nucleoprotamine fraction with 15% of the DNA bound by histones and 85% of the DNA bound by protamines (18, 19). In both mouse and human sperm, histones have been localized to the nuclear periphery in association with LINE/L1 elements (8) and telomeric sequences, respectively (14). It has been postulated that the sequence-specific packaging of human sperm chromatin by histones and protamines may also serve to designate a specific subset of early embryonic expressed genes (7). Studies examining the chromatin structure of the -globin gene cluster in sperm have shown that the embryonic-specific – and -globin genes were histone enriched, whereas the post-natal expressed -globin gene was protamine enriched (20). A nucleosomal-like arrangement has been described for the paternally imprinted IGF-2¹ allele in human sperm (9).

The two human protamines PRM1 and PRM2 and the transition protein TNP2 are clustered together on chromosome 16p13.13 (21). We have shown previously that this cluster exists as a single 28.5-kb chromatin domain flanked by two male, germ cell-specific matrix attachment regions (22, 23). In addition, the human PRM1 PRM2 TNP2 domain forms a DNase I-sensitive, potentiated conformation at the pachytene spermatocyte stage, a requisite event prior to the expression of these three genes in round spermatids (24). Interestingly, this enhanced DNase I-sensitive configuration is retained in mature spermatozoa (22). In both human (9, 14) and mouse (8), increased nuclease sensitivity has been observed to parallel nucleosomal sperm chromatin structure. It was therefore postulated that the retained, enhanced DNase I sensitivity of the human PRM1 PRM2 TNP2 domain in sperm may reflect the preferential association of histones across the locus. To address this tenet, the sperm chromatin from a series of normal, healthy male donors was partitioned into histone or protamine DNA fractions using various restriction enzymes. The distributionofhistonesandprotaminesacrossthehumanPRM1 PRM2 TNP2 domain was then assessed using a PCR-based assay. The results showed that discrete restriction fragments are histone-enriched throughout the enhanced DNase I-sensitive locus. Furthermore, the distribution of histones and protamines was consistent within the same sample and between different individuals. This supports the view that the configuration of the genetic material within the sperm nucleus is not random. A discussion of these results and their functional significance is presented.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Semen Samples—Human semen samples from normal male donors were provided by the in vitro fertilization clinic, Hutzel Hospital (Detroit, MI). Collection and utilization of these samples were performed in accordance with the Human Investigation Committee at Wayne State University (Institutional Review Board number 095701MP2F). The samples were supplied frozen in test yolk buffer freezing medium supplemented with glycerol (Irvine Scientific, Santa Ana, CA) and stored at –80 °C until use.

Fractionation of Sperm Chromatin—This protocol was adapted from that described by Gardiner-Garden et al. in 1998 (20). A total of five fractionations were performed. Semen samples from three individual donors were fractionated by BamHI and EcoRI restriction endonuclease digestion. To assess variation, this procedure was repeated on one individual. An additional sample from a fourth donor was fractionated using PvuII.

Approximately 10⁸ sperm were thawed then combined with Trisbuffered saline (25 mM Tris-HCl, pH 8.0, and 100 mM NaCl) to a final volume of 25 ml. The sperm were then centrifuged at 2000 x g for 5 min at 4 °C. The sperm were washed as above two additional times. After washing, the sperm were resuspended in 4 ml of a freshly prepared solution of a 50 mM Tris-HCl (pH 8.0) buffer containing 10 mM dithiothreitol (Amresco, Solon, OH) and then incubated on ice for 15 min. Subsequent to dithiothreitol treatment, 10% cetyltrimethylammonium bromide (CTAB; Sigma) was added to a final concentration of 0.1%. The sample was then incubated on ice for an additional 30 min. Following incubation, an aliquot was removed and examined using bright field microscopy to verify the complete removal of the tails. The sperm were then centrifuged at 3000 x g for 5 min at 4 °C. The supernatant was decanted, and the pellet was resuspended in 4 ml of Tris-buffered saline supplemented with 0.5% digitonin (Sigma). The sperm nuclei were then centrifuged at 3000 x g for 5 min at 4 °C. This washing procedure was repeated four additional times. Subsequent to washing, the sperm nuclei were resuspended in 10 mM Tris-HCl (pH 8.0) buffer containing 0.65 M NaCl, 1 mM EDTA, and 0.05% digitonin and then incubated on ice for 15 min. Following histone extraction, the nuclei were centrifuged at 3000 x g for 2 min at 4 °C. Extraction of the histone component under these conditions was independently confirmed by immunofluorescence using both histone and protamine antibodies. The extracted pellet of nuclei was then washed in 1 ml of the appropriate 1x restriction enzyme buffer (Invitrogen) supplemented with 0.05% digitonin and centrifuged at 3000 x g for 2 min at 4 °C, and then the final pellet resuspended in 1 ml of the digitonin-supplemented restriction buffer. The exposed histone-free DNA was then cleaved by digestion with 100 units each of BamH1 plus EcoR1 or 100 units of PvuII (Invitrogen) for 1.5 h at 37 °C with gentle rocking. Subsequent to digestion, the cleaved histone-enriched DNA was separated from the protamine-bound DNA by centrifugation at 3000 x g for 2 min at 4 °C. The supernatant containing the histone-enriched DNA was then transferred to a new tube and centrifuged at 16,000 x g for 2 min at 4 °C to remove any contaminating protamine-bound DNA. The pellets containing the protamine-bound DNA were then combined and resuspended in 1 ml of 50 mM Tris-HCl (pH 8.5) buffer containing 50 mM NaCl, 0.5 mM EDTA, and 0.5% SDS. The protamine-bound DNA and histone-enriched DNA fractions were then incubated overnight at 50 °C following the addition of 200 µg/ml proteinase K (Invitrogen). Subsequent to proteinase K treatment, the fractionated DNAs were purified by phenol-chloroform extraction and ethanol precipitation. The DNAs were then resuspended in sterile distilled H₂O supplemented with 0.5 mM EDTA. The protamineenriched DNA was again digested as above and then purified by organic extraction and ethanol precipitation. The relative concentration of both the purified histone-enriched and protamine-enriched DNA was then spectrophotometrically determined.

PCR Amplification—The distribution of histones and protamines across the human PRM1 PRM2 TNP2 domain was evaluated using various primer pairs to specific regions contained within unique restriction fragments across the locus. In addition to the protamine domain, several other loci including acrosin, -globin, and IGF-2 as well as Alu, telomeric, and centromeric sequences were evaluated. Primer sequences are shown in Table I. Equal amounts of template were used for PCR, and each reaction was performed in triplicate to ensure that the differences observed were attributed to the biology and not the variation in PCR efficiency. PCR was performed within the linear range of amplification.

View this table: [in this window] [in a new window]   TABLE I Primers specific to the PRM1PRM2TNP2 domain and other primers GenBankTM accession number for the PRMPRM2TNP2 domain is U15422 [GenBank] . Fwd, forward; Rev, reverse.

Southern Hybridization Analysis—PCR products were resolved on agarose gels, transferred to nylon membranes, and then analyzed by Southern hybridization analysis using the respective [-³²P]dCTP-labeled (Amersham Biosciences) PCR products as probes. The membranes were hybridized at 45 °C overnight in 5x SSPE buffer containing 50% formamide, 5x Denhardt's reagent, 0.5% SDS, 0.1 mg/ml sheared salmon sperm DNA, 10% polyethylene glycol 8000, and 10⁶ dpm/ml of [-³²P]dCTP-labeled probe. Following hybridization, the membranes were washed at room temperature in 2x SSPE containing 0.1% SDS for 30 min. The membranes were then transferred to a circulating bath at 50 °C containing 0.1x SSPE and 0.1% SDS and washed for 15 min. Subsequent to washing, the hybridized signal was visualized by autoradiography.

Computational Analysis—Autoradiographic images were quantitated using the Millipore 60 S version 3.0 whole band image analysis system. The intensity of the hybridized signal was determined, and the mean integrated values were standardized to the GC content of each PCR amplicon. This was defined as the relative optical density (ROD). The mean intensity for each cycle for all similarly fractionated samples was then determined and plotted as a function of cycle number. Statistical data analysis was performed using a paired t test. Repetitive elements within the PRM1 PRM2 TNP2 domain were identified using the CENSOR server at charon.girinst.org.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
To examine the sperm chromatin structure of the human PRM1 PRM2 TNP2 domain, four different semen samples were individually partitioned into BamH1 and EcoR1 histone and protamine-enriched DNA fractions. DNA was subsequently isolated and purified from each fraction, and equal amounts of template were used for PCR. The distribution of histones and protamines across the PRM1 PRM2 TNP2 domain was then assessed by PCR using primers (designated i–x) to specific regions within unique BamH1/EcoR1 fragments for each of the four histone/protamine-purified samples from three individuals. For each sample, PCR was performed in triplicate and remained within the linear range. The PCR products were subsequently analyzed by Southern analysis using the respective [-³²P]dCTP-labeled PCR products as probes. Autoradiographic images were quantitated by densitometric analysis, and the mean integrated intensity values were plotted for the four samples from the three individuals as a function of each cycle. The statistical significance of the mean intensity difference between the two fractions for all four fractionated samples was assessed using a paired t test. A statistically significant difference was considered indicative of a histone or protamineenriched segment. These results are summarized in Fig. 1. As shown, the BamHI/EcoRI restriction fragments interrogated with primer sets i, iv, vii, and x were histone-enriched when the distribution of histones, as indicated by the open circles (p 0.05) in Fig. 1, was compared with that of the protamines (Fig. 1, filled squares). In contrast, BamHI/EcoRI restriction fragments interrogated with primer sets ii and iii were protamine-enriched, as indicated by the filled protamine squares (p 0.05; Fig. 1) in comparison to the open histone circles (Fig. 1). The remaining fragments interrogated by primer sets v, vi, viii, and ix were evenly distributed among the histone and protamine DNA fractions, as the data clearly overlapped. This likely reflects a mixture of histones and protamines throughout these regions. The distribution of histones and protamines that spanned the locus was similar between three different individuals and between different same sample preparations from the same individual for all ten regions examined. This supports the view that histones and protamines are uniquely distributed across the human PRM1 PRM2 TNP2 domain in sperm.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Histone and protamine fractionation using BamHI and EcoRI. Human sperm samples were partitioned into histone-enriched (H) or protamine-enriched (P) DNA fractions using BamH1 and EcoR1 restriction endonucleases. The distribution of histones (open circles) and protamines (closed boxes) across the human PRM1 PRM2 TNP2 domain was then assessed by PCR using primers to specific regions, i–x, contained within these fragments as a function of relative optical density (ROD). This was calculated as the mean hybridization signal, expressed as a function of the GC content of the probe. The BamH1/EcoR1 fragments containing regions i, iv, vii, and x were histone-enriched (open circles, p 0.05). The BamH1/EcoR1 fragments containing regions ii and iii were protamine-enriched (closed boxes, p 0.05). The distribution of histones (open circles) and protamines (closed boxes) in the remaining regions v, vi, viii, and ix overlapped, indicating a mixture of histones and protamines. N.S., not significant.

A similar study examining the chromatin structure of the human PRM2 gene (Figs. 1 and 2, amplicon iv) in sperm from two individuals reported histone enrichment for one subject and protamine enrichment for the other subject (20). This discrepancy was attributed to a restriction site polymorphism. To resolve this apparent discrepancy, the distribution of histones and protamines across regions i–viii of the human PRM1 PRM2 TNP2 domain were evaluated using PvuII-fractionated sperm DNA. The results are summarized in Fig. 2. Regions ix and x were not evaluated, because PvuII bisects these amplicons. As shown, similar to the results of the respective BamH1/EcoR1 fractionation, regions v, vi, and viii displayed no enrichment. In contrast to their corresponding BamH1/EcoR1 results, regions i, ii, iv, vii, and ix also showed no enrichment indicating a mixture of histones and protamines within these PvuII fragments. Interestingly, region iii was protamine-enriched as a 5-kb BamH1/EcoR1 fragment but was histone-enriched (p 0.025) within the 387-bp PvuII sub-fragment. These results further support the view that the deposition of histones and protamines is ordered over discrete and specific regions.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Histone and protamine fractionation using PvuII. Human sperm were partitioned into histone- or protamine-enriched DNA fractions using PvuII restriction endonuclease. The distribution of histones (open circles) and protamines (closed boxes) across the human PRM1 PRM2 TNP2 domain was then assessed by PCR using primers to the specific regions, i–x, contained within these restriction fragments as a function of relative optical density. This was calculated as the mean hybridization signal, expressed as a function of the GC content of the probe. The PvuII fragment containing region iii was histone-enriched (open circles, p 0.05). The distribution of histones (open circles) and protamines (closed boxes) in the remaining regions overlapped, indicating a mixture of histones and protamines. Regions ix and x were not evaluated in the PvuII-fractionated samples because of the coincidence of a PvuII cleavage site within these amplicons.

A comparative summary showing the distribution of histones and protamines across the human PRM1 PRM2 TNP2 domain and the locus is presented in Fig. 3A. The DNase I-sensitive region encompassing the three PRM1, PRM2, and TNP2 genes is flanked by two DNase I-insensitive regions proximal to the two male germ cell-specific matrix attachment regions that designate the physical boundaries of this domain (23). The summarized fractionation data of Fig. 3B show that the bulk of the human PRM1 PRM2 TNP2 domain appears to contain a mixture of histones and protamines. Interestingly there is specific histone-enrichment in association with the relative DNase I-insensitive 5' PRM spMAR region and the DNase I-sensitive promoter regions of the PRM1, PRM2, and TNP2 genes. In addition, histone-enrichment was also observed in association with the CpG island and the DNase I-insensitive SOCS-1 MAR located immediately downstream of the PRM1 PRM2 TNP2 domain. This suggests that, in human sperm, relative DNase I sensitivity does not reflect its histone/protamine status.

View larger version (23K): [in this window] [in a new window]   FIG. 3. The DNase I-sensitive PRM1 PRM2 TNP2 domain in human sperm and the preferential deposition of histones across the locus. The enhanced DNase I sensitivity of the human PRM1 PRM2 TNP2 domain (A) and the distribution of histones and protamines across this region of human chromosome 16p13.13 (B) are shown. There is a marked histone enrichment (open box) in association with the 5' PRM spMAR and the promoter regions for each of the three PRM1, PRM2, and TNP2 genes. Histone-enriched regions indicated by the open boxes colocalized with the CpG island and 5' MAR of the SOCS-1 gene immediately downstream of the PRM1 PRM2 TNP2 domain.

The results reported here and those of others suggest a sequence-specific packaging of sperm chromatin by histones and protamines (7, 20). To globally test this tenet, the histone and protamine distribution of both functional and structural genes was examined. This included the acrosin, -globin, and IGF-2 genes as well as the Alu, centromeric, and telomeric regions, respectively. All were similarly evaluated using the identical four BamH1/EcoR1 fractionated samples. These results are summarized in Fig. 4. As shown in Fig. 4A, the serine protease acrosin, an acrosomal membrane protein (25), showed no enrichment for either histones or protamines. The post-natal expressed -globin gene was enriched in the protamine DNA fraction (p 0.05), as observed similarly by others (20). In contrast, the paternally imprinted, embryonic expressed (26) IGF-2 gene was histone enriched (p 0.05) in accordance with the results of others (9). In comparison, the global distribution of histones and protamines within the various structural sequences is summarized in Fig. 4B. Interestingly, Alu sequences, which are broadly distributed throughout the human genome at a frequency of 1 per 1000 bp (27), were protamineenriched (p 0.05). Given their extensive representation, the protamine enrichment of Alu sequences may simply mirror that of the haploid genome, wherein the majority of the DNA is bound by protamines. Centromeric sequences showed no histone or protamine enrichment. In contrast, telomeres were histone enriched (p 0.05). This is consistent with other studies describing a telomere-histone configuration in human sperm (14). Together, these results indicate that the non-random distribution of histones and protamines also extends to certain repetitive sequences throughout the sperm nucleus.

View larger version (28K): [in this window] [in a new window]   FIG. 4. The distribution of histones and protamines in association with other regions of the human sperm nucleus. Human semen samples were partitioned into histone- and protamine-enriched DNA fractions using BamH1 plus EcoR1 restriction endonucleases. Relative histone/protamine association for the acrosin, -globin, and IGF2 genes (A) and the Alu, centromeric (Cen), and telomeric (Tel) repetitive sequences (B) were determined by comparative PCR of each fraction. The relative optical density (ROD) was calculated as the mean hybridization signal, expressed as a function of the GC content of the probe. Representative ROD values for each PCR are shown. Both IGF2 and telomeric sequences were histone-enriched (open circles, p 0.05). The -globin and Alu sequences were protamine-enriched (closed boxes, p 0.05). Acrosin and centromeric sequences contained a mixture of histones and protamines, as the distribution of the open circle histones and closed box protamines overlapped.

To gain insight into the relationship between structure and function in human sperm, we have examined the distribution of histones and protamines throughout the human PRM1 PRM2 TNP2 domain. We have shown that the region of enhanced exogenous nuclease sensitivity contains several histone-enriched segments. These include the 5' PRM spMAR and the promoter regions for the PRM1, PRM2, and TNP2 genes and the SOCS-1 MAR (28), respectively. In addition, telomeres are also histone-enriched, suggesting that both structural and functional sequences throughout the haploid genome may be packaged by histones. These analyses have established the non-random, sequence-specific packaging of sperm chromatin by histones and protamines.

It is apparent that this arrangement is not only important for the three-dimensional organization of the sperm nucleus but may also provide epigenetic information essential for proper sperm function. It is likely that these discrete regions provide a templating function to initiate, facilitate, and permit the requisite replacement of protamines with histones subsequent to fertilization.

	FOOTNOTES

 
^* This study was supported by National Institutes of Health Grant HD23126 (to S. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a predoctoral fellowship from the Wayne State University Center for Molecular Medicine and Genetics.

^** Charlotte B. Failing Professor and to whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, and Inst. for Scientific Computing, Wayne State University, C. S. Mott Center, 275 E. Hancock, Detroit, MI 48201. Fax: 313-577-8554; E-mail: steve{at}compbio.med.wayne.edu.

¹ The abbreviations used are: IGF-2, insulin-like growth factor 2; SSPE, saline/sodium phosphate/EDTA (buffer); MAR, matrix-attachment region; spMAR, sperm-specific MAR.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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