Applications of X-Ray Microscopy to the Analysis
of Sperm Chromatin
R. Balhorn1, R. E. Braun2, B. Breed3, J. T. Brown4, D. Evenson5, J. M. Heck4,
J. Kirz6, I. McNulty7, W. Meyer-Ilse4, X. Zhang6
1
Biology and Biotechnology Research Program, Lawrence Livermore
National Laboratory, Livermore, CA 94550, USA, E-Mail: balhorn2@llnl.gov
2
University of Washington, Department of Genetics, Seattle, WA 98175, USA
3
The University of Adelaide, Department of Obstetrics and Gynecology,
Adelaide, South Australia
4
Center for X-ray Optics, Lawrence Berkeley National Laboratory,
Berkeley, CA 94720, USA
5
South Dakota State, Department of Chemistry, Brookings, SD 57007, USA
6
State University of New York at Stony Brook, Department of Physics,
Stony Brook, NY 11794, USA
7
Argonne National Laboratory, Chicago, IL 60439, USA
Abstract. Chromatin structure has been particularly difficult to study in
mammalian sperm cells because the DNA molecules are so tightly packed
inside the nucleus that fluorescent probes cannot access the interior of the
nucleus and electrons can only penetrate thin sections of the sperm head. X-rays
can readily be used to interrogate the interior of the intact sperm head without
sectioning or decondensing it. This has made it possible for us to determine the
extent of hydration of sperm chromatin (working at a wavelength inside the
water window), identify the composition of chromatin in the sperm of several
different mammals (using X-ray absorption near edge spectroscopy), visualize
vacuoles located inside the intact sperm head, obtain biochemical information
about the structure of the equatorial segment and perinuclear theca surrounding
sperm chromatin, and examine the nature and uniformity of chromatin
compaction in the marsupial mouse, Sminthopsis crassicaudata, and several
different lines of transgenic mice. These studies have shown that the sperm cell
is a particularly good target for X-ray microscopy studies.
1 X-Ray Microscopy Can be Used to Examine DNA Packing
Inside the Sperm Nucleus
Light, electron and atomic force microscopy have all been used to study how DNA is
organized inside the nucleus of mammalian sperm cells. Conventional light
microscopy has been limited by the small size of the sperm nucleus and the limited
resolution of the technique to providing information about the general morphology of
the sperm head and the uniformity of staining of sperm chromatin using fluorescent
probes. Electron microscopy (EM) has revealed that DNA is packed so densely inside
the nucleus of the mature sperm that electrons cannot penetrate it (Dooher and
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R. Balhorn et al.
Bennett, 1973; Roosen-Runge, 1962). The process of DNA compaction within the
maturing spermatid has been followed by transmission EM of thin sections of the
spermatid nucleus. These studies have shown that the process of condensation is
initiated at the apical end of the nucleus and progresses toward the tail as the nucleus
elongates and takes shape. During this process, the diffuse chromatin characteristic of
all somatic cells is completely reorganized. A family of very arginine-rich proteins,
called protamines, are synthesized and bind to DNA, replacing the histones and other
chromosomal proteins during mid spermiogenesis (Balhorn, 1989). Upon binding,
these small proteins coil the DNA into toroidal structures that contain up to 50Kb of
DNA (Hud et al., 1993). Once the process is completed, each sperm nucleus contains
approximately 50,000 of these structural subunits. The coiling of DNA into toroidal
structures in vitro has been examined by EM (Hud et al., 1993; Hud et al., 1995) and
their existence in vivo has been confirmed by atomic force microscopy (AFM)
(Balhorn et al., 1993).
While X-ray microscopy cannot achieve a resolution comparable to EM or AFM,
the ability of X-rays to penetrate the sperm nucleus makes it possible to probe the
interior of the sperm nucleus and obtain structural and compositional information that
reflects the entire nucleus. We have used this technique to obtain structural
information about sperm chromatin organization that could not be obtained using
other forms of microscopy. These studies have provided new information in three
different areas: 1) the extent of chromatin hydration, 2) the biochemical composition
of sperm chromatin and associated structures, and 3) the uniformity of chromatin
packing inside the nucleus.
2 Extent of Sperm Chromatin Hydration
Sperm cells are extremely unusual in that they, as a terminal differentiation product of
an organ (the testis), are not destined to undergo apoptosis and cell death after
performing their function. Each sperm cell instead carries the complete genomic
blueprint of the individual that produced it, and the process of sperm development has
been designed to temporarily "deprogram" the genome it carries so when it is
combined with the genome of the egg (following fertilization) and reactivated, it can
be reprogrammed to function as an embryonic cell, not a testis cell. This temporary
inactivation of an entire genome and the attendant condensation of the sperm's DNA
into a biochemically inert, highly compacted state has not been observed to occur in
any other type of cell. In an effort to estimate the extent of this compaction, data
obtained from earlier biochemical studies performed in our laboratory and estimates
of sperm nuclear volumes obtained by serial section EM were used to calculate the
concentration of DNA and the density of its packing inside the sperm nucleus
(Pogany et al., 1981). The results indicated that the volume of the sperm nucleus, and
the physical volume of the DNA molecule packed inside it, were essentially identical.
Thus the DNA, packed at a concentration of approximately 750mg/ml, appeared to
fill the entire volume of the sperm nucleus.
These calculations suggested that the sperm chromatin must contain very little
water. Only the minor groove of the helix appeared to contain possible sites for water
binding, because the protamine molecules fill the major groove (Hud et al., 1994,
Prieto et al., 1997). While the data were convincing, the results seemed inconsistent
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
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with the way the sperm chromatin was known to decondense both in vitro and in vivo
after fertilization. In both cases, the highly compacted sperm head are observed to
decondense rapidly, with the entire mass of sperm chromatin swelling relatively
uniformly throughout. Since this decondensation requires reduction of a series of
intermolecular disulfide bonds that interlock protamine molecules around the DNA
helix, and this reduction could only be achieved if the protamine is hydrated and
accessible to the reducing agent, the two findings appeared inconsistent.
Working inside the water window at wavelengths (4.483 nm) where protein and
DNA absorb strongly but water does not (Fig. 1), we were able to use X-ray
microscopy, combined with atomic force microscopy, to obtain reasonably accurate
estimates for the water content of air-dried rat sperm chromatin (DaSilva et al., 1992).
Fig. 1. X-ray transmission through 1 micron of DNA, protein and water.
Accurate thickness measurements were obtained for individual rat sperm nuclei
air-dried onto silicon nitride windows using the atomic force microscope (Fig. 2A).
Transmission images were subsequently taken of the same sperm nuclei (Fig. 2B)
using LLNL's pulsed X-ray laser microscope. Using the known composition of the
protamine-DNA complex, the density of the complex, and the transmission of
4.483nm X-rays through a particular region of the nucleus, we were able to calculate
the thickness of the DNA-protamine complex (470nm) inside the rat sperm nucleus.
Since the actual thickness of the nucleus in this region was determined to be 700nm
by AFM, the results indicated that ~33% of the volume of the dried rat sperm nucleus
must be occupied by water. Subsequent studies using the AFM to monitor changes in
mouse sperm nuclear volume upon dehydration provided similar results. In these
studies, the volume of the air-dried nucleus was found to be 26–36% greater than the
volume of the completely dehydrated nucleus (Allen et al., 1996).
While the pulsed-X-ray laser studies could not provide information about the
amount of water inside the nucleus of fully hydrated sperm, they did provide the first,
clear evidence that sperm chromatin must be extensively hydrated, even in its highly
compacted state. The subsequent AFM studies that confirmed the result extended the
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R. Balhorn et al.
A
B
Fig. 2. AFM and X-ray microscopy image of rat sperm. Similar images were used to
determine the extent of hydration of the nucleus by measuring the thickness of the head
with the AFM and determining the thickness of DNA and protein present in the same
head from the transmission of X-rays (4.483nm) through it.
analysis to include fully hydrated sperm and revealed that water comprises as much
as 64–69% of the volume of sperm chromatin (Allen et al., 1996).
3 Composition of Sperm Chromatin (XANES Imaging)
Biochemical studies of the proteins that package DNA in mammalian sperm have
shown that two different types of protamines bind to DNA and work together to
package it inside the nucleus of the sperm cell (Balhorn, 1989). The smaller protein,
protamine 1, is found bound to DNA in the sperm of all species of mammals. A larger
histidine-rich protamine 2 molecule is only present in the sperm of selected species
(predominantly rodents and primates). Unlike the histone proteins that package DNA
in all other cells, which are always present in the same proportion, electrophoretic
analyses of the isolated protamines have shown that the relative proportion of the two
protamines bound to sperm DNA differs dramatically among species (Balhorn, 1989).
These studies were not, however, able to provide both DNA and protamine content
information for the same sperm cell. Consequently, the absolute amount of protamine
1 and 2 bound to DNA in sperm chromatin could not be determined accurately.
Even though the amount of protamine 2 is highly variable among species, several
studies have indicated that its presence is critical for male fertility (Balhorn et al.,
1988; Bach et al., 1990; Belokopytova et al., 1993; de Yebra et al., 1993). To understand the significance of this variation, we must first understand how the two proteins
package DNA. A first step in this process requires that we know the mass ratio of
protamine to DNA in sperm chromatin. Because semen contain normal, abnormal and
immature sperm cells as well as cells from supporting tissues, accurate determinations
of the protamine to DNA mass in sperm can only be obtained by analyzing individual
cells. This allows the investigator to select normal, fully matured sperm cells for
analysis and avoid including data obtained from defective, immature or supporting
cells.
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
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Linear Absorption Coefficient
(inverse microns)
5
4
3
2
DNA
Protamine 1
Protamine 2
1
4.10
4.20
4.30
4.40
4.50
X-ray Wavelength (nm)
Fig. 3. X-ray absorption spectra of DNA, protamine 1 and protamine 2 at the carbon edge.
Images of sperm heads were obtained at the wavelengths indicated by vertical lines and the
spectral differences between DNA and protamines were used to map out these components
separately.
Previous studies have shown that X-ray absorption near edge spectroscopy
(XANES) can be used in combination with scanning transmission X-ray microscopy
to discriminate between the protein and DNA components of individual Chinese
hamster ovary cells (Kirz et al., 1994). X-ray absorption spectra obtained for DNA,
protamine 1 and protamine 2 (Fig. 3) revealed spectral differences between DNA and
the protamines that could be used to map (and quantitate) these components
separately inside the sperm nucleus of four different species of mammals. To
accomplish this, individual dried sperm were imaged at six different wavelengths
(4.100, 4.279, 4.297, 4.318, 4.339, and 4.400nm) that represent specific peaks in the
DNA or protein absorption spectra using the Scanning Transmission X-ray
Microscope at Brookhaven National Laboratory. The optical density of the sperm
nucleus at each wavelength was obtained from these images and the data were used to
calculate the mass of DNA and protamine present in the nucleus using the Singular
Value Decomposition method (Zhang et al., 1996). Sperm from four species were
chosen as representatives of the range of protamine 2 variation that is known to occur
among different mammalian species. Bull sperm contain only protamine 1. Stallion,
hamster and mouse sperm contain increasing amounts of protamine 2 (14%, 34% and
67% respectively). Attempts were also made to obtain data for human sperm (~50%
protamine 2), but the nuclei proved to be too thick for quantitative analysis.
Using this method, DNA and protein maps were obtained for the sperm nuclei of
all five species (Zhang et al., 1996). In each case, as shown in Fig. 4 for hamster, the
DNA was found to be confined to the nucleus, as expected. Occasionally the
midpiece of the tail appeared in the DNA images, suggesting the analysis picks up the
small amount of mitochondrial DNA located in this region of the tail. Protein maps
indicate the protein is distributed fairly uniformly throughout the majority of the
head. The extra protein present in the acrosome is also apparent as additional material
surrounding the anterior end of the nucleus. Quantitative analyses were performed on
sperm nuclei from each species treated to remove the acrosome and tails, leaving only
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R. Balhorn et al.
the sperm chromatin. The results show that the mass of protamine in the sperm
nucleus, relative to DNA, is constant for all four species irrespective of the protamine
2 content. This has allowed us to discriminate between two possible scenarios for
DNA packing by protamine in mammals (Fig. 5). One possibility is that a protamine
1 molecule binds to each turn of DNA in each species, and those species that contain
protamine 2 have an increase in protamine above that found in bull sperm (a species
that contains only protamine 1). A second is that all the DNA is covered uniformly by
protamine, and when protamine 2 is present, it replaces protamine 1. The XANES
data show that the total protamine content of the sperm nucleus is constant in each
species. If the amount of protamine 2 used to package DNA increases, the amount of
protamine 1 decreases proportionately.
A
B
Fig. 4. XANES images were used to produce DNA (A) and protein (B) maps
of hamster sperm heads. This particular head was not attached to a tail.
Stallion
Hamster
Mouse
Stallion
Hamster
Mouse
Fig. 5. Possible scenarios for protamine 1 and protamine 2 binding to DNA. Each
protein is assumed to cover approximately one turn of DNA (rectangle represents
protein aligned along the DNA molecule). White rectangles are protamine 1,
black are protamine 2. The XANES studies indicate the total mass of protamine
bound to DNA in the species is constant, ruling out the possibility that protamine
2 is present in addition to protamine 1.
3.1 Analysis of Vacuoles in Human Sperm Chromatin
Qualitative analyses of DNA and protein maps of human sperm nuclei have provided
additional information about defects or inhomogeneities in the packing of DNA in
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
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human sperm. A structural feature often observed in human sperm chromatin are
small voids or vacuoles (Fig. 6). These vacuoles appear to be present at a higher
frequency in the sperm of infertile individuals. EM studies have suggested that these
regions are simply voids in the chromatin, regions of the nucleus that appear to be
empty. These vacuoles are visible in DNA maps of the human sperm nucleus
obtained by XANES imaging (Fig. 7), but they appear to be obscured or missing in
the protein maps of most nuclei (Fig. 8). This observation has provided the first
evidence that the vacuoles are not really empty, and suggests that while they do not
contain DNA, they do contain significant amounts of protein.
Fig. 6. The densely packed chromatin that fills the human sperm head occasionally contains
voids or vacuoles, as shown here by electron microscopy.
Fig. 7. DNA maps of human sperm nuclei
obtained by XANES imaging. These two
sperm heads have vacuoles (holes) that
do not contain DNA.
Fig. 8. Protein maps of human sperm
nuclei obtained by XANES imaging.
The holes that were visible in the DNA
maps of these same sperm heads do not
appear to be empty, but contain protein.
3.2 Biochemical Composition of the Equatorial Segment
In certain species, a structure called the equatorial segment becomes visible when the
acrosome is removed from the head (Allen et al., 1995). While the structure and
function of this component of the nucleus remain a mystery, it is known to be the first
part of the nucleus that comes in contact with the egg upon fertilization (Bedford et
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R. Balhorn et al.
al., 1979). The equatorial segment shows up clearly in AFM images of bull sperm
heads (Fig. 9) as a triangular belt wrapped around the nucleus. This structure is not
visible in DNA maps of sperm chromatin (Fig. 10). But it stands out clearly in protein
maps of the sperm heads obtained by XANES imaging (Fig. 11), providing evidence
that this structure contains predominantly protein.
Equatorial
Segment
Fig. 9. AFM image of a bull sperm head with the acrosome disrupted. The
equatorial segment appears as a triangular belt wrapped around the nucleus.
Fig. 10. DNA maps of bull sperm heads obtained by XANES imaging.
The equatorial segment is not visible in these images.
Fig. 11. Protein maps of bull sperm heads obtained by XANES imaging.
The equatorial segment is clearly visible in these images.
Scanning transmission X-ray microscopy images of the sperm chromatin stained with
a maleimide derivative of nanogold (Fig. 12) show the perimeter of the equatorial
segment is stained intensely, at a level that is well above the background staining
achieved for the rest of the nucleus. This suggests that at least the edges of the equa-
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
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torial segment are extremely rich in cysteine. Cysteine is the only amino acid present
in proteins that reacts with maleimide under the conditions used to prepare the nuclei
for analysis.
Fig. 12. Scanning transmission X-ray microscopy images of two
amembraneous bull sperm nuclei stained with a maleimide derivative of
nanogold. The edges of the equatorial segment are stained more densely
than the rest of the nucleus, indicating that the equatorial segment may
contain cysteine rich proteins.
3.3 Perinuclear Theca
Both EM and biochemical studies have indicated that a thin layer of proteinaceous
material covers the surface of sperm chromatin, lying immediately underneath the
plasma membrane (Longo and Cook, 1991; Bellve, 1992; Oko and Maravei, 1994).
A
B
Fig. 13. XANES images of
bull sperm heads were used
to plot the protein to DNA
ratio of the head. A. Ratio
for an intact head showing
the acrosome and a protein
rich ring around the head.
B. Bull sperm head treated
with the detergent mixed
alkyltrimethyl ammonium
bromide, which removes the
acrosome and perinuclear
theca, a protein rich layer
surrounding the chromatin.
The function of this material, referred to as the perinuclear theca, is not known.
But it appears to be present in all mammalian sperm. Treatments of the sperm heads
with certain detergents, such as mixed alkyltrimethylammonium bromide (MTAB), in
the presence of a reducing agent dissolve the membranes that surround the chromatin
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R. Balhorn et al.
as well as the proteins that make up the perinuclear theca (Balhorn et al., 1977). Two
dimensional plots of the XANES data obtained for bull sperm, as the ratio of protein
to DNA (Fig.13), show the presence of a very protein rich, DNA deficient layer
surrounding the chromatin (Zhang et al., 1996). This layer, which is not present in
MTAB treated nuclei, is too wide (~200nm) to be the plasma and nuclear membranes
and appears to be the perinuclear theca.
4 Uniformity of Chromatin Organization
Because the mammalian sperm nucleus is not much more than a micron thick, the
transmission of X-rays through the densely packed DNA-protein complex that makes
up sperm chromatin can be used to map the uniformity of chromatin condensation
throughout the entire nucleus without having to examine individual thin sections of
the head as is required by EM. This allows the investigator to examine large numbers
of individual sperm cells and obtain information on chromatin organization in each
cell in a relatively short period of time. We have used this capability to examine how
alterations in protamine synthesis in transgenic mice affect the uniformity of DNA
compaction inside the maturing sperm head. The method has also been used to
examine the chromatin of the marsupial mouse Sminthopsis crassicaudata and
confirm the existence of two different regions inside the nucleus that appear to differ
in the nature of their organization.
4.1 Early Expression of the Protamine 1 Gene in Mice
The final stage of DNA compaction in differentiating spermatids occurs when the
histones and transition proteins bound to DNA in spermatid chromatin are displaced
by the two arginine and cysteine rich protamines, protamine 1 and protamine 2. The
synthesis and deposition of these two protamines onto DNA occurs during step 11 in
mouse spermatids, approximately the same time the nucleus begins to develop its
characteristic hook-like shape (Balhorn et al., 1984). Protamine deposition onto DNA
and chromatin compaction proceeds in a specific, highly ordered fashion, being
initiated at the apical end of the sperm nucleus and progressing inward and toward the
implantation fossa. Once complete, the chromatin is so tightly packed that the
individual DNA molecules are separated by only 5-7Å (Hud et al., 1994).
As part of a study designed to examine the DNA sequence domains that control
the timing of expression of the protamine 1 gene, several lines of transgenic mice
were produced by Lee et al (1995) that express the protamine 1 gene beginning in
step 7 spermatids, several days earlier than normal. Sperm produced by two of these
lines were examined by X-ray microscopy using the XM-1 microscope at the
Advanced Light Source, Lawrence Berkeley to determine if early expression of the
protamine 1 gene disrupts the process and the uniformity of chromatin compaction
that normally occurs in the mouse sperm nucleus (Fig. 14 and 15).
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
Fig. 14. X-ray microscopy images of sperm produced by a line of transgenic mice
(Line 6) that express the protamine 1 gene beginning in step 7 spermatids, several days
earlier than normal. These images show the chromatin is not uniformly dense and the
edges of the nuclei appear to be folded, taking on the appearance of a flower.
Fig. 15. X-ray microscopy images of sperm produced by a second line of
transgenic mice (Line 13) that also express the protamine 1 gene beginning
in step 7 spermatids. The chromatin is not uniformly dense in these nuclei
either, and the edges of these nuclei also appear to be folded.
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R. Balhorn et al.
While heterozygotes from both of these transgenic lines appear to be fertile, X-ray
microscopy of the sperm heads show that the majority of the sperm produced by these
animals (Lines 6 and 13) exhibit dramatic differences in the uniformity of DNA
compaction and sperm head morphology. In contrast to the sperm heads obtained
from control mice (Fig. 16), the head shapes of sperm produced by both Line 6 and
Line 13 animals are grossly distorted. In most cases, the chromatin in the transgenic
lines appear to be convoluted and folded, and the head appears to be shaped like a
flower (Fig. 14 and 15). Within the nucleus, dramatic differences are also observed in
the density of chromatin packing. These results suggest that the early synthesis and
deposition of protamine 1 onto DNA has a dramatic effect both the shaping of the
sperm head and the pattern of chromatin condensation. Other than the general flowerlike shape of the nucleus and apparent folding observed in thinner regions of most
nuclei, the specific shape and pattern of condensation appeared to be different for
each nucleus.
Fig. 16. X-ray microscopy images of sperm produced control mice that
express the protamine 1 gene at the proper time (step 11).
4.2 Co-expression of Mouse and Chicken Protamine Genes
In an effort to examine how the synthesis of abnormal protamines in transgenic mice
might compete for binding to DNA and affect the process of DNA condensation,
sperm maturation, and male fertility, Rhim et al. (1995) generated several lines of
transgenic mice expressing the chicken protamine gene in addition to the normal
mouse protamine 1 and protamine 2 genes. The chicken protamine molecule was chosen because it is nearly twice as large as mouse protamine 1 and because it does not
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
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contain any cysteine residues, the amino acids that form the disulfide bonds that
crosslink neighboring protamine molecules together during the final stages of sperm
maturation in mammals. Biochemical and immunological studies of the sperm
produced by transgenic animals expressing the chicken protamine gene revealed that
the chicken protamine was incorporated into sperm chromatin. Staining of sperm
nuclei with antibodies to the chicken protein suggested that every sperm cell contains
a detectable amount of chicken protamine. Preliminary EM studies also indicated that
the chromatin in a number of mature sperm contained regions of the chromatin that
are less condensed than normal. This suggested that the DNA complexed with the
chicken protamine was not packed as tightly as the DNA packaged by the mouse
protamines. Based on the combination of immunological and EM data, the
investigators concluded that all sperm produced by the transgenic males contained
regions of chromatin that are not properly packaged. And yet at least a subpopulation
of the sperm produced by these transgenic males were fully functional (the males
were fertile).
The extent of chromatin condensation observed in certain regions of the sperm
head by EM was significantly less than normal, and our previous studies with mouse
A
B
C
D
Fig. 17. X-ray images of sperm produced by transgenic mice expressing both
the mouse and chicken protamine genes. A small percentage of the sperm heads
contain less densely compacted regions of chromatin as shown in A. Electron
microscope images of similar sperm show these regions to have less densely
packed chromatin similar to that found in chicken sperm. B-D. The majority of
the sperm appear to exhibit normal patterns of chromatin condensation.
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R. Balhorn et al.
sperm indicated that sperm containing these regions could be easily detected by X-ray
microscopy without having to resort to sectioning and analyzing multiple sections
through each nucleus. Using the transmission X-ray microscope at the Advanced
Light Source, Lawrence Berkeley Laboratory, we could also examine relatively large
numbers of intact fully hydrated sperm to determine what percentage of sperm in the
population actually contained these “pockets” of less condensed chromatin. Although
the analysis of sperm from these transgenic animals is not yet complete, the
preliminary results suggest that the majority of the sperm produced by transgenic
males expressing the chicken protamine gene contain normally condensed chromatin.
Only a very small percentage of the sperm (Fig. 17A) appear to contain pockets of
lesser condensed chromatin similar to those observed by EM.
4.3 Heterogeneity of Chromatin Organization inside the Nucleus
of Marsupial Mouse Sperm
The normal fertile sperm produced by most mammalian species contain chromatin
that is, for the most part, uniformly condensed throughout the nucleus. Alterations in
this uniformity usually signal that the process of DNA repackaging that occurs during
spermatid maturation is defective. Even in human sperm, where as much as 10-15%
of the DNA remains packaged by histones (Gatewood et al., 1987), EM analyses of
sections through the nucleus of a normal sperm cell show it to be uniform in
compaction.
A
B
Fig. 18. Sperm chromatin organization in heads of the marsupial mouse
Sminthopsis crassicaudata. A. Transmission electron microscopy (TEM)
image of stained sections of the nucleus showing the two different types of
chromatin organization in regions C1 and C2. B. Transmission X-ray
microscopy images suggest that the unusual chord-like organization in
region C1 may be real, and not an artifact caused by tissue dehydration and
imbedding for TEM.
Transmission EM studies performed by Breed et al. (1994) have suggested that
the marsupial mouse Sminthopsis crassicaudata may be an exception to the rule.
Sections of the nucleus stained with uranyl nitrate and lead citrate revealed what
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
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appeared to be two different types of chromatin. One region located at the apical end
of the nucleus under the acrosome contains chromatin that appears to be more
electron dense that the remainder of the chromatin, which is more granular and
appears less condensed. While both regions have been shown to contain DNA by
their staining with fluorescent DNA binding dyes (Soon and Breed, 1996), it has not
been possible to confirm that the two regions of chromatin are condensed to different
degrees by EM. These regions can only be observed after staining. Consequently, the
observed differences might be attributed to biochemical differences in the chromatin
that affect their intensity of staining with uranyl nitrate and lead citrate.
In an effort to attempt to confirm the existence of two distinct regions of
chromatin in Sminthopsis sperm that differ in their condensation state, transmission
X-ray microscopy images were obtained of air-dried sperm using XM-1 at the
Advanced Light Source. While the results are very preliminary, and only a few nuclei
have been examined to date, the images (Fig. 18) do suggest that two different types
of chromatin are actually present in the sperm of these mice. Additional experiments
will be conducted to confirm this result by imaging cells in fluid and at the oxygen
edge. If the crevices in the apical chromatin actually exist, the additional water
present should help make the crevices stand out when imaged at the oxygen edge.
5 Future Applications of X-Ray Microscopy to Sperm
Because some of the results we have presented are preliminary, we must focus our
initial efforts on completing the studies we have just described. However, the
intriguing successes we have had in combining x-ray microscopy with XANES and
applying it to the analysis of individual sperm cells indicates this technique may
prove to be extremely useful for examining the content and distribution of particular
proteins within the sperm nucleus. Consequently, we hope to focus a significant
portion of our future efforts on the analysis of a variety of proteins in mammalian
sperm, including the distribution of protamine 2 precursors in mouse spermatids, the
localization of histones in human and marsupial mouse sperm, and the co-localization
of chicken protamine and the lesser condensed chromatin domain in the sperm of
transgenic mice. Our ultimate goal will be to apply the various X-ray microscopy
methods we have described to the analysis of sperm from infertile men. The purpose
of these studies will be to determine if these males produce a population of normal
sperm that can be identified and distinguished from defective sperm based on 2D
hydration maps, histone and protamine 2 precursor contents and distributions, and the
extent of vacuolization. Such studies will help us identify the physical or biochemical
causes for certain types of male infertility, as well as provide new information that
can be used to help clinicians select normal, fully functional sperm cells produced by
infertile individuals for use in vitro fertilization or related techniques of fertility
intervention.
6 Conclusions
Perhaps one of the most obvious conclusions to be drawn from this work is that the
sperm cell appears to be an ideal target for study by X-ray microscopy. Because its
DNA is so densely packed inside the nucleus, most other techniques cannot obtain
information about its organization without resorting to sectioning or decondensing the
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R. Balhorn et al.
sperm head prior to analysis. The ability to obtain structural or compositional
information on large numbers of individual cells also makes it possible to examine
variation within the population and obtain reasonable statistical data. The availability
of fluid cells for obtaining images in water and the development of cryo-techniques
will allow us to investigate structure in its fully hydrated state, eventually under
conditions that minimize X-ray damage.
The examples we have described clearly demonstrate that X-ray microscopy can
be used to obtain new information about biological structure without having to push
the resolution beyond its current limit. In certain cases, data obtained by X-ray
microscopy may need to be combined with data obtained by other techniques to
provide the results we need. It is also clear that the biological systems we study
should be picked carefully so they can actually provide new structural information. In
the early stages of X-ray microscope development, which we have experienced in the
last few years, this has not been as important. Groups needed objects to study that
had been well characterized by light and electron microscopy so they could compare
the quality and resolution of their X-ray images with the state-of-the-art offered by
other methods. But if future projects using X-ray imaging or analysis are to be
funded, we must direct our energies toward studies that put more emphasis on the
attainment of new information about biological structure, not only microscope
development. It is not necessary, however, that we identify and pose questions that
only X-ray microscopy can answer. In biology, as in the other sciences, it is critical
that any important finding be confirmed using more than one technique. Two of the
studies we have just described are good examples. The results we’ve obtained on
sperm chromatin hydration, as determined initially by X-ray microscopy, were later
confirmed using a very different approach and technique, atomic force microscopy.
Our determination of the protamine and DNA contents of sperm chromatin from
different species, and the observation that the mass ratio of protamine to DNA is
constant irrespective of the cell’s protamine 2 content, was achieved both by XANES
and particle induced X-ray emission spectroscopy (PIXE). In this latter case, the two
techniques provided corroborating as well as complementary information; XANES
identified the total protein content of the nucleus, while PIXE provided information
about the protamine 1 and protamine 2 content of sperm chromatin (Bench et al.,
1996).
Acknowledgments
We thank all the unnamed individuals that helped make these studies possible, either
by providing materials for analysis, assistance in sample preparation, or various other
types of support. This work was supported by the United States Department of
Energy, Office of Basic Energy Sciences and the Office of Health and Environmental
Research under contracts W-7405-ENG-48, FG02-89ER60858, and DE-AC 0376SF00098 and the National Science Foundation grant BIR-9316594.
Applications of X-Ray Microscopy to the Analysis of Sperm Chromatin
II - 45
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