Human Reproduction vol 11 no 7 pp 1457-1462, 1996
Changes in chromatin condensation of human
spermatozoa during epididymal transit as determined
by flow cytometry
R-Golan1-5, T.G.Cooper2, Y.Oschry1,
F.Oberpenning 3 , H.Schulze4, L.Shochat 1 and
L.M.Lewin1
'Department of Clinical Biochemistry and Interdepartmental
Equipment Facility, Sackler Medical School, Tel Aviv University,
Ramat Aviv 69978, Israel and 2Institute of Reproductive Medicine
and 3Urology Clinic of the University, D-48149 Muenster,
Germany, and 4Urology Clinic, Manenhospital n, D-44627 Heme,
Germany
^ o whom correspondence should be addressed
Inasmuch as caput epididymal and even testicular spermatozoa are now being used to generate pregnancies by
direct injection into the oocyte, differences in the chromatin
of spermatozoa from proximal and distal locations in the
epididymis were studied. Acridine Orange staining was
used to investigate chromatin structure in human spermatozoa which had left the testis and were undergoing maturation in the epididymis. Measurement of green and red
fluorescence intensities of human spermatozoa by flow
cytometry demonstrated a decrease in binding of Acridine
Orange to DNA as the spermatozoa traversed the epididymis. Using spermatozoa from the cauda epididymis as the
standard, the percentages of spermatozoa from the efferent
duct, proximal corpus epididymis, midcorpus epididymis,
distal corpus epididymis, proximal cauda epididymis and
distal cauda epididymis that had matured with regard to
chromatin condensation were 28 ± 5, 39 ± 3, 49 ± 5, 64
± 5, 69 ± 6 and 74 ± 4% respectively. It may be
concluded that eggs fertilized by ejaculated spermatozoa
receive a more highly condensed form of chromatin than
that received by eggs inseminated with proximal epididymal
or testicular spermatozoa.
Key words: Acridine Orange/chromatin condensation/epididymis/flow cytometry/human spermatozoa
Introduction
Recent advances in the treatment of infertility have utilized
micromanipulation to place spermatozoa obtained from the
testis or caput epididymis in or near fertilizable ova. The
success of such treatment has led to some disagreement as to
whether human spermatozoa undergo significant maturational
processes during their passage through the epididymis (Cooper,
1993). Potential changes in the structure of sperm chromatin
might be particularly worth considenng in the context of
intracytoplasmic sperm injection (ICSI), in which even testicular spermatid nuclei can decondense and generate male pronuclei (Uehara and Yanagimachi, 1977).
© European Society for Human Reproducuon and Embryology
The chromatin of spermatozoa first undergoes dramatic
changes during spermatogenesis. The histones which bind to
DNA in somatic cells and in germinal cells through the
spermatocyte stage become replaced, first by 'transition proteins' and then by protamines in spermatids during spermiogenesis (Green et al., 1994). The protamines, small basic
proteins containing much arginine, bind more tightly to DNA
than do histones and this results in compaction of chromatin
in the sperm nucleus, a process which is termed 'sperm
chromatin condensation'. The tightness of fit between protein
and DNA can be assessed by the degree of exclusion of the
dye Acndine Orange, which binds to DNA (Evenson et al.
1986; Evenson 1990). This dye produces green fluorescence
when bound to double stranded nucleic acids and red fluorescence with single stranded nucleic acid (Evenson et al, 1986).
With this technique it has been demonstrated that a significant
portion of chromatin condensation in hamster spermatozoa
occurred during passage of the spermatozoa through the
epididymal lumen (Yossefi et al., 1994). In the present study
this flow cytometric method has been used to search for
possible changes in chromatin of human spermatozoa that
occur during their transit through the epididymis.
Materials and methods
Source and handling of spermatozoa
Six human epididymides, provided following informed consent from
five pauents, were removed at operations for prostauc carcinoma
(n = 4) or testicular cancer and were brought to the laboratory on
ice within 90 min of the operation. Information about these patients
is provided in Table I. Epididymides were separated from the testis
and the capsule was removed to expose loops of the epididymal
tubule in six regions: 1, efferent ducts, 2, proximal corpus epididymis,
3, mid corpus epididymis, 4, distal corpus epididymis, 5, proximal
cauda epididymis, and 6, distal cauda epididymis (see Figure 1). The
tissues were excised with cleaned indectomy scissors and minced in
1.0 ml TNE buffer (0.01 M Tris(hydroxymethyl)-aminomethane,
0 15 M NaCl, 1 mM EDTA, pH 7 4) in a small Petri dish to release
sperm cells. Each dish was shaken on an Eppendorf shaker or 96well plate shaker for 15 min at room temperature to allow the tubule
contents to be dispersed. All tissues with medium were transferred
to three layers of TNE-moistened gauze in a small filter funnel to
remove tissue debris and the filtrate was collected in a 15 ml plastic
tube The Pern dish and gauze were washed with 1 ml TNE, the
suspension added to the tube before centnfugaUon (800 g, 5 min,
room temperature) The supernatant fluid was discarded, the pellet
with residual TNE transferred to Eppendorf tubes and stored frozen
at -80°C until used for flow cytometry Seven semen samples
(obtained by masturbation) which conformed to the criteria for normal
semen (World Health Organization, 1987), were diluted 1 10 (v/v)
with TNE buffer and pooled to form a reference standard. This pooled
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R-Golan et aL
Table I. Information on the patients providing epididymal material for this
study
Patient code no
Age (Years)
Operation
Anti-androgen
pretreatment
H199
53
PCA
H206
H207
77
56
PCA
PCA
H210
H211
38
60
TC
PCA
CPA 3 X 50 mg/day
for 3 days
No ann-androgens
CPA 1 X 50 mg/day
for 1 day
No treatment
No anu-androgens
PCA = prostatic carcinoma; TC = tesncular carcinoma; CPA =
cyproterone acetate.
buffer: citnc acid 0 1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl
0.15 M, pH 6). After 3 min the chilled sample was aspirated into a
flow cytometer (Becton Dickinson FACS IV Flow Cytometer, San
Jose, California, USA). Green fluorescence (BP 530 filter) and red
fluorescence (BP 630 filter) were measured for 10 000 counts/sample
after excitation with a 488 run argon laser.
The flow cytometer was standardized for each analysis session
using ejaculated human spermatozoa stained with Acndine Orange
from the reference pool mentioned above. The cytometer was adjusted
such that the peak channel and total window of the pooled spermatozoa
coincided for each experiment, thus allowing results from different
experiments to be compared reproducibly. The Consort 40 (Becton
Dickinson) and/or WINMDI data processing programs (J.Trotter,
http.//facs.scripps.edu) were used to analyse the resulting data.
Determination of mature forms
As previously reported (Yossefi et al, 1994), a quantitative measure
for this maturation process can be obtained by displaying red versus
green fluorescence values for individual sperm heads as shown in
Figure 2. This procedure was conducted as follows: on a scattergram
of red versus green fluorescence, a maturation zone ('window M')
was drawn which contained the bulk of sperm nuclei from the
distal cauda epididymis (which, by definition, have completed their
epididymal sperm maturation). Another window, labelled 'window
T' (total spermatozoa), was drawn around the broad sperm cell band
(Figure 2). Sperm nuclei whose fluorescence values fell in window
M were considered to have completed their maturation process. For
spermatozoa from each epididymal region the flow cytometer program
determined the most frequent coordinate m window T on the redgreen scatter plot as an index of the position of the mode (or 'peak
height') in this area.
Results
Figure 1. Photomicrograph of a human epididymis showing the
location of the six regions from which spermatozoa were obtained
in this study: 1, efferent ducts; 2, proximal corpus epididymis; 3,
mid corpus epididymis; 4, distal corpus epididymis; 5, proximal
cauda epididymis and 6, distal cauda epididymis.
material was divided into 0.2 ahquots and held at -80"C, until used
as reference standard.
Flow cytometry
The method for flow cytometry of spermatozoa stained with Acridine
Orange followed the procedure of Evenson et aL (1986) and Evenson
(1990). Spermatozoa, obtained as described above, were thawed,
centrifuged (500 g, 10 min), and the pellet was resuspended in
HEPES buffer (1.5 ml, 25 mM HEPES, 0.15 M NaCl, pH 7.0) to
~10 7 spermatozoa/ml and held in ice until staining. The suspension
(200 ml) was mixed with 400 ml of chilled lysis solution (0.1%
Triton X-100 [v/v], 0.15 M NaCl, 0.08 M HC1, pH 1.4), held for 30
s, and mixed with 1.2 ml dye solution (6 mg Acridine Orange/ml in
1458
The spermatozoa obtained from various locations in the human
epididymis were stained with the intercalating dye Acridine
Orange and flow cytometry demonstrated that both green and
red fluorescence were emitted by all sperm cells but at different
intensities depending upon their maturational status. In all of
the samples that were tested the sperm nuclei from the efferent
ducts differed from those from the cauda epididymis by
generating greater intensity of total green and red fluorescence
signals. This shift in fluorescence to lower intensities upon
maturation led to an increasing proportion of spermatozoa
entering the window defined as mature (Figure 2, window M).
The contour lines in Figure 2 show that most of the spermatozoa
obtained from the efferent ducts of patient H206 were far
removed from the mature region, those from the proximal
corpus epididymis had started entering the mature zone, and
spermatozoa from the distal cauda epididymis were concentrated in the M window. The percentages of spermatozoa with
mature chromatin that were obtained from each patient for
each region of the epididymis are shown in Table II and the
increase in percentage maturation of the sperm chromatin can
be seen in Figure 3.
It can be seen that condensation of chromatin occurred
during transport of spermatozoa through the proximal portions
of the epididymides. In two (H211L, H210) epididymides a
plateau had been reached in values of percentage matured cells
by the distal corpus epididymis, in two (H211R, HI99)
Chromatin condensation during epididymal transit
Figure 2. A dispersion diagram of spermatozoa stained with Acndine Orange from patient H206 (see Table I): A = from distal cauda
epididymis, B = from proximal corpus and C = from efferent ducts. The window 'M' encloses the spermatozoa with matured chromatin.
The window 'T' encloses the region in which normal spermatozoa are found, y axis = red fluorescence, arbitrary units, x axis = green
fluorescence, arbitrary units.
Table II. Chromatin condensation in human epididymal spermatozoa.
Values are percentages of spermatozoa with mature chromatin recorded for
samples from each patient for each epididymal site
Epididymal site
Efferent duct
Proximal corpus
Mid corpus
Distal corpus
Proximal cauda
Distal cauda
Human epididymal spermatozoa samples
H199
H206
H211L H211R H210
41
46
42
68
81
85
14
38
37
45
46
67
31
46
64
82
82
78
44
41
53
73
80
83
22
31
35
57
54
56
H207
15
30
65
61
71
74
Percentage mature = no. of spermatozoa in window MXlOO/no of
spermatozoa in window T, where M and T are windows shown in Figure 2
enclosing regions where spermatozoa with matured chromaun and normal
spermatozoa are found respectively
maximum maturity had been reached by the proximal corpus
and two (H206, H207) reached the maximal values in the
distal cauda epididymis. The mean (± SEM) percentage of
cells, from all epididymides, in the mature (m) window for
spermatozoa entering the epididymis from the testts was
27.3±5.0 in contrast to 74.7±4.5% of cells in the distal cauda
epididymis.
Further study showed that the peak height value of the red
and green fluorescence intensities of the sperm nuclei that had
entered the mature window was lower in spermatozoa from
more distal epididymal locations but these spermatozoa
remained in the window. It was also determined that whereas
both the total amount of red and green fluorescence decreased
as spermatozoa matured, the ratio between them did not alter
significantly.
Mature epididymal spermatozoa from three of the patients
contained satellite populations of cells with higher red: green
fluorescence ratios than those in zone T (Figure 4). These
were rarely found in populations of spermatozoa from the
efferent ducts (6.8, 5.9, and 8.8% of all cells in patients H206,
H207 and H210 respectively) but their numbers increased in
the mid- or distal corpus epididymis and reached 32.5, 38.4,
and 18.3% of the total fluorescent cells in the cauda epididymis.
With the few samples analysed, no clear association could
be seen between the ages of the patients or their pre-operation
anti-androgen treatment and the degree of chromatin condensation that was achieved. The window drawn to confine the
majority of spermatozoa from a normal ejaculate pool (Figure
5A) clearly enclosed most of the population of spermatozoa
obtained from distal cauda epididymides of patients H207,
H211L, H210, and HI99. Most of the spermatozoa from the
distal cauda epididymal region of patient H206 fell near but
not completely in the indicated window.
Discussion
The reduced access of Acndine Orange to the DNA has been
used to measure tightness of DNA-protein binding in sperm
chromatin (Evenson et al., 1986). This reflects the process of
protamine binding to the external groove of DNA (Ward and
Coffey, 1991). Other dyes such as propidium iodide have also
been used for this purpose (Evenson et al. 1986; Molina
etal. 1995).
The major conclusion from this study is that the DNA
packaging process in human spermatozoa is not completed
during sperm production in the testis. Further condensation
occurred in the majority of the nuclei as they passed through
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R.Golan et aL
3
4
REGION OF EPIDIDYMIS
Figure 3. Chromatm maturation in human epididymal spermatozoa. The percentage of spermatozoa with mature chromatin (% mature =
lOOXno. of spermatozoa in window M/no. in window ' T \ where M and T are windows shown in Figure 2 enclosing regions where
spermatozoa with matured chromatin and normal spermatozoa are found respectively) is recorded for samples from each patient for each
epididymal site: 1, efferent ducts; 2, proximal corpus epididymis; 3, mid corpus epididymis; 4, distal corpus epididymis; 5, proximal cauda
epididymis and 6, distal cauda epididymis. Patient code: H199 • , H206 O, H207 A, H210 A, H211L • , H211R Q
Figure 4. Dispersion diagram of Acndine-Orange-stained spermatozoa showing satellite(s) populations. Spermatozoa were from distal cauda
epididymides of: A, patient H210; B, patient H207; C, patient H206 and D, patient H211 (without satellite zone), y axis = red fluorescence,
arbitrary units; x axis = green fluorescence, arbitrary units. See Figure 2 for definition of regions M and T.
the epididymal lumen. One previous report on changes in
human chromatin within epididymal spermatozoa, using image
analysis of transmission electron micrographs, indicated that
caput spermatozoa were little different from testicular spermatozoa but that a change occurred between the caput and cauda
epididymis (Auger and Dadoune, 1993). In another report, in
which Aniline Blue staining of histone was taken as a measure
of chromatin condensation, Haidl et al. (1994) demonstrated
that the majority of spermatozoa in the caput epididymis were
normal (Aniline Blue negative) and that only 20% of them
matured between the caput and cauda epididymis. They suggested that chromatin condensation was apparently not com1460
pleted before the epididymal passage in a minor percentage
of spermatozoa. These results are similar to those presented
in this paper, which clearly show that 14-44% of spermatozoa
entering the epididymis contain fully condensed chromatin
and that approximately 70% of the spermatozoa had entered
the mature zone by the time they had reached the distal cauda
epididymis.
Although the epididymal samples were from patients with
carcinoma of the prostate or the testis, the same phenomenon
of condensation is likely to occur in the normal reproductive
tract because the location of the matured spermatozoa from
these samples fell in the same flow cytometric zone as did
Chromatin condensation during epididymaJ transit
Figure 5. Dispersion diagram of spermatozoa stained with Acridine Orange from pooled semen sample (A) and samples of spermatozoa
obtained from the distal cauda epididymides of patients H207 (B), H211L (C), H210 (D), H206 (E), and H199 (F). y axis = red
fluorescence, arbitrary units; x axis = green fluorescence, arbitrary units. See Figure 2 for definition of region M.
spermatozoa from a pool of normal ejaculates obtained from
men in whom no epididymal dysfunction was anticipated
(Figure 5). A majority of these epididymal spermatozoa reached
a mature status, comparable with that found in ejaculated
spermatozoa from younger men. This suggests that epididymal
function in these older men was normal, at least in regard to
chromatin condensation.
In other species, Evenson et al (1989) have shown a loss
in the ability of Acridine Orange to stain DNA in mouse and
rat spermatozoa passing from caput to corpus epididymides,
and Yossefi et al. (1994) found similar results with the hamster.
In the latter study flow cytometry was used to dissect the
process of chromatin condensation in hamster spermatozoa
into two phases: (i) a caput epididymal process which may
involve dephosphorylation of protamine and (ii) sulphydryl
oxidation which occurs mostly in the cauda epididymis. In
man protamines are known to form disulphide bonds upon
maturation in the epididymis (Saowaros and Panyim 1979;
Seligman et al. 1991) but dephosphorylation events have not
been clarified.
In our study, in addition to the spermatozoa whose staining
values fell in the range of red-green fluorescence marked 'zone
T in Figure 2, the spermatozoa from some samples contained
satellite populations of cells with abnormal staining character-
istics. The nature of these cells is unknown but it is interesting
to note that similar cells were also found in semen samples
from certain infertile patients (Evenson et al. 1991; Soffer
etal. 1995).
These observations may have relevance to the success of
in-vitro fertilization programmes using epididymal spermatozoa. In many species immature spermatozoa, despite performing competently with respect to fertilizing eggs, suffer
some disability with respect to the generation of viable embryos
(see Mieusset, 1995). There is some evidence for this in the
human, where pregnancy rates following fertilization in vitro
by spermatozoa from more proximal regions of occluded
human epididymides are lower than those obtained with
spermatozoa from more distal regions of the occluded ducts
(Patrizio et al., 1994; Schlegel et al, 1994) It may be
pertinent to mention the changes in the methylation status
of spermatogenic genes in mouse sperm chromatin during
epididymal transit (Ariel et al, 1994). That this occurs within
the spermatozoon indicates that the methylase must be within
the nucleus, but what activates the enzyme at a certain region
in the epididymis is not known. Structural rearrangements of
chromatin such as those reported by Rodriguez-Martinez et al.
(1990) might modify access of enzyme to substrate, thus
activating or deactivating methylation. On the other hand, the
1461
R-Golan et al
role of the egg cytoplasm may be merely to decondense the
highly condensed chromatin of the mature sperm cells and
remove protamine by nucleoplasmin (Montag et al., 1992). If
the sole function is to reverse the changes undergone by sperm
chromatin in the testis and epididymis, the significance of the
epididymal condensation may be merely to render the sperm
cell more hydrodynamically efficient or the sperm head more
resilient to forces experienced during penetration of the zona
pellucida (Bedford et al, 1972). In the hamster, decondensation
of sperm nucleus chromatin and pronucleus formation after
ooplasmic injection occurs with less delay for testicular than
for mature epididymal spermatozoa and spermatid nuclei show
little lag time (Perrault et al. 1987; Ogura and Yanagimachi,
1993). In the human, apparently healthy babies are born after
mtracytoplasmic injection of spermatid nuclei into ova (Tesarik
et al., 1995). Thus the significance of the degree of sperm
chromatin condensation at the time of sperm injection for
normal fertilization and embryo development needs to be
explored further.
Acknowledgements
This work was supported, in part, by a grant to L.M.L. and T.G.C.
from the German-Israeli Foundation for Scientific Research and
Development No I.-225-002.02/92
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Received on February 7, 1996, accepted on April 18, 1996.