BIOLOGY OF REPRODUCTION 56, 42-49 (1997)
Atomic Force Microscope Analysis of Chromatin Volumes in Human Sperm with
Head-Shape Abnormalities'
J.D. Lee IV,3 M.J. Allen,4 and R. Balhorn2,3
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550
Digital Instruments,4 Santa Barbara, California 93103
ABSTRACT
sperm with abnormal chromosome complements were not
correlated. This study could not, however, rule out the possibility that an individual class of abnormal head-shape
morphology might be caused by alterations in chromosome
or chromatin content. Alterations in the relative proportion
of DNA packaged by protamines and histones in the sperm
head might also affect the shape of the nucleus, since nucleosomal organized DNA occupies much more volume
than does protamine-compacted DNA [12]. It is not generally believed, however, that either of these factors dictates
or has a direct impact on the shaping of the human sperm
nucleus. Shaping begins well before protamine synthesis
and the final stage of chromatin compaction are initiated.
Cytoskeletal or intra-nuclear factors, such as the manchette,
perinuclear theca, and nuclear matrix, appear to be more
likely candidate regulators of nuclear morphogenesis (see
review by Meistrich [13]).
High-resolution scanning probe methods such as atomic
force microscopy have been used to image and obtain information about the structure of a variety of biological samples, ranging from intact cells [14, 15] to individual DNA
molecules [16-18]. Sperm cells, nuclei, and chromatin particles have been demonstrated to be excellent subjects for
imaging with the atomic force microscope (AFM) [19-22]
because of their rigidity and relatively small size. In this
study, we used the AFM to obtain volume measurements
for amembranous air-dried human sperm nuclei representing eight of the ten classes of head-shape morphology. The
result, which revealed that the volume of the nucleus is
constant and independent of the head shape, was unexpected and has been interpreted to indicate that neither the
nature of chromatin organization nor the extent of chromatin compaction differs dramatically in sperm of the eight
head-shape classes examined.
We used the atomic force microscope to perform volume
measurements on individual human sperm nuclei under ambient
conditions. Data obtained for normal sperm and for sperm from
seven of the nine classes of head-shape abnormality revealed
that the nuclear volumes were essentially identical for sperm in
each headshape class, even though the projected areas and
shapes of the nuclei have were shown to vary widely. These
results indicate that the abnormal sperm head morphologies
found at a rate of 25-40% in fertile males are not caused by
factors that affect the volume of sperm chromatin, such as the
DNA content of the sperm nucleus, differences in chromatin
organization, or the extent of DNA compaction.
INTRODUCTION
Human sperm cells, as a population, have been shown
to exhibit a wide range of head shapes [1, 2]. In studies of
normal human males, nine different classes of abnormal
head morphology have been identified by Moruzzi et al.
[2]. These studies have shown that the percentage of sperm
with abnormally shaped heads present in the semen of normal males is generally high (25-40%) and variable among
different individuals. Longitudinal studies performed using
semen samples obtained from a number of different individuals have also demonstrated that each individual has a
unique "pattern" of head-shape abnormalities in which certain classes of head shape are more predominant in the
semen [3], and this pattern does not change significantly
with time. If the male is exposed to toxic chemicals or other
environmental insults, however, the proportion of sperm
with these abnormalities changes dramatically in semen [46]. In addition, infertile males often exhibit high percentages of abnormal sperm [2, 3].
The factors that cause these observed differences in human sperm head morphology are unknown. Attempts have
been made to compare the DNA contents of sperm obtained
from normal and infertile individuals with different percentages of head-shape abnormalities, but these studies
have been inconclusive [7-10]. One hypothesis is that the
shape of the head might be affected to some extent by the
volume of material that is packed inside it. Since DNA and
the histone and protamine proteins that package it as chromatin constitute the bulk of the material packed inside the
head, differences in the DNA content of sperm resulting
from various abnormal meiotic events might lead to distortions in head shape caused by a smaller or larger volume
occupied by the chromatin. A study performed by Martin
and Rademaker [11] has provided evidence that the percentage of abnormal sperm and the percentage of total
MATERIALS AND METHODS
Isolation of Amembranous Nuclei
Human semen samples were obtained from local volunteers and stored frozen at -200 C until they were used to
prepare nuclei. Aliquots (100 il) of semen were diluted in
4 ml 0.01 M Tris-HCI buffer (pH 8), 0.15 M sodium chloride (Tris-saline) and centrifuged at 4000 x g to pellet the
cells. The sperm were washed in Tris-saline a second time,
centrifuged, and then resuspended in 0.05 M Tris-HCI buffer (pH 8), 10 mM dithiothreitol (DTT), and 1% mixed alkyltrimethyl ammonium bromide (MTAB) to remove the
tails, acrosome, and membranes, as described previously
for the mouse [23]. After setting on ice for I h, the amembranous sperm nuclei were centrifuged at 4000
g for 3
min. The pellet of nuclei was washed in Tris-saline to remove residual detergent, centrifuged again (4000
g, 3
min), and resuspended in 2 ml Tris-saline by brief sonication.
To prepare samples for imaging with the AFM, a single
Accepted August 15, 1996.
Received January 31, 1996.
'Financial support: Office of Health and Environmental Research, Department of Energy, Contract No. W-7405-ENG-48.
'Correspondence. FAX: (510) 422 2282; e-mail: [email protected]
42
AFM ANALYSIS OF ABNORMAL HUMAN SPERM
43
FIG. 1. Technique used to determine the
volume of the sperm nucleus. The process
for identifying the masks and identifying
the points to be used in calculating the
volume is described in detail in the Materials and Methods section. A) Example of
actual AFM data obtained from a typical
image displayed in gray scale (enhanced
for easier viewing of background debris).
B) Binary mask of the nuclear data. C) Binary mask of the background data. D) Final data used to calculate the volume of
the sperm nucleus.
drop of the sperm nuclei suspension was placed on an ethanol-cleaned glass coverslip (12-mm diameter, Fisher #12545-80; Fisher Scientific Co., Pittsburgh, PA), and the nuclei were allowed to settle onto the glass for 15 min. The
fluid was "flicked" off the surface, and the coverslip was
immediately rinsed in distilled water to remove residual salt
and buffer. The coverslips were then allowed to dry overnight at ambient humidity.
Imaging Sperm Nuclei with the AFM
Topographical images of the sperm nuclei were obtained
in a contact mode using a Nanoscope II AFM (Digital Instruments, Santa Barbara, CA) equipped with a "J" scanner. The AFM acquires topographical information of a sample by raster scanning the sample under a sharp contact
probe. The probe is attached to a cantilever that deflects in
the direction of the z-axis as the probe is drawn over the
surface of the sample. As the scanning probe (and the cantilever on which the probe is mounted) is deflected by
changes in sample thickness (z height), the AFM adjusts
the position of the sample to maintain a minimal set point
deflection of the cantilever. These adjustments are recorded
by the computer, and they are used to generate a highresolution topographical map of each nucleus.
The silicon nitride probes used in this study have a pyramidal tip with a 5-Rpm-square base and an opening angle
of 70 ° . This tip is attached to a 200-ptm-long triangular
cantilever with a spring constant of 0.12 N/m. The cantilevers are attached to a substrate (-2 x 3 x 1 mm), which
is mounted in the AFM at a 10° angle (orientation shown
in Fig. 4B).
Before the nuclei were imaged, the AFM scanner (initially calibrated by the manufacturer) was recalibrated using
various carbon, gold, and silicon gratings, with periods
ranging from 0.463 to 10 microns. Deviations between the
actual and AFM-measured distances on the gratings indicated that the maximum calibration error in calculated volume measurements could be + 5%. This was reduced significantly (to < 3%) by keeping the AFM scanner offset
less than 15 pxm from the neutral position (0,0 point).
Analysis of Data and Calculation of Nuclear Volumes
The topographical images of individual sperm nuclei obtained in this study each contain 160 000 data points. These
data are distributed in a square array over an image window
measuring 8 pxm on a side for the majority of the images.
The data in each image were leveled (plane fit by the Nanoscope II software using 0° polynomials) by choosing a
vertical and horizontal line that did not intersect data points
comprising the nucleus. Height data from the image that
fell on the chosen lines were used to calculate slopes of the
background image plane. The image was then leveled by
setting the background slopes to zero (Fig. 1A). Data points
corresponding to the topography of each nucleus were isolated from background (the substrate plane) by forming a
top profile binary mask of the nuclear data (Fig. B). These
masks were prepared using basic image-processing algorithms such as z-thresholding and small object removal.
44
LEE ET AL.
FIG. 2. A) Top-view AFM images of nuclei representing each of the head-shape morphology classes analyzed in this study. The three-dimensional
image in the inset shows the data obtained for a normal sperm nucleus as an example.
This was possible because the nuclei were significantly
thicker and had larger top profile areas than any of the
background debris. The mask was subsequently overlaid on
the original image and used to extract those data points that
comprised the nucleus, excluding the background (substrate
and contaminants). The area of the image window divided
by the total number of topographical data points (160 000)
was used to obtain the area in the x-y plane represented by
each data point. The volume for each data point was obtained by subtracting the average background from the z
component of each data point and multiplying it by the data
point area. The average background level for each image
was calculated by forming a binary ring mask around the
top profile mask for each nucleus (Fig. C). Data points
from the original image that fell within this ring were used
to calculate the average background level. The volume of
the nucleus was then calculated by summing up the volumes of all data points that lay within the nuclear mask
(Fig. D). SCIL Image, the image-processing software
used in this study, was developed by the Computer Systems
Group of the University of Amsterdam.
RESULTS
Our classification of sperm nuclei as normal or abnormal
in this study was based on the head-shape classification
scheme of Moruzzi et al. [2]. This scheme relies only on
the shape (top profile) of the sperm nucleus and does not
AFM ANALYSIS OF ABNORMAL HUMAN SPERM
45
FIG. 2. B)Three-dimensional images of nuclei representing each class of abnormal head-shape morphology shown in A.
require the presence of an acrosome or other information
derived from the intact sperm. Images of air-dried MTABtreated sperm nuclei, rather than intact dried or hydrated
sperm, were used for volume measurements to avoid con-
tributions to volume by the acrosome and other surface
structures. In addition, sperm heads do not bind as tightly
to the substrate in fluid, and the tip often dislodges the
nucleus during imaging. This is avoided by imaging in air.
46
LEE ET AL.
9
TABLE 1. Volumes of human amembranous sperm nuclei determined by
ATM.
8-
E
c
E
o
Heat morphology
Normal
Narrow at base
Narrow
Pear
Small
Irregular
Round
Decondensed
7.
634)
21-
A
V
2
4
6
Scan
Number
8
12
10
8
;
7-
6-
E
a-
,
-
C
._
oE
4-
0
3-
E
2-
Working Range
1-
B
0
....
6
-4
-2
I
0
.
.
.·
..
2
4
6
NanoNewtons
FIG. 3. A) Volume measurements obtained from successive scans of a
single human sperm nucleus. The amembranous nucleus was prepared
and imaged in air as described in the Materials and Methods. B) Volumes
obtained for an amembranous human sperm nucleus imaged with the
AFM using varying cantilever deflections that contributed -4 to 7 nN to
the overall probe-sample force. Negative forces indicate that the cantilever is deflected such that it is trying to pull the probe away from the
sample, while the dominant meniscus force keeps the probe in contact
with the sample.
Initially, images of intact sperm cells were also found to be
unacceptable for nuclear volume analysis because the additional height of the intact sperm head and steepness of
its edges produced large amounts of probe-induced error
(tip effect) in the images. This error could be minimized
by removing the acrosome and membranes with MTAB,
decreasing the edge slope to satisfactory levels. Previous
analyses of amembranous nuclei prepared by treatment with
MTAB have shown that the removal of the tail, acrosome,
and membranes does not change the morphology of the
head significantly (personal communication with A.J. Wyrobek, Lawrence Livermore National Laboratory) when
viewed by light microscopy. Biochemical analyses performed in our lab have also shown that neither DNA, his-
No. cells
32
36
35
10
18
3
2
20
Volume
5.02
5.02
5.15
5.09
5.30
4.93
5.39
4.68
+ 0.37
+ 0.45
+ 0.47
+ 0.55
+ 0.49
+
0.27
+ 0.34
+
0.53
tones, or protamine are extracted from the nuclei by MTAB
treatment (unpublished data).
While the AFM provides very detailed information
about the shape of the sperm nucleus in three dimensions
(Fig. 2A, lower right insert, and Fig. 2B), only top profile
views (looking straight down the z-axis) of the AFM data
were used to categorize the nuclei into each head-shape
abnormality class. Images of the sperm nuclei were examined and their morphologies identified independently by
two individuals. Representative examples of nuclei from
each class are shown in Figure 2A. While the same categories of normal and abnormal head shapes as assessed by
light microscopy and described by Moruzzi et al. [2] were
used to identify particular nuclei in this study, the categorization of each nucleus into a particular morphology class
was not always straightforward. For example, images of
some nuclei fell into intermediate "gray" areas between
classes. Data from these nuclei and any others that were
not identified to be in the same morphology class by both
examiners were not used in this study.
Partially decondensed or incompletely condensed (we
cannot distinguish between these two possibilities) nuclei
were frequently encountered on coverslips and imaged (Fig.
2A). Nuclei in this class appeared as though they had lost
(or not gained during maturation) their rigidity and spread
onto the surface, and their top profile areas appeared somewhat larger than the nuclei in other classes. These nuclei
may correspond to the ghost category of Moruzzi et al. [2],
or they may simply represent nuclei in the large morphology category that never fully condensed during maturation
or decondensed slightly as a result of the DTT treatment.
Volumes were measured for 148 nuclei distributed
among 8 of the ten classes of head-shape morphology (Table 1). Measured volumes of nuclei from all the morphology classes were essentially identical. The average volume
measured for all 148 nuclei was 5.07 jtm 3 with a standard
3
deviation of 0.47 ~pm
.
To determine whether the probe might remove chromatin
material from the nucleus during imaging and change its
volume, a single nucleus was repeatedly imaged (eleven
times), and the volume of the nucleus was determined from
data collected in each image. The results (Fig. 3A) show
that no measurable amount of material is removed from the
nucleus during the course of imaging. The data collected
from this experiment did, however, reveal a system-associated 1% random variation in the volume measurements
obtained for the same nucleus.
Deviations in AFM cantilever deflection (a small component of the overall force applied to the sample by the
probe during imaging) were determined to have no significant effect on measured nuclear volumes (Fig. 3B). Cantilever deflections used during imaging normally result in
a contribution of 0-3.5 nN to the overall force applied to
AFM ANALYSIS OF ABNORMAL HUMAN SPERM
the sample by the AFM probe. Electrostatic and/or surface
tension forces from the adsorbed gas layer were measured
to contribute a force varying between 0 and 100 nN to the
overall probe-sample force (calculated by extrapolating
curves from force calibration plots) during a typical scan
[24, 25]. For this reason, the cantilever deflection could not
be set to counteract the electrostatic and/or surface tension
forces; otherwise the probe would lose contact with the
sample partway into the image. Since the cantilever deflection contribution to the probe-sample force (0-3.5 nN) is
small compared to the electrostatic/surface tension contribution (0-100 nN), slight drift or changes in cantilever deflection (set point) do not significantly change the overall
probe-sample force. Multiple measurements revealed that
with each additional nN of force applied to the sample by
way of cantilever deflection, the volume of the nucleus was
not significantly reduced. A linear interpolation of the data
indicated a decrease in the cell volume measurement of less
than 0.05% per nN of additional force.
Other measurements were made to estimate the magnitude of their effect on the volumes determined for nuclei.
Because the nuclei were not always imaged at the same
data point resolution ("magnification"), it was necessary to
determine how the ratio of the nuclear top profile area
(NTPA):image area (IA) affected the calculated volumes.
This was determined by imaging a single nucleus multiple
times using different window sizes. It was found that within
the range of NTPA:IA used (20-30%), nuclear volume
measurements did not change more than the observed random system variation (1%).
The masking method of distinguishing nuclear data from
background data points proved stable to variations in the
z-threshold level used in making the binary nuclear mask.
Nuclear volume calculations were constant for masking
z-threshold levels ranging from 1% to 5% of maximum
nucleus height above background. The masking z-threshold
levels used in this study were well within this range with
a target value of 2.5%.
DISCUSSION
The AFM data obtained for amembranous human sperm
nuclei clearly show that the volume of the normal human
sperm nucleus and nuclei in seven of the abnormal morphology classes are essentially identical. This was not expected, particularly since the top profile areas for the different morphology classes vary so markedly. The most dramatic example involved the nuclei classified as "small."
Even though these nuclei have only half to three quarters
of the top profile area of the other nuclei (including normal)
[26, 27], the AFM images revealed that these nuclei contained the same volume of chromatin. The small nuclei
were simply much thicker than those in the other classes.
Because these measurements were performed on sperm
nuclei treated with the detergent MTAB, the acrosome,
membranes, and other structures that contribute to the structure of the sperm head were removed and only the sperm
chromatin remained. While this highly compacted chromatin retained the characteristic shape of the nucleus, AFM
measurements indicated that the isolated chromatin was less
constrained and was not as thick as when packed inside the
nucleus. Since the volume of normal and abnormal chromatin were indistinguishable, these results suggest that neither the DNA content of the nucleus nor differences in the
extent of chromatin compaction contribute to the production of human head-shape abnormalities. Subtle differences
47
FIG. 4. A) An example showing how tip effect is generated during the
imaging of a hypothetical nucleus. In this example, the AFM probe is
travelling from right to left, with the left side of the probe approaching
the object. In this case, the edge slope of the nucleus is greater than the
45° angle between the left side of the probe and the sample substrate
(horizontal). The tip of the probe loses contact with the surface of the
sample while the left side of the probe is in contact with the right side of
the object. During this time, the AFM records erroneous height data because the instrument assumes that only the very tip of the probe is in
contact with the sample. This is tip effect and is shown as a gray region
in the resulting AFM image. The very tip of the probe remains in contact
with the left side of the object because the 55° edge slope is less than
the 65° angle between the right side of the AFM probe and the horizontal.
In this example, there is no tip effect on the left side of the object. B)
Illustration showing the AFM tip orientation during data acquisition (not
to scale).
in nuclear volume produced by alterations in protein content or chromatin organization cannot be detected by this
method. However, the technique is sufficiently sensitive to
detect gross differences in chromatin compaction such as
those that might be present if the normal protamine displacement of histones did not occur during spermiogenesis.
Significant changes in volume would also be detected if
chromosome gains or losses contributed to the formation
of abnormal head shape. Aneuploid sperm, on the other
hand, would normally have DNA contents that differ from
normal sperm by only a few percent, well within the nucleus-to-nucleus variation observed in this study.
We would not expect a difference in chromosome number to be responsible for the production of a particular
head-shape abnormality since numerous studies [28-31]
have shown that the frequency of sperm with abnormal
chromosome complements (4-5% of total sperm contain a
numerical abnormality, while 0.04-0.43% of sperm are
48
LEE ET AL.
aneuploid for any particular chromosome) is significantly
lower than the frequency of abnormal sperm. However, we
were somewhat surprised by the lack of a difference in
chromatin volume since a number of different studies have
suggested that a significant population of human sperm exhibit differences in chromatin compaction or organization
as revealed by their sensitivity to decondensation by SDS
[32, 33] or heparin [34], stabilization by zinc [35], chromomycin A3 staining [36], and extent of chromatin condensation as determined by electron microscopy [37].
The application of the AFM to measuring the volume of
the sperm nucleus appears to work reliably. Similar measurements could not be made by conventional light or confocal microscopy because of their limited resolutions (the
dimensions of the sperm are only 4 x 8 x 0.5-1 ~Lm).
Volume measurements have been made by serial section
electron microscopy [38], but the effort required to analyze
the number of cells we measured here would be unreasonable. Repeated imaging of the same nucleus did not result
in the loss of any material. The volume measured for the
nucleus was also found to be independent of the cantilever
deflection contribution to the probe-sample force. This may
not, however, prove to be typical of all biological structures.
The DNA in sperm chromatin is known to be organized
inside the nucleus in a highly compact state, and the condensed structure of sperm chromatin would be expected to
be resistant to compression by the AFM probe.
Other factors were also examined to determine whether
they contributed significant error to the nuclear volume
measurements. The largest error resulted from contributions
of the AFM probe to the image, inflating the measured
nuclear volume [39]. This error, often referred to as tip
effect, is defined as data that are acquired by the AFM
while one of the sides or edges of the pyramidal probe
(rather than the tip of the probe) is in contact with the
sample (Fig. 4A). This is a problem because it causes the
AFM to record erroneously high z-values (height) for locations on the sample adjacent to features with steep edges.
Because of the geometry of the AFM tip and its orientation during data acquisition, the maximum measurable
edge slope of a sample depends on which face of the AFM
probe is in closest proximity to the sample edge to be measured. This edge slope resolution is 0-45 °, 0-55 ° , and 065 ° for the front, side, and back faces of the probe, respectively (Fig. 4B). Attempts to measure sample edge slopes
that exceed these ranges will result in tip effect. Tip effect
was rarely observed along edges of the nuclei where the
back face of the AFM probe dictated the maximum measurable edge slope (65°). This indicates that the edge slope
of nuclei must normally be less than 65 °. In contrast, edges
of nuclei measured where the front face of the AFM probe
dictated the maximum measurable edge slope (45 ° ) showed
tip effect more often (see hypothetical example in Fig. 4A),
suggesting that the average edge slope of the nucleus is
closer to 45 ° .
Errors in volume calculations caused by tip effect were
measured for eleven of the nuclei imaged in this study. To
be conservative, on those regions of the edge of the nucleus
that exhibited signs of tip effect, a 90° nucleus edge slope
was used in the error calculations, which gave higher estimates of error than the more realistic 65° edge slope. This
error appeared to be greatest for nuclei falling into the small
head-shape category. While these nuclei had much smaller
top profile areas than nuclei in other classes, they were
much thicker than the others. This increase in height coupled with the decrease in top profile area was responsible
for increasing the edge slopes, which in turn increased the
amount of tip effect. Estimates of tip effect revealed that
this effect could account for only an average of 6.8% (+
3.4%) of the measured nuclear volume. Realistically, the
average inflation in volume calculations caused by the tip
effect is approximately 3.4% (+ 1.7%) given the 65 ° maximum edge slope argument. Attempts to decrease the tip
effect were made by using AFM probes with higher aspect
ratios. Unfortunately, these attempts were unsuccessful because of additional problems that significantly decreased
image quality.
In addition to factors described earlier, volume measurements performed on nuclei are also influenced by humidity.
Increased humidities induce nuclear swelling and may also
increase probe-sample forces because of a thicker adsorbed
gas or liquid layer on the sample. Humidity was not strictly
controlled in this study, but all AFM measurements were
performed in an environmentally controlled laboratory
where the relative humidity was maintained near 40%.
ACKNOWLEDGMENTS
We thank M. Wagner, D. Ow, and L. Mascio for their computer support
and assistance, and Nick Hud for his ideas and encouragement. This work
was performed under the auspices of the U.S. Department of Energy by
the Lawrence Livermore National Laboratory under contract W-7405ENG-48.
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