Download Localization of Three Genes in the Hook

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
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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-
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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.
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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-
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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.
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