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BIOLOGY OF REPRODUCTION 54, 1271-1278 (1996) Localization of Three Genes in the Hook-Shaped Hamster Sperm Nucleus by Fluorescent In Situ Hybridization' W. Steven Ward, 2 '3 John McNeil, 4 Joy de Lara,3 and Jeanne Lawrence 4 Departmentof Surgery/Urology,3 Robert WoodJohnson Medical School New Brunswick, New Jersey 08903- 0019 Department of Cell Biology,4 University of Massachusetts, Worcester, Massachusetts 01655 ABSTRACT We mapped the positions of three different genes inthe flat, hook-shaped hamster sperm nucleus to determine the specificity of sperm DNA positioning. The positions of the 5S rRNA gene cluster, the CAD gene, and the class 11.6 gene were determined by fluorescent in situ hybridization (FISH) in over 50 hamster sperm nuclei for each gene. We first demonstrated by FISH with mitotic chromosomes that the latter two genes were localized on the same chromosome. Within the sperm nuclei, we found that the precise position was variable for each of the three genes, but that there were two areas of preferred localization that contained 26-31% of the nuclear area and within which 80% of the signals were located. Nuclei were then hybridized to two genes simultaneously, using either two genes located on the same chromosome or two genes located on different chromosomes. We found no preference for orientation of one gene relative to the other for either pair of genes examined. This suggested that the relative arrangements of chromosomes within the sperm nucleus are flexible. These data demonstrate that the topographical arrangements of genes within the hamster sperm nucleus have a limited plasticity allowing for a relatively large range of possible localization. INTRODUCTION A major question regarding formation of the sperm nucleus is the specificity involved in the order of chromosome packaging. This problem is particularly interesting in the hook-shaped sperm nuclei of many rodents, in which specific chromatin packaging might be expected to contribute to the unusual shape. These nuclei provide a unique opportunity to examine this question because their flat, asymmetric shape allows us to readily compare the locations of individual genes in different nuclei, thereby testing how specific the positioning of the DNA is. The specificity involved in the locations of individual genes and of whole chromosomes within the eucaryotic nucleus is not a problem that is limited to spermatozoa. Several investigators have previously addressed this problem in somatic cells with varying results. Comings [11 provided a rationale for at least a limited specificity for the three-dimensional organization of chromosomes in eucaryotic cells on the basis of functional considerations. The most clearly defined such structure is the nucleolus where rRNA is transcribed. It is composed of both nucleolar proteins that make up the nucleolar matrix [2] and DNA from the ends of the short arms of five chromosomes in the human [3-5]. More recent data have defined the pres- ence of transcription foci and domains enriched in splicing factors and poly(A) RNA, with which transcription and AcceptedJanuary 18, 1996. Received December 1, 1995. 'This work was supported by a Research Career Development Award toJ.B.L. and NIH grants GM49254 (to J.B.L) and HD28501 (to W.S.W.). 2 Correspondence: W. Steven Ward, Ph.D., Division of Urology, Robert Wood Johnson Medical School, MEB-588, 1 RWJ Pl., New Brunswick. NJ 08903-0019. FAX: (908) 235-7013: e-mail: [email protected] splicing of some genes and pre-mRNAs are associated [69]. Another recently identified type of structure within the nucleus with which DNA is associated is represented by the 300- 1000 discrete areas within the nucleus where DNA replication takes place, termed "replication foci" [10-12]. These domains may be related to the association of both transcription [13-17] and replication [18-201 with the nuclear matrix, a proteinaceous structural support of the nucleus [21]. Lawrence et al. [22] have previously noted a nonrandom, though variable, localization of genes relative to overall nuclear space in cultured fibroblasts. However, given that fibroblast nuclei are ovoid and exist at different stages of the cell cycle, the conclusions from this work were preliminary. Finally, work in Drosophila identified several packaging motifs of the four chromosomes that were shared in all the nuclei examined, some of which were tissue-specific [23-25]. There was, however, a wide range of configurations for each chromosome such that no individual chromosome had the same structure in any two nuclei. In this work, we have addressed the question of the specificity of DNA packaging in the hamster sperm nucleus. DNA within the sperm nucleus is transcriptionally inactive and is not being replicated [26], so that these functions are unlikely to have direct effects on the positioning of the DNA. However, the unique shape of this nucleus suggests that a specific order of chromosome packaging may be required for spermiogenesis. The hamster sperm nucleus is asymmetric along all axes except one (side to side), with a length of 8 jpm and a maximum width of only 0.5 gm [27]. It is a flat, hook-shaped nucleus with a blunt posterior end and a bent, pointed anterior end (Fig. 1). Thus, unlike most somatic cell nuclei, the asymmetry of the hamster sperm 1271 1272 WARD ET AL. A. The Hamster Spermatozoon Nucleus Tail Acrosome B. The Hamster Sperm Nucleus t _______________________ three were localized to only one point in the hamster karyotype (see below and Fig. 2, C and D). Also, the probes for all three genes included the complete genomic sequences. The first gene, a single-copy CAD gene, is a gene for a multifunctional protein that catalyzes the first three steps of uridine biosynthesis [29]. The second gene was a single-copy gene from the major histocompatibility complex (MHC) class I family, the class 1 1.6 gene [30]. The third gene was the tandemly repeated 5S rRNA gene, which comprises a 2.2-kb sequence that is repeated up to 1350 times in the haploid sperm genome [311. The 5S rRNA gene cluster is expressed in all cell types that are making RNA [31], and the CAD gene is expressed in all cell types that are replicating DNA [29]. The class I 1.6 gene, however, is tissuespecific and is expressed only in certain lymphoid cell types [30]. Thus, we have examined tissue-specific and more generally expressed genes and found no real difference between the two. We found that for each gene, there was a surprising degree of variability in position, both independently and with respect to each other. Longitudinal Cross Section FIG. 1. Diagram of hamster sperm nucleus. A) Hamster spermatozoon, shown with complete head and tail. B) Diagram of hamster sperm nucleus, based on electron micrographic studies (after Yanagimachi and Noda 1271). Lower diagram is a sagittal cross section through center of nucleus. nuclear structure together with its marked degree of compression in the third dimension allows one to readily compare the localization of in situ hybridization signals in different nuclei, using the nuclear shape as an independent reference. Mammalian sperm nuclei have three additional advantages for studying the specificity of the three-dimensional organization of DNA within eucaryotes. The first is that mature sperm nuclei are terminally differentiated cells and are therefore not heterogeneous with respect to aspects of chromatin structure that might be affected by cell cycle stage. The second is that mammalian sperm chromatin packaging appears to be more homogeneous than somatic chromatin, in that it does not contain, as revealed by electron microscopic examination [27, 28], prominent blocks of heterochromatin and euchromatin. This eliminates another potential complicating factor: the distribution of the DNA into regions of euchromatin and heterochromatin. Finally, sperm nuclei are haploid, so the localization of a single gene is not complicated by the presence of two signals. The hamster sperm nucleus, therefore, provides a unique biological system for examining the specificity of DNA organization at its most basic level, in highly compacted, biologically "silent" DNA that is packaged within an asymmetric nucleus. We examined the position of three different genes within condensed hamster sperm nuclei using fluorescent in situ hybridization (FISH). All three genes used as probes in this study were cloned from the Syrian golden hamster, and all MATERIALS AND METHODS Preparationof Hamster Sperm Nuclei Hamster nuclei were prepared as described in detail previously [32]. Briefly, spermatozoa were extracted from the caudae epididymides and washed immediately in cold buffer that contained 50 mM Tris (pH 7.4) and 0.5% SDS. This treatment separates the heads from the tails. The suspension was then layered onto Beckman (Palo Alto, CA) SW-28 tubes with a double step gradient; the upper layer contained 2 M sucrose, 25 mM Tris (pH 7.4), and 5 mM MgCl, and the bottom layer contained the same solution plus 0.075 g/ml CsCl. The tubes were centrifuged at 25 000 rpm for 1.5 h. The supernatants were aspirated, and the pelleted nuclei were resuspended in 300 mM CaCl 2. The nuclei were then incubated for 30 min on ice with 10 mM dithiothreitol to extract some of the protamines, and 20 1tlof this suspension was then placed on a pre-chilled slide and incubated at 4°C for 20 min. This allowed the nuclei to attach to the slides. The slides were dipped in 10 mM Tris (pH 7.4) and dried overnight. The next day, the slides were fixed in ice-cold ethanol for 20 min, then two times in 3:1 methanol:acetic acid for 20 min each. The slides were then kept at - 70°C until they were used for in situ hybridization. This method of extraction of the hamster sperm nuclei prevented any gross distortion of shape. The extracted nuclei have a slight increase in width but are the same length as unextracted nuclei and retain the same flat hook shape that is characteristic of unextracted nuclei. Preparation f Mitotic Chromosomes Primary hamster tail skin fibroblasts were obtained by cutting the tail and stripping the skin with dissecting scis- 1273 GENE POSITIONING IN HAMSTER SPERM NUCLEI sors. Both the tail and the skin were incubated with 1 mg/ ml of collagenase in a-MEM medium without serum for 1 h at 37 0C, and then incubated in medium with 10% fetal bovine serum in a tissue culture flask until the cells were growing. The cells were replated onto slide chambers, and when they reached approximately 50% confluence, they were treated with 0.0225 g/ml colcemid for 4 h. The medium was then washed out, 4 ml per slide of 75 mM KCI was added, and incubation proceeded at 37°C for 35 min; 2 ml of 3:1 methanol:acetic acid was then added to each slide chamber, and slides were incubated for 2 min at room temperature. Slides were then fixed in four washes of 3:1 methanol:acetic acid, the first two at 1 h and the last two at 45 min, all at room temperature. Chromosomes were spread by gently forcing a stream of humidified air onto the slide. DNA Probes All three genes used as probes in this study were cloned from the Syrian golden hamster. The single-copy CAD gene was the generous gift of Geoffrey Wahl (Salk Institute, La Jolla, CA) [29]. The clone used, termed cCAD-1, is a 47-kb cosmid that contains the complete genomic sequence of the CAD gene. The second gene, a single gene from the MHC class I family, was the kind gift of Philip Tucker (Dalhouse University, Halifax, NS, Canada) [30]. The probes from this gene actually comprised three different plasmids-pHm1.65, pHm-1.6i, and pHm-1.63-that together span 7.8 kb of genomic sequence including all seven exons and six introns. The third probe, the 2.2-kb repeated unit that makes up the 5S rRNA gene cluster, was the kind gift of William Folk (University of Michigan, Ann Arbor, MI) [31]. This gene is repeated up to 2700 times in the hamster, all at one point in the karyotype (data shown in this paper). FluorescentIn Situ Hybridization Details of the procedure have been previously described [33, 34]. In brief, probes were prepared by nick translation with digoxigenin-16-dUTP or biotin-16-dUTP. Calcium-extracted preparations of hamster sperm nuclei were denatured in 70% formamide and double-strength sodium citrate buffer (SSC) for 2 min and were incubated overnight with 10 gl hybridization buffer (50% formamide, double-strength SSC, 10% dextran sulfate, 1 mg/ml BSA, 5 mg/ml hamster DNA, 1 mg/ml Escherichia coli tRNA) containing 10 ng of probe. Probes were detected with tetramethylrhodamine isothiocyanate (TRITC), a-digoxigenin and fluorescein isothiocyanate avidin (FITC); sperm nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). The dUTP materials, E. coli tRNA, TRITC, and FITC were obtained from Boehringer Mannheim (Indianapolis, IN), and the hamster DNA was purified, using standard methods, from Syrian golden hamster spleen. Analysis Samples were analyzed on an epifluorescence-equipped microscope (Zeiss, New York, NY) with a Plan-apochromat 100X, 1.4 NA objective and a multibandpass filter (Chroma, Brattleboro, VT) and were photographed with a cooled CCD camera (Photometrics, Tucson, AZ). Distance and intensity measurements were gathered from digital images by means of WHIP image-processing software (GW Hannaway & Assoc., Boulder, CO). Distribution of genes within the nuclei were recorded relative to nuclear landmarks and plotted on a standardized sperm nucleus. RESULTS Integrity of the Structure of the PartiallyExtracted Sperm Nuclei Electron micrograph studies of hamster spermatozoa revealed that fully condensed hamster sperm nuclei have a characteristic, asymmetrical hook-shaped nucleus that is very flat [27]. Yanagimachi and Noda [27] determined that the nucleus is only 0.5-0.6 gm at its thickest point and 0.20.25 m at the thinnest section (Fig. 1). Interestingly, the thinnest part of the nucleus is not at the anterior hook but at a site that covers a wide band across the nucleus approximately one third the distance from the anterior end. This slight constricture of the hamster sperm nucleus is also the location of a cytoplasmic element termed the acrosomal ring, which may be partly responsible for the condensation. Assuming that the concentration of DNA is relatively homogeneous throughout the nucleus and that, therefore, DNA concentration is reflective of nuclear volume, the shape of the nucleus can be examined by the DAPI-stained image (Fig. 2, A and B). Examination of such a DAPI-stained hamster sperm nucleus that has been partially extracted, fixed, and hybridized to one of the probes, as described in Materialsand Methods, illustrates that both of the characteristic features described above-the hooked shape of the nucleus and the anterior constricture-are preserved during the FISH procedures (Fig. 2, A and B). While the preservation of the general structure of the sperm nucleus does not in itself prove that structural changes in the chromatin did not occur, nuclear structure is one necessary predictor. That is, chromatin structure would definitely not be expected to be preserved in a distorted nucleus. The false color image showing digital image analysis of the DAPI concentration located throughout the nucleus (Fig. 2B) demonstrated that the highest concentration of DNA is at the posterior base, while the lowest concentration is at a wide band that spans the nucleus at the site of the acrosomal ring. The retention of the characteristic hooked shape, as well as the unique distribution of DNA concentration throughout the hamster sperm nucleus, suggests that the extraction procedures used to examine these nuclei do WARD ET AL. FIG. 2. A and B) Structure of sperm nuclei used for FISH. A) DAPI-stained extracted sperm nucleus. B)False color intensity map of the same nucleus showing distribution of DNA based on a digital image (intensity is highest from white and blue, and lowest at red). Note band of decreased intensity at site of acrosomal ring (Fig. I ) . C and D) Localization of three genes in Syrian golden hamster metaphase spreads. C) Double localization of 5s rRNA gene cluster (green) and class 1 1.6 gene (red) to q arm of chromosome B6. D) Localization of CAD gene on p arm of chromosome B9 (see text). E and G) FISH in sperm nuclei with three different genes. E) 5s rRNA gene cluster. F) Class 1 1.6 gene. G) CAD gene. Arrows point to two genes localized to pointed, anterior end of nucleus; arrowheads point to examples where gene is located near posterior end. H) Double localization of two genes located on same chromosome--5s rRNA gene cluster (red) and class 1 1.6 gene (greenhlose together with 5s more basal. I) The two genes on same chromosome; 5s and class I 1.6 farther apart with 5s more apical. J) Double localization of two genes on different chromosomes, showing CAD gene (red) and 5s rRNA gene cluster (green) far apart with CAD gene more apical. K) Second example showing the two genes on same chromosome closer, with CAD gene more basal. A-D and H-K, x 50 000; E-G, x 25 000. 1275 GENE POSITIONING IN HAMSTER SPERM NUCLEI not significantly alter the higher-level in vivo arrangement of the DNA sequences. Mapping of Each Gene to Its Chromosome Before these genes could be mapped within the sperm nuclei, their relative chromosome localization had to be determined. Using FISH, we first determined that the class I 1.6 gene and the 5S rRNA gene cluster were located on the q arm of the same chromosome, with the class I 1.6 gene located closer to the centromere (Fig. 2C). On the basis of the relative lengths of the p and q arms of this chromosome, we have tentatively identified it as B6 of the hamster karyotype. Of the three genes used in this study, only the CAD gene had been previously mapped in the Syrian hamster genome, on another chromosome, B9 [29]. We confirmed that the CAD gene was located on a chromosome with the same relative dimensions as B9, on the basis of the relative lengths of the p and q arms, and that this chromosome was different from the one on which the other two genes were located (Fig. 2D). Thus, our study employed three genes located on two of the larger chromosomes of the hamster karyotype. This allowed us to compare the spatial distribution of two DNA sequences on different chromosomes, and of two genes located on the same chromosome. Localization of the Three Genes in HamsterSperm Nuclei We next mapped the positions of each of the three genes within hamster sperm nuclei using FISH, with biotin-labeled probes for the genes. The position for each gene was determined in 50-80 nuclei with respect to the well-defined morphology of the sperm nucleus. The apical end was defined as the pointed hook, where the acrosome is located, and the basal end was the blunted, opposite end where the implantation fossa is located (Fig. 1). The ventral position was defined in this study as the side of the nucleus to which the hooked end was pointing, and the dorsal side was the opposite side (Fig. 1). No attempt was made to identify positions of the genes along the z-axis since the sperm nucleus is only 0.5 pm thick. The position of each gene in different nuclei varied greatly, though some definite restrictions were noted (Fig. 2, E-G). For example, two nuclei shown in Figure 2G (arrows) contain a CAD gene near the pointed apical end of the nucleus, while two others (arrowheads) are localized towards the basal end of the nucleus. The distributions of the positions for each gene in all nuclei examined are diagrammed in Figure 3. These patterns were variable and uneven, with identified locations for each gene spread throughout the sperm nucleus. In all three cases, the genes were concentrated in preferred areas of localization at the upper and lower peripheries of the flat nucleus, which contained a majority of the total possible locations for the gene. The areas of preferred localization for each gene are also diagrammed in Figure 3. The solid lines indicate the areas within which over 60% of the genes Gene Distributions Preferred Area of Localizations - 5SrRNA Class 11.6 FIG. 3. Distributions of genes. Composite diagrams for localization of genes within all nuclei mapped are shown on left. Each dot in diagram represents position of gene in one single nucleus. Areas of preferred localization for each gene are diagrammed on right. Solid lines indicate areas in nucleus within which over 60% of signals were located and represented 11% (CAD), 12% (class 11.6), and 17% (5S rDNA) of total nuclear area. Areas outlined by dashed lines indicate extended areas where > 80% of genes were located; these areas represented 31% (CAD), 26% (class 11.6), and 30% (5S rRNA) of total nuclear area. were localized. In all three cases, these areas represented very narrow margins close to the periphery of the sperm nucleus, which contained only 11% (CAD), 12% (5S rRNA), or 17% (class I 1.6) of the total nuclear area. The dashed lines indicate extended boundaries for these areas that increase the percentage of gene locations to > 80%. These extended areas of preferred localization still represented a relatively small (26-31%) portion of the total sperm nuclear area. When compared with the distribution of the total DNA within the hamster sperm nucleus (Fig. 2B), the areas of preferred gene localizations did not correspond with the areas with the highest concentrations of DNA, located at the center and basal end of the sperm nucleus. This suggested a variable though nonrandom distribution of the genes. Relative Localization of Different Genes to Each Other We next examined the relationship of the positions of individual genes to each other using double-labeling techniques, allowing us to visualize more than one gene within each nucleus. Given the chromosomal localizations of the three genes chosen for this study, we were able to make two types of comparisons. First, we examined the relationship of the positions of the two genes that were located on the same chromosome, the 5S rRNA gene cluster and the class I 1.6 gene. This allowed us to test the flexibility of folding of a single chromosome within the sperm nucleus. In the second double-labeling experiment, the relative positions of two genes that were located on different chro- 1276 WARD ET AL. A. Same Chromosome Same Chromosomes 5SrRNA- e 53% 47% Class 11,6- o 38% 9% n = 36 31% 22% n = 32 B. Different Chromosomes 5SrRNA - 0 c ) U ' a 'Different a) % 04 0 IU CAD- o IZ Chromosomes L- 48% 52% 33 n=31 3 n=24 U. FIG. 5. Relative orientations of genes located on the same and on different chromosomes. Relative orientation of two genes on the same chromosome-5S rRNA and class 11.6 genes-were compared to each other with respect to shape of sperm nucleus. Along the apical-basal plane, nuclei were scored as to which gene was located more apically. Along the dorsal-ventral plane, nuclei were scored in four categories; 5S rRNA located dorsal to class 1 1.6, the 5S rRNA gene located more ventral, both genes located dorsally, both genes located ventrally. The same analysis was performed for two genes located on different chromosomes-the 5S rRNA gene cluster and the CAD gene. Micrometers FIG. 4. Histogram of distances between different genes in the same nucleus. A)Distribution of distances between the 5S rRNA gene cluster and class 11.6 genes, measured by double hybridization experiments. These two genes are located on the same chromosome. Inthis experiment, the 5S rRNA gene cluster was oriented more basally in47% of nuclei and more apically in53%. BI Distribution of distances between the CAD gene and the 5S rRNA gene cluster, two genes located on different chromosomes. In this experiment, the 5S rRNA gene cluster was oriented more basally in 52% of nuclei and more apically in 48%. mosomes were examined. This allowed us to- examine the flexibility of the positioning of different chromosomes within the sperm nucleus. We first measured the distances between these two pairs of genes and confirmed that the two located on the same chromosome were positioned closer together, on average, than the two genes on different chromosomes. The 5S rRNA gene cluster and the class I 1.6 gene, present on the same chromosome arm, were located closer than 4 gm to each other in 83% of the nuclei (Fig. 4A). The CAD gene and the 5S rRNA gene cluster, present on different chromosomes, were located closer than 4 gum in only 65% of the nuclei and had a wider distribution range (Fig. 4B). Unlike the 5S rRNA and the class I 1.6 gene, the 5S rRNA gene and the CAD gene were almost never less than 1 lm apart. We next compared the relative orientations of these two sets of genes with respect to the shape of the sperm nucleus and found these to be highly variable. The two genes located on the same chromosome could be oriented in such a way that either gene was located more basally relative to the other one (Fig. 2, H and I). The same was true for the two genes located on different chromosomes (Fig. 2, J and K). We analyzed these data in two ways. We first examined whether there was any preference for either gene to be located more towards the basal or apical end of the sperm nucleus, that is, with respect to the longitudinal axis. We found that for either case, whether the two genes were located on the same chromosome or on two different chromosomes, approximately half of the nuclei had one orientation and half had the other (Fig. 5). With respect to the dorsal-ventral axis, the orientations appeared to be more varied, so nuclei were scored in four categories; nuclei with the 5S rRNA gene cluster located more ventrally or more dorsally, and nuclei with both genes ventral or dorsal (Fig. 5). Again, we found no clear preferential orientation for either pair of genes located on the same chromosome or on different chromosomes. The only possible orientation that may have been under-represented by random distribution was that in which the 5S rRNA gene cluster was located more dorsal to the class I 1.6 gene, found in only 9% of the nuclei examined (Fig. 5A). DISCUSSION A major conclusion from these data is that the three genes examined are not positioned into rigid three-dimensional coordinates within the hamster sperm nucleus. Rather, the sperm nucleus seems to allow a large degree of plasticity in the location of these DNA sequences. The data GENE POSITIONING IN HAMSTER SPERM NUCLEI therefore suggest that highly precise positioning of these genes is not necessary for proper sperm structure or function. However, the data also demonstrated that there are significant limitations to this plasticity. The gene distributions observed are clearly not random; hence some structural constraints do exist for the three-dimensional positioning of genes within the sperm nuclei (see below.) These conclusions are especially pertinent in sperm nuclei in which the DNA is not biochemically active, that is, where there is no DNA replication or transcription ongoing. The question of the specificity of DNA packaging is therefore not complicated by differences in cell cycle or gene expression, such as the association of DNA with splicing factor/poly(A) RNA-rich domains [6, 7] or replication foci [1012], which are factors in other cell types. It is important to consider that the FISH procedures could impact chromatin structure to some degree. While some impact cannot be ruled out, we believe the influence on our conclusions was minimal for several reasons. First, the procedures used might be expected to affect the details of fine chromatin structure, not the higher-level positioning of genes in the nucleus studied in this work. In other work, these procedures, in fact, preserved the sequence-specific location of genes with respect to internal nuclear reference points [7]. In addition, the overall structure of the sperm nucleus was preserved, as shown in Figure 2, A and B. Furthermore, the sperm nuclei did not change shape or increase in size during the extraction, so that chromatin would not have much opportunity to shift. Finally, the large variation in positions of the individual genes, from the base of the nucleus to the apical hook (Fig. 3), would require a tremendous reorganization during the FISH procedure that seems very unlikely. Another possible source of error, background hybridization, was minimized, particularly with the 5S rDNA, which because of its large repetition of sequences provides a uniquely strong FISH signal. If the three-dimensional packaging of genes within the sperm nucleus is not rigidly specific, our results show that the distribution is clearly not random. For each of the three genes examined, we identified two "preferred areas of localization" encompassing only 30% or less of total nuclear area, within which 80% of the signals were found. Interestingly, the preferred area of localization was essentially the same for all three genes studied. The fact that each of these areas of preferred localization was not identical with the highest concentrations of DNA suggests that the genes were positioned in those areas for a reason, probably related to the arrangement of chromosomes or higher-level folding of chromatin. This could be related to packaging constraints on the whole chromosome or for specific classes of DNA. For example, the localization to the nuclear periphery of the CAD gene and the 5S rRNA gene, neither of which was close to the telomere or centromere, could be the result of telomere and centromere sequences being localized in a 1277 specific part of the nucleus, as has been proposed [35]. This could result in the exclusion of other gene-coding chromatin from these regions, potentially contributing to the preferred regions of localization for the three genes observed here. In the case of somatic cells, more precise localization was observed when transcriptionally active genes were mapped with respect to internal nuclear structures related to pre-mRNA splicing [6, 7]. Similarly, it is possible that more precise sequence localization will be observed in spermatozoa as more sperm nuclear structures are identified that can serve as internal reference points, such as the nuclear annulus. The relative positions of the two genes that were located on the same chromosome arm (the 5S rDNA and the class I 1.6) were also variable. This suggests that the chromosome is variable in the way it is positioned within the sperm nucleus, and that it may be more extended than in mitotic chromosomes. A more extended fiber may still occupy less volume. Consequently, the packaging ratio of sperm DNA could still be higher than in somatic cells. One recent report using whole chromosome painting probes has provided evidence for such extended and variably positioned chromosomes in mouse sperm nuclei [36]. As shown in the present work, the two genes on the same chromosome arm were positioned at various distances to each other in different sperm nuclei (Fig. 2, H and I, and Fig. 4A). This suggests fairly extended chromosome fibers in sperm nuclei. The double localization experiments that simultaneously compared the positions of two genes on different chromosomes suggest that the conclusion that individual genes are placed variably within the sperm nucleus may be extended to include the relative positions of chromosomes with each other. The distance between the CAD and 5S rRNA genes was variable, and the orientation of the two genes to each other was evenly divided between one being more apical than the other and being more basal. There was also no clear preference for the position of the genes with respect to the dorsal-ventral axis. The data therefore suggest that the two chromosomes on which these genes are located are not positioned the same way with respect to each other in every sperm nucleus. It should be noted that these two genes, however, were almost never located such that the FISH signals seemed to be touching (Figs. 2J and 4B). Another type of DNA structure that is very specific is the organization of DNA into loop domains. Previous data from Gerdes et al. [16] described differential packaging of genes relative to the higher-level loop domains. For seven RNA polymerase II transcribed genes investigated, the active genes were more closely associated with the nuclear matrix, or nuclear structure, whereas inactive genes were on the extended portion of the loop. These data supported earlier reports that reached similar conclusions but employed different methodologies [37-391. Mammalian sperm DNA is also organized into loop domains that exhibit a similar spec- 1278 WARD ET AL. ificity [40, 41]. The 5S rRNA gene cluster, for example, has recently been shown to be organized into three small loop domains by the hamster sperm nucleus, while the same DNA is organized into one large loop domain in adult somatic cells [41]. In both somatic and sperm nuclei, the position of the genes with respect to the nuclear matrix was highly specific-certain genes were associated and others were not. These data, together with those presented in this paper, suggest that there are nonrandom structural arrangements of genes at different levels and imply that the association with nuclear substructure may be more rigid than the position of individual genes within overall nuclear space. One area that was devoid of the three genes examined was at the posterior end, at the implantation fossa where the tail is attached to the sperm nucleus. DAPI staining showed that this area contains a high concentration of DNA, but in our studies we were unable to find a single nucleus with any of the three genes located in this area. This area is the site of a sperm-specific nuclear structure termed the nuclear annulus, which anchors the sperm genome when the nucleus is decondensed [42]. The complete absence of genes in this area suggests that some specific chromosomal packaging constraint exists in this area. Such a constraint could be the presence of unidentified DNA sequences that are attached to the nuclear annulus. It may also be the result of packaging constraints of the two chromosomes. In conclusion, our data suggest that the positioning of DNA within the hamster sperm nucleus is not rigidly specific. The data support a model of chromatin packaging in which structural constraints do exist for the positioning of the centromeres and telomeres, but not for the order of the chromosomes with respect to the nucleus. REFERENCES 1. 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