BIOLOGY OF REPRODUCTION 49, 166-175 (1993)
Nuclear Status of Human Sperm Cells by Transmission Electron Microscopy and Image
Cytometry: Changes in Nuclear Shape and Chromatin Texture during Spermiogenesis
and Epididymal Transit
JACQUES AUGER 2 and JEAN-PIERRE DADOUNE 1
Groupe d'Etude de la Formation et de la Maturation du Gam&te male, UFR Biomddicale des Saints-Pdres
University Rene Descartes, Paris, France
ABSTRACT
Computer-assisted transmission electron microscopy (TEM) image cytometry was used to investigate changes in nuclear shape
and chromatin texture of human sperm during normal spermiogenesis and epididymal transit. Analysis was performed on a large
series of micrographs of longitudinal sections of nuclei from spermatids and epididymal caput and cauda sperm. Thirteen parameters characterizing nuclear shape and chromatin texture were measured. Quantitative data showed that from early spermiogenesis to the end of epididymal transit, a decrease in nuclear area and width occurred concomitantly with not only an increase in
chromatin condensation but also an increase in heterogeneity of the degree of condensation. The oriented spatial arrangement
of chromatin along the major nuclear axis as measured by gradient parameters indicated that in humans, condensation of chromatin begins in the posterior pole and proceeds apically; this is an important difference between humans and other mammalian
species. Most parameters did not differ substantially in sperm from testis and caput epididymis, but did change as the cells moved
from the caput to the cauda epididymis, indicating completion of nuclear maturation. Discriminant functions of basic parameters,
as followed by canonical transformation and cluster representation, automatically classified the nuclei in a sequence that was
found to concur with the biological maturation sequence during normal spermiogenesis and epididymal transit.
INTRODUCTION
Dramatic biochemical and ultrastructural changes in nuclei occur as mammalian spermatids develop into mature
spermatozoa. Histones of spherically shaped nuclei possessing fine granular chromatin are replaced by more basic
proteins, the transition proteins, in elongating and condensing nuclei; and these basic nucleoproteins are then replaced by even more basic sperm-specific nucleoproteins,
the protamines, in mature spermatids with compact chromatin [1-3]. Nuclear maturation continues in the epididymis through an increase in formation of protamine disulfide bonds [4]. In the mouse and rat, spermatid nuclei
abruptly undergo developmental stages characterized by
increased resistance to disruption by various mechanical and
chemical agents, and these alterations are correlated with
changes in the basic nuclear proteins [5]. It has been suggested that the functional status of ejaculated sperm nuclei
could result from these complex structural and biochemical alterations. Thus, penetration through the oocyte vestments could be facilitated for elongated spermatozoa with
dramatically condensed chromatin [6]. Subsequently, the early
stages of embryogenesis might depend on nuclear proteins
and chromatin organization [7, 8]. Various reports in mammalian species support the hypothesis of a close relationship between nuclear maturity and fertility of ejaculated
sperm [9-11]. This relationship could depend on events that
occur during spermiogenesis and epididymal maturation.
In the present investigation, changes in nuclear shape
and chromatin texture of human sperm were analyzed
through use of a new quantitative approach combining
transmission electron microscopy (TEM) computer-assisted
image cytometry, and multivariate statistical analysis. Similar methods have proven useful in other cell types for
quantitative assessment of nuclear changes occurring during cell cycling [12, 13]. Size, shape, and chromatin texture
parameters were found to be salient indicators of stage-related nuclear transformations from early spermiogenesis to
the end of epididymal transit. Furthermore, a combination
of the most discriminant parameters, as followed by cluster
representation, automatically classified the nuclei in a sequence that was found to concur with the biological sequence of normal differentiation and maturation of human
sperm.
MATERIALS AND METHODS
Collection of Tissue and Cell Samples
Normal testicular and epididymal tissue samples were
obtained using two methods: 1) testicular biopsies from four
healthy patients aged 20-25 who were undergoing operations for hydrocoel, and 2) removal of testis and epididymis
from four men aged 25-40 with proven brain death. Epididymal spermatozoa were collected from caput and cauda
epididymis by cutting and mincing into fragments in Hanks'
solution.
Accepted March 1, 1993.
Received February 19, 1992.
'Correspondence: J-P. Dadoune, Laboratoire d'Histologie, UFR Biomedicale des
Saints-P&res, 45 Rue des Saints-P6res, 75270 Paris cedex 06, France. FAX: 42-86-8512
2
Present address: CECOS (Centre d'Etude et de Conservation du Sperme Humain), H6pital de Bicetre, 94270 Le Kremlin Bictre, France.
166
NUCLEAR CHANGES IN SPERMATIDS AND EPIDIDYMAL SPERM
167
FIG. 1. Negative prints (left corner inset) of early (step 2) (1), intermediate (steps 3/4) (2), and late (step 8) (3)nuclei of spermatids selected according
to the classification of Holstein and Roosen-ROnge, with related computer-generated three-dimensional representations of chromatin texture illustrating its
complex temporal evolution during spermiogenesis. The impression of relief is provided by a perspective projection of the image with artificial lighting
from the right and by the z dimension depicting grey-level value of the pixel composing the nuclear image (from black to white). This representation, in
contrast to two-dimensional images, gives a more incisive visual insight into modifications in the overall condensation process and distribution of the
different degrees of chromatin condensation within the nucleus concomitant to shrinking and flattening. The progression of chromatin condensation from
early to late spermiogenesis is illustrated by an increase in the relative distance between the plane of the nucleus outline and the plane of the higher
peaks (arrows), while evolution of chromatin distribution within the nucleus is illustrated by gradual modifications in relief. x4500.
Preparationfor (TEM)
Image Acquisition and Processing
All samples were fixed by immersion in 1% glutaraldehyde in 0.1 M collidine buffer, postfixed in osmic acid, and
embedded in Epon. Ultrathin sections (80 nm) were contrasted with uranyl acetate and lead citrate and observed
with a JEOL 120 CX electron microscope eol Ltd., Tokyo,
Japan).
The configuration of the image cytometric device and
the algorithm of the dedicated software have been previously reported [15]. Briefly, the image cytometric device
consisted of 1) a macroviewer equipped with a light box
(for transmission of the negative image of the nucleus), 2)
a I2S CCD video camera with a photo objective, 3) a Matrox
PIP 1024 digitized card (Electronics Systems Ltd., Dorval,
PQ, Canada), and 4) a Hexagone 386 AT host computer with
a 60-megabyte disk and a floppy disk unit. Prior to analysis,
each micrograph was centered and oriented in the field,
with the posterior region of the nucleus on top and its major axis orthogonal to the scanning lines (the major axis of
the nucleus was defined as the distance from the Golgi
structures to the diametrically opposite side in early steps,
and as the distance from the acrosomal apex to implantation fossa in later steps in epididymal sperm nuclei). The
image was digitized into 256 grey levels in a frame of 512
x 512 pixels at 0.032-lpm pixel size.
The objective description of nuclei was obtained by
computation of extractable information related to size, shape,
and chromatin texture. A pilot study using multiple regression (not shown) indicated a high level of correlation (>0.70)
between some of the 22 parameters available in the dedicated software. These parameters were excluded from subsequent data processing, and only the remaining 13 least
correlated parameters in the correlation matrix were used
in the study (Table 1). Geometric parameters were measured after segmentation of the nucleus outline and exclusion of the large chromatin vacuoles. Because of random
sectioning of nuclei, measurement of the minor axis of the
nucleus, the "width" parameter, did not correspond with
the true width but rather with an average between the width
Nuclei Selection
The nuclei of spermatids were selected on the basis of
ultrastructural features belonging to morphologically normal spermatids and spermatozoa. The spermatids were divided into eight steps according to the classification of Holstein and Roosen-Riinge [14]. Because of the difficulty in
distinguishing step 3 from step 4, these steps were considered together. To avoid bias in subsequent analysis, only
nuclei of spermatids and epididymal spermatozoa exhibiting visible anterio-posterior structures (acrosomal vesicle,
cap or granule, mature acrosome sliced longitudinally, neck
structures) were selected and photographed at a magnification of 7200x. For similar reasons, the analysis was conducted entirely from negative prints. Figure 1 presents three
typical aspects of nuclei on negative prints at steps 2, 4, and
8 with a corresponding computer-generated three-dimensional nuclear texture representation. This representation
(where the z dimension shows the various grey levels of
chromatin) illustrates the complexity of the chromatin texture by enabling sharper visual appreciation. The data set
consisted of a total of 450 negative prints of nuclei: 50 micrographs of spermatid nuclei at each of steps 1, 2, 3/4, 5,
6, 7, and 8, and 100 micrographs of sperm nuclei from epididymis-50 from caput and 50 from cauda epididymis.
168
AUGER AND DADOUNE
TABLE 1. List of the 13 nuclear parameters measured by TEM-image cytometry.
Grouping
Abbreviation
A) Size and shape
2
1. Area (m )
2. Form factor"
3. Length (m)
4. Width (m)b
B) Chromatin texture
5. Relative area within the nucleus occupied by large vacuoles (%)
Global level of condensation
C
6. Mean grey level (a.u.)
Distribution of the different degrees of chromatin condensation
7. Standard deviation of the mean grey level (a.u.)'
8. Skewness'
9. Kurtosis c
10. Run-length percentaged
11. Correlation (r-coefficient) grey level x length of sectiond
Organization of chromatin condensation along the major axis
12. Gradient of the mean grey level
13. Gradient of the grey-level variance
A
FF
L
W
LV
MGL
SDGL
SKGL
KUGL
RLP
R
GGL
GVGL
aThe form factor parameter is computed from the perimeter (P) and the area (A) of the nucleus as follows: FF = P2/
4HA. This value is 1 for a perfect circle, and it increases with the complexity of the outline.
bThe width parameter is an average between the true width and the thickness of the nucleus because the sperm were
not sectioned in a specific plane.
CParameters computed from the grey-level distribution of pixels composing the nuclear image.
dParameters derived from computation of the run-length matrix: see Materials and Methods and [16]. Briefly, RLP is
the ratio of the total number of run lengths within the nucleus to the area of the nucleus and R evaluates, for each
nucleus, the relationship between the grey level and the length of isodensity segments.
eSee Materials and Methods for the computation principle of these original parameters.
and the thickness of the nucleus. Measurement of the greylevel value of the pixels composing the nuclear image enabled subsequent calculation of parameters describing the
various attributes of chromatin texture. These parameters
were related to the overall level of condensation. They were
computed from the grey-level distribution and from the
measurement of run lengths, according to the run-length
section matrix method previously described [16]. Briefly,
sections were defined as segments perpendicular to the
major axis direction and composed of adjacent pixels with
identical grey-level values after the dynamics of the greylevel scale were reduced to avoid minimal local variations
in chromatin texture. In addition, in order to determine
whether there was a peculiar spatial arrangement of the
different degrees of chromatin condensation and distribution during spermiogenesis, two original parameters were
introduced. These two parameters, the gradient of mean
grey level and the gradient of the variance of the mean
level, were computed from the grey-level values in crosssections of equal width, orthogonal to the major axis of the
nucleus, versus the distance of the cross-sections to the base
of the nucleus.
StatisticalAnalysis
Univariate statistical analysis was performed using the
STAT2005 statistical software package (Alcatel, TITN, Grenoble, France). Comparison of mean values of parameters
made use of parametric or nonparametric tests according
to the distributions profile. Stepwise linear discriminant
analysis of nuclei populations was performed with the DIS-
CRI program (Alcatel, TITN). This software provided automatic selection of the most discriminative functions and the
percentage of good classification of nuclei populations. After canonical transformation following discriminant analysis
(program CLUSTER, Alcatel TITN) nuclei from the various
steps were projected into the canonical space composed of
the first two canonical variables (linear functions of parameters). Therefore, the classification of nuclei was done automatically by computer without the need for human intervention and subjective decisions. In addition, the program
computed and yielded, in the canonical parameter space,
the tolerance ellipse (95% interval around the mean nucleus type) and vectors of the major nuclear parameters.
The importance of their contribution to the linear combination of parameters was indicated by the length of their
orthogonal projection on the axis depending on their weight
(length) and direction.
RESULTS
Evolution of Nuclear Parametersduring Spermiogenesis
Variations in size and shape parameters according to
spermiogenesis chronology were found to objectively describe the gradual narrowing of the nucleus (Fig. 2): dimensional parameters (area, length, and width) decreased
from step 1 to step 8, while the nondimensional parameter,
FF, increased mainly during the intermediate steps (2-5).
Large chromatin vacuoles were measurable from step 6, and
from this step on, their relative area, LV, did not significantly vary (=1%). Changes in basic grey-level parameters
169
NUCLEAR CHANGES IN SPERMATIDS AND EPIDIDYMAL SPERM
E:.
1
2 34 5 6
7 8
1 2 3/4 5 6
7
8
I
1
2 3/4 5
6
7
8
1
2 3/4 5
6
7 8
FIG. 2. Evolution of size (area, A; length, L; and width, W) and shape (form factor, FF) of nuclear parameters
during spermiogenesis. Mean values with standard deviation are plotted against the steps selected and the dynamic
pattern of parameters is depicted by the midline joining the points of mean values. Significant differences between
two successive steps are denoted by one, two or three dots (p < 0.05, 0.01, and 0.001, respectively).
corresponded with the progressive condensation and narrowing of the nucleus (Fig. 3). The mean grey level, MGL,
increased regularly throughout spermiogenesis and epididymal maturation, but most dramatically during steps 2-6.
The standard deviation of the mean grey level, SDGL, followed a similar profile, indicating a concomitant increase
in heterogeneity in the degrees of chromatin condensation.
The parameter skewness, SKGL, measured the form (symmetry) of the nuclear grey-level histogram. The evolutive
profile of skewness from positive to negative values indicated some fine aspects of the condensation process. At steps
1 and 2, the grey-level histogram was skewed to the right;
but when nucleoli disappeared, at steps 3/4, symmetry was
restored (SKGL near zero). After the beginning of chromatin condensation, a proportion of the chromatin remained dispersed, leading to negative SKGL values. Figure
4 presents the evolution of gradient parameters and parameters calculated from the run-length sections matrix. The
grey-level gradient of chromatin condensation along the
longitudinal axis of the nucleus, GGL, became negative from
step 5 on, while a negative gradient of the grey-level variance, GVGL, was measured earlier, from steps 2 to 3/4.
Evolution of the gradient values during spermiogenesis indicated, for a majority of cells: 1) chromatin condensation
beginning in the posterior part of the nucleus and remaining prevalent in this region until the end of spermiogenesis
(GGL) and 2) a more heterogeneous distribution of the different degrees of chromatin condensation in the posterior
part of the nucleus from the early steps until the end of
spermiogenesis, with this heterogeneity increasing as compaction progressed (GVGL). The increasing values of runlength percentage, RLP, indicated increasing heterogeneity
in the degrees of chromatin condensation, concomitant with
the rise in condensation as spermiogenesis progressed.
However, the phenomenon reached a plateau in the later
steps. The correlation between grey level and length of sections measured by R changed from negative to positive at
the transition from early to intermediate steps. However, R
became statistically significant (positive correlation) only in
later steps.
170
AUGER AND DADOUNE
1
1 2 3/4 5
6 7 8
1
2 3/4 5 6 7 8
7 8
1
2 314 5
-
1
2 3/4 5
6
6 7 8
FIG. 3. Evolution of parameters computed from grey-level distribution of the different degrees of chromatin condensation (standard deviation of the mean grey level [SDGLI, skewness [SKGL] and kurtosis [KUGL] of the grey-level
distribution) during spermiogenesis. Mean values with standard deviation are plotted against the steps selected and
the dynamic pattern of parameters is depicted by the midline joining the points of mean values. Significant differences
between two successive steps are denoted by one, two or three dots (p < 0.05, 0.01, and 0.001, respectively).
Evolution of Nuclear Parametersbetween Testis, Caput,
and Cauda Epididymis
For most parameters, no significant differences were
measured between testis and caput epididymis, while significant differences were found between the caput and cauda
epididymis (Table 2).
DiscriminantAnalysis of Major Nuclear Parametersand
Automated Classification of Nuclei during Differentiation
and Maturation
The program DISCRI discriminated between the seven
steps of spermiogenesis with a good classification rate of
75% with only one parameter selected (SKGL was found to
be the most discriminant parameter) and of 83% with five
parameters selected (Table 3). With the program CLUSTER,
each nucleus was projected into the canonical plane generated by the two first canonical variables (Fig. 5). From
the position and shape of confidence ellipses in this representation, it can be inferred that 1) the nuclei were placed
TABLE 2. Comparison of mean values (SEM) of nuclear parameters
between testis (step 8), caput, and cauda epididymis.
Parameters*
A
FF
L
W
LV
MGL
SDGL
SKGL
KUGL
RLP
R
GGL
GVGL
Testis
4.05
1.76
3.77
1.72
(0.13)'
(0.04)'
a
(0.08)
(0.04)'
1.08 (0.35)
80 (2)"
29 (1)"
a
-0.60 (0.04)
'
2.05 (0.06)
18.7 (0.5)b
a
0.42 (0.01)
a
-1,8 (0.3)
d
-1.2 (0.1)
Cauda
Caput
4.20
1.61
3.67
1.71
d
(0.15)
c
(0.03)
(0 .07 )b
(0.04)d
1.06 (0 .2 8 )b
d
78 (2)
31 (1)d
-0.64 (0.0 7 )b
2.06 (0.06 )b
20.2 (0 .5 )bd
0.42 (0.0 1)b
-1.6 (0 .3 )b
-0.8 (0.1 )b
3.39
1.75
3.46
1.49
d
(0.14)
c
(0.05)
c
(0.09)
d
(0.04)
C
1.64 (0.30)
d
90 (2)
37 (1 )d
C
-0.62 (0.04)
C
2.06 (0.06)
d
26.5 (0.8)
0.41 (0.01)'
c
-2.2 (0.3)
-1.2 (0. 1)b
'See Table 1 for definition and unit of nuclear parameters.
"-dDifferent superscripts denote no significant difference from preceding
column; same superscripts denote significant difference from preceding
column at p < 0.05 (b), p < 0.01 (c), and p < 0.001 (d).
171
NUCLEAR CHANGES IN SPERMATIDS AND EPIDIDYMAL SPERM
2
GGL
1
2
1
0
-
-1
-
5
-2
1 2 3145 6 7 8
1 23145
6 7 8
2 3/4 5 6 7 8
1 2 3/4 5
6 7 8
-
-
1
FIG. 4. Evolution of parameters depicting the chromatin organization along the major axis of the nucleus (gradient of the mean level [GGL] and gradient of the variance of the mean grey level [discriminant GVGL]; see Materials
and Methods) and parameters computed from the run-length matrix (see Materials and Methods and [161) depicting
the distribution of the different degrees of chromatin condensation (correlation coefficient between grey level and run
length of section R and run-length percentage, RLP). Mean values with standard deviation are plotted against the
steps selected, and the dynamic pattern of parameters is depicted by the midline joining the points of mean values.
Significant differences between two successive steps are denoted by one, two, or three dots (p < 0.05, 0.01, and
0.001, respectively).
in a sequence concurrent with the spermiogenic process,
2) the decrease in size of ellipses from step 5 to step 8
accounted for a decrease in interindividual variations between nuclei of successive cell populations from the beginning of the process of compactness, and 3) the direction
of morphological changes from steps 1 to 2 was almost orthogonal to that observed from steps 6 to 8 and accounted
for major changes in the nuclear profile at intermediate steps
3/4 and 5. Vector projections in the canonical space indicated the weight and direction of the major nuclear parameters that best separated the different nuclear populations.
The program CLUSTER also provided a representation of
cell stages from the beginning of compactness (step 5) to
the end of epididymal transit (Fig. 6). The confidence ellipses of steps 6-8 and the caput epididymis population in
the canonical plane were not found to be significantly separated. Together, they were significantly distant from step
5 and the cauda epididymis population. Vector projection
in the canonical space indicated the weight and direction
of the major nuclear parameters in this sequence (SDGL,
RLP, W, and R).
DISCUSSION
The method used in the present study made it possible
to depict the metrics of shaping and to measure changes
TABLE 3. Ranking of the five best nuclear parameters that discriminate
the seven steps of the spermiogenesis and classification rate.
Rank
1
2
3
4
5
Parameter
selected
F-value
Number of
parameters
Classification
rate %)
SKGL
A
RLP
SDGL
MGL
31.6
14.0
4.3
5.8
2.5
1
2
3
4
5
75
75
79
78
83
172
AUGER AND DADOUNE
17.89
13.81
13.81
18.5Z
18.52
UJ
I
T
< 7.237
3.952
y
z
?7.237
4 3.952
.J
.6673
R .6673
0
9 -2.618
86 .
o
, -2.618
0
-9. 88
-12.47
-12.47
I
-I
! I
-8.018
-.1393
7.739
15.6
23.50
11.96
-. 78
3.888
11.68
19.56
27,.44
11.96
.878
3.888
CANONICAL VARIABLE 1
11.68
19.56
27.44
CANONICAL VARIABLE 1
FIG. 5. a) Representation of sperm nuclear differentiation sequence in the canonical parameter space generated by the first two canonical variables
(linear combinations of nuclear parameters). Ellipses delineating 95% confidence intervals around the center of gravity of each population are assigned to
a step in spermiogenesis (according to the Holstein and Roosen-Runge classification [14]), and the nuclear profile evolution (arrows) concurs with the
evolution in spermiogenesis. b) Vectors representing the weight and direction of the major nuclear parameters corresponding to the canonical parameter
space shown in (a). Note that these parameters (except A) measure chromatin texture.
in the chromatin pattern of human sperm cells during normal differentiation and maturation. Furthermore, by combining nuclear parameters through discriminant and canonical analysis it was possible to obtain an automated
12.26
I
17 X
-
,
9.297
9.297
1
6.332
6.332
1
m 3.367
I
classification of selected nucleus populations placing the
nuclei on a trajectory concurrent with the entire evolutive
process of spermiogenesis and nuclear maturation in late
spermiogenesis and in the epididymis.
.4820
; -2.563
Lu
6o
°2
SDGL
-1.40
RLP
-. 4
RLP
SKGL
SKGL
O -S.528
-5.528
Z0
C.
-8.493
3/4
C -8.493
-8.493
-11.46
-11.46
-14.42
3.367
i
.
I
.
I
.
.
I
.
I
.
.
-28.57
-3.371
21.83
47.83
72.23
41.17
-15.97
9.228
34.43
59.63
84.83
CANONICAL VARIABLE 1
-14.".2
I
I
-28.57
41.17
I
I
I
-3.371
21.83
-15.97
9.228
I
I
I
47.03
34.43
I
72.23
59.63
W.83
CANONICAL VARIABLE 1
FIG. 6. a) Representation of sperm nuclear maturation sequence from late steps of spermiogenesis (according to Holstein and Roosen-Runge classification [141]) to the end of epididymal transit (nuclear populations from the head [HI and tail [TI of epididymis) in the canonical parameter space generated
by the first two canonical variables (linear combinations of nuclear parameters). Ellipses delineate 95% confidence intervals around the center of gravity
of each population. Each population is assigned to a step in spermiogenesis or to an epididymal localization, and the nuclear profile evolution (arrows)
concurs with the entire evolutive process of sperm nuclear maturation. b) Vectors representing the weight and direction of the major nuclear parameters
corresponding to the canonical parameter space shown in (a). Note that the major nuclear parameters of the maturation process are slightly different from
those depicting differentiation.
NUCLEAR CHANGES IN SPERMATIDS AND EPIDIDYMAL SPERM
Metrics of Nuclear Size and Shape during Sperm
Differentiation
Area (A) and width (W) depicted the visually obvious
geometrical changes in nuclear shrinking and flattening
during spermiogenesis. A is related to the nuclear volume
[17], and its decrease accounted for the chromatin packaging process. The pattern of form factor, FF, indicated that
the process of elongation was initiated early, between steps
2 and 3/4, and its subtle regular increase in the late steps
monitored the completion of nuclear modeling.
Measurement of Changes in Chromatin Texture during
Sperm Differentiation
Changes in the chromatin pattern concomitant with nuclear shaping were measured by parameters depicting the
evolution of overall chromatin condensation, of distribution of the different degrees of chromatin condensation,
and of the distribution and organization of degrees of condensation along the major nuclear axis.
Sperm Chromatin Condensation
Our data take into account the concomitant processes of
nuclear shaping and chromatin condensation: the 7-fold decrease in the nuclear area that paralleled a regular increase
in chromatin condensation (MGL) is consistent with the
predicted minimum nuclear volume calculated on the basis
of typical packing of sperm nuclear DNA [18]. The MGL depicted a three-step pattern of chromatin condensation, suggesting a discontinuous progression of this process that had
previously been related to nucleoprotein transitions in various animals [2] and in humans [19].
The present data confirm the remarkable heterogeneity
of chromatin condensation in human sperm [20, 21]. The
standard deviation of MGL and SDGL roughly depicted the
degree of variation in condensation within the nucleus from
areas with highly compact patterns to tiny areas devoid of
chromatin (which differed from the relative proportion of
large vacuoles, LV, measured from step 6). SDGL was found
to be a salient indicator of temporal changes in the homogeneity of chromatin condensation (Table 3, Figs. 5 and
6b). Furthermore, the profile of SDGL followed a pattern
similar to that of MGL, indicating that as the chromatin
globally condenses, it becomes more heterogeneous in distribution. This result demonstrated that in humans, the process of sperm chromatin condensation is indissociable from
concomitant heterogenization of the degrees of chromatin
condensation, as previously suggested [22]. Heterogeneity
in chromatin condensation was also depicted by other parameters including two discriminatory parameters, SKGL,
the skewness of the grey-level histogram, and RLP, a texture
parameter indicating the relative number of different chromatin motifs within the nucleus. These two parameters
reached a plateau from steps 5 to 6, like that observed for
MGL and SDGL, indicating a slowdown in the mechanisms
173
responsible for chromatin reorganization that might be related to the replacement of basic transition proteins by protamines [19]. Similarly, the concomitant heterogenization in
the degrees of chromatin condensation could depend on
the ratio of protamine 1 to protamine 2 and/or on the level
of the phosphorylation state [22]. Such a relationship has
been indirectly suggested in a study of chemically induced
sperm decondensation [23].
The grey-level gradient parameters (GGL and GVGL)
provided evidence for nonrandom chromatin organization
in mature spermatids and epididymal spermatozoa. Our data
showing oriented reorganization of chromatin along the
major axis of the nucleus during spermiogenesis suggest
that in humans, chromatin condensation begins posteriorly
and proceeds upwards; this constitutes a major difference
from other species [2], presumably due to differing nucleoprotein equipment [24]. Nevertheless, it is commonly
admitted from ultrastructural observations that in humans,
normal mature spermatids and spermatozoa have chromatin that is homogeneously distributed within the nucleus; and cytochemical studies have failed to ascertain a
particular modality of rearrangement of chromatin related
to nucleoprotein content and transitions [19]. These discrepancies in results may be related to the very subtle greylevel transitions, measured by gradient parameters, that were
not accessible to the human eye-brain axis: the average 1020 grey-level differences between the anterior and posterior part were calculated on a 256-grey scale, while the human eye grey scale consists of only some thirty grey levels.
Modifications in Parametersin Testis Caput and Cauda
Epididymis
The present study indicated significant changes in nuclear parameters during the transport of spermatozoa between the testis (step 8) and the cauda epididymis. Most of
these changes occur in the unique environment of the epididymal lumen: between caput and cauda epididymis, the
significant decrease in geometrical parameters (A, W, L) that
paralleled a significant increase in the mean grey-level value
probably traced the increase in the amount of disulfide crosslinks known to occur during epididymal transport [4]. In
the rat, a decrease in sperm head area during epididymal
transit has recently been described in a study using similar
methods [26]. Conversely, most parameters were not found
to be significantly different between testis and caput epididymis except for the form factor; the gradient of the greylevel variance, which decreased (and subsequently increased in the epididymal lumen); and the run-length percentage, which increased slightly between testis and caput
epididymis (p < 0.05) and markedly in the epididymal lumen (p < 0.001). Together with the significant increase in
the standard deviation of the grey level in the epididymis,
these data could reflect subtle changes in the reorganization of chromatin related to the topology and not the num-
174
AUGER AND DADOUNE
ber of cross-links, depending on the nature and localization
of nucleoproteins within the nucleus.
Automated Classification of Nucleus Populations during
Differentiation and Maturation
Automated classification of nuclear sequences on the basis of a combination of nuclear parameters is consistent with
the description of successive steps in human spermiogenesis [14,19]. Light microscopy enables a fine analysis of the
nuclear chromatin pattern. It has been successfully used to
define the functional status of certain cell types [13, 27] and
for automatic cell-cycle phase recognition [28], showing a
correlation between the nuclear image and the functional
status of the cell. In contrast to previous reports, in the
present study representations of nuclear sequences were
based on a combination of nonredundant ultrastructural
parameters that depicted the fine organization of chromatin
and nuclear shape. In spite of the inert nature of the chromatin of differentiating sperm cells, present results indicate
the usefulness of such a representation to describe the cell
differentiation and maturation processes; they suggest that
the molecular reorganization of the sperm nucleus involving protein replacement can be indirectly traced by highresolution image cytometry of the nucleus. Such a method
could be of interest for probing the effect of toxic agents
known to alter sperm differentiation and maturation.
FactorsInvolved in Nuclear Shaping
Various ultrastructural, biochemical, and cytochemical
studies in different species have suggested a role for the
microtubular manchette [29, 30], the perinuclear material
(i.e., the perinuclear theca [31] and the calyx [32]), and nucleoprotein-DNA aggregation [33]. However, there is presently no evidence for attribution of a predominant role to
any of these factors.
Observations on the role of the manchette in nuclear
shaping in rodents after chemical treatments or in mutant
mice have suggested that chromatin condensation reinforces the shape changes already brought about by the
manchette or another structural element acting on the nucleus [34]. Among other cytoskeletal components, the perinuclear theca has been postulated to be involved in the
morphogenesis of the sperm head. Previous investigations
have led to the hypothesis that the perinuclear theca may
function as a dense web connecting the acrosome, nucleus,
and postacrosomal membrane, thereby stabilizing the association of these organelles and maintaining the overall
shape of the sperm head [35]. Moreover, perinuclear theca
proteins, denoted as thecins, undergo dynamic changes
during spermiogenesis and epididymal maturation [36]. Recently, it was shown that after removal of protamines and
DNA, the mouse sperm nucleus was reduced to a skeletal
structure, the perinuclear matrix, consisting of the perinuclear theca and an internal network of transverse fibers
and retaining the original shape of the nucleus [37]. Thus,
in light of the observations made in rodents, it is evident
that the perinuclear theca as well as the manchette may
exert an influence on nuclear shaping.
However, the possibility cannot be excluded that sperm
nuclear shape may also be defined intrinsically by edification of the DNA nucleoprotein complex [33]. DNA aggregation depends on the nature and the ratio of protein associated with DNA. In normal human sperm, most DNA is
bound to protamines, while a small portion (15%) of chromatin is packaged into histones [38]. Our cytometric data
indicated that concomitant with the net decrease in geometric parameter values related to chromatin condensation, grey-level parameter values increased at the beginning
of steps 3/4. There exists no proof of a direct relationship
between initiation of nuclear narrowing and the modalities
of chromatin condensation. However, in human step 3
spermatids, chromatin appears to be totally condensed following PTA staining-suggesting the placing of new nucleoproteins-while spreading of the acrosome is unfinished, the theca is unformed, and the nuclear ring and
associated manchette are still located opposite the anterior
pole of the nucleus [19]. These observations, together with
cytometric findings, are consistent with the hypothesis that
the DNA-nucleoprotein complex may play a role not only
in the chromatin texture pattern but also in nuclear shape.
Moreover, the significant parallel changes in morphometry
and texture parameters observed in sperm cells from the
epididymal caput and cauda, respectively, support this assumption.
A well-condensed and typically outlined sperm nucleus
could therefore result from a normal interaction between
the perinuclear structures and the nucleoprotein-DNA complexes. Conversely, a defect in these structures and/or abnormal protein equipment associated with DNA could then
explain possible concomitant or dissociated alterations in
outline and chromatin texture that are frequently encountered in ultrastructural observations of human sperm nuclei. Correlations have previously been found between sperm
head morphology and nuclear maturity [11,39], sperm
chromatin organization and nuclear functional activity [40],
and nuclear maturity and fertility [9-11,41, 42]. Our data
redirect attention to humans to determine whether correlations exist between nucleoprotein equipment, nuclear
morphology, organization of the sperm DNA within the nucleus, and the fertilization process and early stages of development.
ACKNOWLEDGMENTS
The authors would like to thank Pr. G. Brugal for his expertise and thoughtful
comments on this study, M.F. Alfonsi for her skillful technical assistance, and S. Teste
for preparation of the manuscript.
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