Human Reproduction vol.12 no.8 pp.1693–1698, 1997
Disintegration of chromosomes in dead sperm cells as
revealed by injection into mouse oocytes
A.Rybouchkin1, J.Benijts, P.De Sutter and M.Dhont
IVF Laboratory, Department of Gynaecology and Obstetrics,
University Hospital of Ghent, Ghent, Belgium
1To
whom correspondence should be addressed at: IVF Laboratory,
Dept of Gynaecology and Obstetrics, University Hospital,
De Pintelaan 185, B-9000 Ghent, Belgium
Intracytoplasmic sperm injection of immotile (dead) ejaculated human spermatozoa has been carried out by several
centres for the treatment of infertility caused by severe
asthenozoospermia (necrozoospermia). No healthy pregnancies have been reported as yet, suggesting irreversible
damage to sperm DNA, centrioles and/or other important
structures. We investigated this hypothesis by injection of
immotile human spermatozoa obtained from several male
infertility patients into mouse oocytes and analysis of the
oocyte activation rate and sperm chromosome integrity.
Motile spermatozoa of the same sample were used as a
control. The proportion of living spermatozoa among the
immotile was also assessed in each sample and was related
to the results of the mouse oocyte injection test. The
oocyte activation rate after injection of immotile human
spermatozoa into mouse oocytes was the same or only
slightly lower than after injection with initially motile
spermatozoa (87–100% versus 100% respectively). The
rate of normal sperm chromosome spreads correlated
significantly (r J 0.90, P < 0.05) with the proportion of
living immotile spermatozoa in a given sample. It varied
from 4 to 48% for samples containing respectively 8 and
40% of living spermatozoa. Most of the mouse oocytes
injected and activated with immotile human spermatozoa
were arrested during a prolonged period of time at the
interphase of the first cell cycle (from 22 to 60%). Others
underwent a delayed nuclear envelope breakdown but
showed signs of abnormal structure of both male and
female or only the male pronuclear chromosomes. Our
data demonstrate an irreversible damage of chromosomes
in dead ejaculated human spermatozoa and provide an
experimental basis for recommending the use of testicular
or epididymal spermatozoa for treatment of male infertility
due to necrozoospermia.
Key words: DNA damage/heterospecific fertilization/necrozoospermia/oocyte activation/sperm karyotype
Introduction
Attempts have been made in several laboratories to use
ejaculated, immotile (frequently dead) human spermatozoa for
© European Society for Human Reproduction and Embryology
intracytoplasmic injection (ICSI) for treatment of infertility
due to severe astheno- or necrozoospermia (Dozortsev et al.,
1995; Nagy et al., 1995; Liu et al., 1995; Kahraman et al.,
1996; Nijs et al., 1996; Hassan and Hisham, 1996). In fact,
the use of immotile spermatozoa was even reported as being
mandatory in the first reports on ICSI (Palermo et al., 1993).
In most of the cases immotility of sperm cells was registered
directly in the ejaculates or after sperm washing. Supravital
staining for membrane integrity was also applied. Apart from
the cases where the immotility is due to defects in the
sperm locomotion apparatus (e.g. Kartagener’s syndrome) most
immotile spermatozoa were found to be membranously dead
(Eliasson, 1977). The results of ICSI with these spermatozoa
were variable, ranging from total absence of oocyte activation
(Dozortsev et al., 1995) to a fertilization rate comparable to
that following injection of motile spermatozoa (Kahraman
et al., 1996; Nijs et al., 1996; Hassan and Hisham, 1996). The
last authors, however, did not report the results of any vitality
test on the spermatozoa. It is, therefore, unclear whether human
spermatozoa, which are membranously dead at the moment of
ejaculation or soon afterwards, have any oocyte activating
capacity. Even if some of them can activate injected oocytes,
it remains unclear whether these activated oocytes can give
rise to healthy embryos. No pregnancies have as yet been
reported after injection of ejaculated immotile (dead)
spermatozoa.
By electron microscopy it was found that some spermatozoa
of patients with necrozoospermia show signs of karyolysis
(Zamboni, 1987). Hence, it is possible that membranously
dead human spermatozoa are also dead in terms of their
reproductive capacity (i.e. have disintegrated DNA). On the
other hand, however, it is widely accepted that the sperm
nucleus is a very stable structure with very tightly compacted
and highly protected DNA (Zamboni, 1987; Yanagimachi, 1994
and references therein). This generally accepted hypothesis
provided a rationale for the aforementioned attempts of using
dead human spermatozoa in human ICSI. It is important to
note, however, that this hypothesis is mostly founded on
experiments which have considered male pronucleus formation
as the main sign of sperm nuclear stability. Dead spermatozoa
killed by unprotected freeze–thawing were also used in assisted
human and animal reproduction (Goto 1993; Hoshi et al.,
1995). With the application of new methods for chromosomal
analysis, we have shown recently that more than half of human
spermatozoa immobilized by unprotected freeze–thawing bear
gross structural chromosomal aberrations (Rybouchkin et al.,
1996a). These aberrations can also be minor, and are thus
compatible with survival of the fetus to term. This finding
should warn against the use of spermatozoa after unprotected
1693
A.Rybouchkin et al.
freeze–thawing in ICSI. It also indicates that a detailed analysis
of the chromosomal integrity of dead spermatozoa in human
ejaculates is required. The absence of such an analysis hitherto
might be due to the lack of methods to analyse the chromosomal
structure of immotile spermatozoa. Indeed, immotile spermatozoa cannot be studied by the standard hamster zona-free egg
fertilization method as they are immotile. Also the injection
of human spermatozoa into hamster oocytes is as yet not
very productive (Martin et al., 1988). Injection of human
spermatozoa into mouse oocytes has recently been proposed as
a useful experimental model and has been used for chromosome
analysis of the spermatozoa in male infertility patients
(Rybouchkin et al., 1995a, 1996b; Lee et al., 1996). In the
present paper we report on our investigation of the oocyte
activating capacity and chromosome integrity of immotile
human spermatozoa, most of which were dead at the moment
of ejaculation or soon thereafter.
Materials and methods
Sperm collection and preparation for microinjection
Five sperm samples obtained from each of five patients enrolled in the
intracytoplasmic sperm injection (ICSI) programme of our infertility
centre were used for this study. Patients were selected according to
their sperm analysis: to be included the percentage of motile spermatozoa in a sample had to be ,21%, i.e. severe oligoasthenoteratozoospermia. The proportion of immotile spermatozoa in ejaculates
obtained on the day of ICSI varied from 79 to 99%. After washing
the ejaculates in Earle’s balanced salt solution spermatozoa were
freed from round cells and cell debris by centrifugation on a 50%
isotonic Percoll column for 20 min at 600 g. They were then washed
twice in calcium-free M2 medium (Sigma, Bornem, Belgium) by
centrifugation at 300 g for 10 min. The membrane integrity of
immotile spermatozoa was assessed by the eosin staining test (World
Health Organization, 1992). After a final wash the pelleted cells were
resuspended in calcium-free M2 medium and immediately used
for injection.
Oocyte collection and microinjection
Mouse oocyte collection and microinjection were performed as
described elsewhere (Rybouchkin et al., 1996b) with some modifications. These included the preparation of separate drops of spermatozoa
and of sperm injection medium [4% polyvinylpyrrolidone (PVP), 360
kDa in calcium-free M2] on the cover of a Petri dish before injection.
This was performed to ensure a better differentiation between initially
motile and initially immotile spermatozoa, since PVP suppresses
sperm motility. A motile or an immotile spermatozoon was pickedup head-first from the drop without PVP and transferred to the drop
with injection medium. Sperm tail squeezing was applied to cells in
this drop whether or not they were motile. Afterwards spermatozoa
were aspirated into the injection needle with their tail first. The
pipette was transferred into the drop with oocytes incubated in M2
medium and injection was performed as fast as possible to reduce
the mixture of calcium-free injection medium and calcium-containing
incubation medium. To assess the procedure-induced activation rate,
part of the oocytes were injected with mineral oil. All injected
oocytes were incubated in a humidified incubator at 37°C before
cytological analysis.
Cytological analysis
Mouse oocytes injected with initially motile or immotile spermatozoa
or mineral oil were scored for pronucleus (PN) formation at 10 h
1694
after injection and those with pronuclei were considered to be
activated. The oocytes that were not activated after the injection with
spermatozoa were fixed to assess the presence of sperm chromosomes.
The fixation procedure was as described by Rybouchkin et al. (1996b).
If no traces of the sperm cell were found in non-activated oocytes
they were excluded from further consideration. The oocytes which
had displayed pronuclei after injection with initially motile or immotile
spermatozoa were fixed for sperm chromosome analysis by the
fixation technique mentioned above. To prevent the formation of a
common metaphase plate and facilitate chromosome analysis, activated oocytes with two or more pronuclei were incubated for a few
hours before the expected entry into first mitosis in M2 medium with
0.2 µg/ml of nocodazole (Sigma, Bornem, Belgium). Fourteen hours
post-injection oocytes incubated in nocodazole were checked every
30–40 min for the disappearance of pronuclei, and those without
pronuclei were fixed 1–1.5 h later.
G-banding of chromosomes and inclusion criteria
Fresh metaphase spreads were treated for 10 min at 100°C followed
by treatment with 0.4 N HCl for 40 s and then incubated in 13SSC
at 50°C for 8 min. If slides were left for ageing for longer than 5
days, they were additionally treated with a 0.01% trypsin solution
(Cat. No. 25050-014, GibcoBRL, Life Technologies, Merelbeke,
Belgium) in a phosphate buffer with pH 6.8 for 2–4 s at 37°C. Finally
the slides were washed with distilled water, air-dried and stained
under the control of an inverted microscope (3400 magnification)
with a drop of a fresh Wright stain solution (Sigma, Bornem,
Belgium). Mouse and human chromosome spreads can be easily
distinguished by their chromosome size (more uniform in mice), the
number of chromosomes (20 in mice and 23 in the human), the
position of centromeres (all mouse chromosomes are acrocentric) and
the G-banding pattern. The normality of human sperm chromosomes
was assessed according to the ISCN (1985) criteria.
Statistics
The differences in the numbers of chromosome spreads with normal
morphology between the different categories of patients were statistically analysed using the χ2-test and Spearman one-tailed correlation
test with 95% confidence interval.
Results
Sperm samples from five patients were analysed. Every sample
contributed to the initially immotile and the immotile groups.
The samples were divided in three categories, A (n 5 1), B
(n 5 3) and C (n 5 1), according to the proportion of living
spermatozoa among the immotile sperm cells (Table I). The
mouse oocyte survival and activation rates, as well as the
number of pronuclei in activated oocytes following injection
with motile or immotile spermatozoa and mineral oil are
summarized in Table I. Since the results of injection of initially
motile spermatozoa were comparable for the sperm samples
from different patients they were joined together. This was also
the case for the results following the injection of mineral oil.
The survival rates of mouse oocytes after injection with
immotile and motile spermatozoa and with mineral oil were
in the range from 74 to 85% and did not differ significantly
among the categories. The oocyte activation rate for immotile
spermatozoa showed a tendency to decrease reciprocally with
the proportion of living immotile spermatozoa in the samples.
This, however, did not reach statistical significance. The
Dead spermatozoa and ICSI
Table I. Mouse oocyte activation after injection with motile or immotile ejaculated human spermatozoa
Groupa
% aliveb
No. injected
No. surviving
(%)
MII
1PN
2PN
3PN
AR (%)
II-A
II-B
II-C
IM
Oil
8
17–25
40
NA
NA
40
114
41
73
53
30
92
32
54
45
4
8
0
0
44
8
15
5
2
1
16
64
27
50
–
2
5
–
2
–
87
91
100
100
2
(75)
(81)
(78)
(74)
(85)
aII-A included one
bShows proportion
sample, II-B three samples, II-C one sample, IM five samples.
of living spermatozoa among immotile as determined by eosin test. n 5 number of
samples; II 5 initially immotile; IM 5 initially motile spermatozoa; AR 5 activation rate; NA 5 not
applicable; PN 5 pronuclei; MII 5 metaphase II.
Table II. Cytological findings in mouse oocytes injected with initially immotile and motile spermatozoa of
the same patients
Group
%
alive
No.
analysed
No. arrested at
interphase (%)
Mouse chromosomes 1
Normal (%)
II-A
II-B
II-C
IM
8
17–25
40
NA
25
69
27
43
15
35
6
1
(60)
(51)
(22)
(2)
S-PCC SH Nothing
human chromosomes
1
6
13
39
(4)b
(7)
(48)ab
(91)a
Abnormal (%)
6
9
3
2
(24)
(13)
(11)
(5)
–
13
4
–
1
1
1
1
2
5
–
–
a,bDifference between groups marked with the same letter is significant at P , 0.001.
II 5 initially immotile; IM 5 initially motile; S-PCC 5 premature chromosome condensation in S-phase;
SH 5 swollen sperm head.
activation rate of mouse oocytes after injection with mineral
oil was negligible (Table I). In oocytes that remained arrested in
meiosis after injection with immotile spermatozoa, prematurely
condensed sperm chromosomes (G1-PCC) were found along
with well-preserved meiotic mouse chromosomes. In half of
these spreads (seven of 12) signs of sperm chromosome
fragmentation could clearly be observed (data not shown).
The results of the cytological analysis of mouse oocytes
displaying one or two pronuclei after injection with initially
immotile or motile human spermatozoa are shown in Table II.
A high proportion of the oocytes activated after injection with
immotile spermatozoa (from 22 to 60%) remained arrested at
the first interphase for at least 30 h after injection. In contrast,
the vast majority of the oocytes injected and activated with
initially motile spermatozoa entered mitosis 14–16 h following
injection. Similar to the activation rates, arrest at the first
interphase was inversely related to the proportion of living
immotile spermatozoa. On the other hand, the number of
normal human sperm chromosome spreads, obtained after
fixation of oocytes having entered mitosis, showed a positive
correlation (r 5 0.90, P , 0.05) with the proportion of living
spermatozoa (Table II).
The two abnormal chromosome spreads obtained after
injection of motile human spermatozoa into mouse oocytes
included a disomy of chromosome 14 and a double disomy of
chromosomes 1 and 6. Both of them were found in the
spermatozoa of the patient with the lowest rate of living
immotile spermatozoa (group A). The chromosome abnormalities were structural in all cases where an immotile spermatozoon
was injected and consisted mostly of various numbers of
chromosomal fragments. They frequently were of the rejoined
type with the formation of rings and dicentrics (Figure 1A).
When activated mouse oocytes injected with immotile spermatozoa entered mitosis late (20–24 h) after injection, the male
pronuclei displayed chromatin figures resembling premature
chromosome condensation in S-phase (S-PCC) (Figure 1B). It
is interesting that in these cases mouse chromosomes frequently
had a curved, puffed structure with separated chromatids,
looking like meiotic rather than mitotic chromosomes.
In a few cases, when oocytes with one pronucleus were
analysed, a swollen sperm head was observed close to wellformed mitotic mouse chromosomes, whereas in other cases
no traces of sperm chromatin was found (Table II).
Discussion
When no motile (living) spermatozoa are found in the ejaculate
at the time of ICSI, there are two possibilities to address this
problem. An attempt can be made to retrieve motile (living)
spermatozoa by microsurgical epididymal sperm aspiration
(MESA) or testicular sperm extraction (TESE) or one can
proceed with the injection of immotile, possibly dead ejaculated
spermatozoa. Both approaches seem to have their pros and
cons. The use of immature gametes and the invasiveness of
the procedure are two potential drawbacks of MESA and
TESE. In addition, a legislative ban on the use of immature
spermatozoa for assisted reproduction exists in some countries
(e.g. The Netherlands). The injection of ejaculated immotile
(dead) spermatozoa could overcome these problems. Indeed,
equal activation rates have been reported for ICSI with immotile
1695
A.Rybouchkin et al.
Figure 1. Cytological findings in mouse oocytes injected and activated with initially immotile human spermatozoa following fixation at first
mitosis. (A) Normal mouse chromosome spread (M) and human sperm chromosome spread (H) with ring (arrow head), dicentric (arrow)
and multiple fragments (small arrows). (B) Morphologically abnormal mouse mitotic chromosomes (M) and S-PCC (premature chromosome
condensation in S-phase)-like structure of human sperm chromosomes (H). Initial magnification for both sets was 31000.
and motile ejaculated human spermatozoa (Kahraman et al.,
1996; Nijs et al., 1996; Hassan and Hisham, 1996). It is
generally accepted that sperm chromatin is highly stable,
protecting DNA from disintegration (for references see Zambony, 1987; Yanagimachi, 1994). Even if a low activation rate
can be expected to occur, it could be amended by assisted
oocyte activation (Tesarik, 1995; Hoshi, 1995). However, no
pregnancies have been reported after injection of immotile
(dead) ejaculated human spermatozoa.
In this paper we provide experimental evidence that dead
human spermatozoa are really dead, not only in terms of their
membrane integrity, but also in terms of their reproductive capacity. Indeed, in our experiments the number of chromosomal
aberrations or interphase arrests showed a high positive correlation with the proportion of dead spermatozoa among the immotile ones in the samples studied. This means that in dead human
spermatozoa DNA undergoes a fast degradation, so that only
few dead spermatozoa can retain an intact DNA. The mechanism
of DNA degradation in spermatozoa is presently not clear.
Apoptosis-like death of human sperm cells was recently suggested (Gorczyca et al., 1993; Baccetti et al., 1996) and DNase
I was demonstrated to be present in mature rat spermatozoa
(Stephan et al., 1996). It is, however, very tempting to question
what event comes first: spermatozoon membranous death or
DNA degradation and whether these two events have a causal
link? It is, indeed, unknown whether the spermatozoon’s motility
disappears first due to membrane disintegration and degradation
of DNA starts soon afterwards or whether inherited abnormalities in DNA packaging and a high rate of strand breaks lead to a
1696
fast death of the spermatozoon (Bianchi et al., 1993). The latter
scenario would have a profound biological adaptive meaning by
preventing oocyte wastage due to fertilization with spermatozoa
bearing grossly fragmented chromosomes. It is noteworthy, in
this context, that a great increase in the rate of chromosomal
aberrations was found in a few remaining motile mouse spermatozoa after storage in vitro for 48 h, while the vast majority of
them are already dead at this time (Munné and Estop, 1991). In
contrast, human spermatozoa can remain motile after storage in
vitro for 2 weeks without a significant increase in the rate of
chromosomal abberrations (Munné and Estop, 1993). Whatever
the relationship between membrane and DNA integrity in human
spermatozoa might be, our data prove once again the high sensitivity of sperm DNA to various deteriorating agents.
The findings concerning the presence of oocyte activating
capacity in membranously dead human spermatozoa are less
clear. Indeed, we obtained a rather high activation rate after
injection of immotile spermatozoa, even in cases where most of
them were dead. This seems to be in agreement with findings in
human ICSI of some authors (Kahraman et al., 1996; Nijs et al.,
1996; Hassan and Hisham, 1996). On the other hand, the activation rate tends to be lower than after injection of motile spermatozoa and is close to the activation rate found after ICSI with
spermatozoa from patients with sperm-related oocyte activating
deficiency (Rybouchkin et al., 1995b). In that study we observed
that, when using the same sperm sample, the activation rate of
mouse oocytes was higher than in human oocytes. The interspecies difference in the sensitivity of oocytes to the spermassociated oocyte activating factor (SAOAF) has also been
Dead spermatozoa and ICSI
reported by other authors (Parrington et al., 1996). This means
that the capacity of immotile (dead) human spermatozoa to
activate mouse oocytes does not necessarily involve a similar
activating effect on human oocytes. This could explain the
absence of oocyte activation after ICSI of human oocytes with
dead spermatozoa reported by other authors (Dozortsev et al.,
1995; Nagy et al., 1995). Anyway, since dead sperm cells are
able to activate a high proportion of mouse oocytes some SAOAF
activity should be left over. This is, however, very puzzling since
SAOAF seems to be a cytosolic protein (Dozortsev et al., 1995;
Parrington et al., 1996) and is supposed to leave the spermatozoon after membrane disintegration. Whether the remnants of
the membranes of the dead human spermatozoa still interfere
with the loss of all SAOAF or the activation of mouse oocytes
with dead spermatozoa is the result of action of the specific noncytosolic SAOAF fraction should be the subject of further
research.
The other interesting finding we would like to comment on
is the formation of S-PCC-like structures in some of the mouse
oocytes injected with dead human spermatozoa. One of the
possible explanations for this phenomenon is a borderline
damage of sperm DNA. At a higher rate of damage the DNA
integrity surveillance mechanisms would completely stop the
cell cycle at the S or G2 phase as it is reported for mouse
zygotes fertilized with X-irradiated spermatozoa (Boerjan and
Saris, 1991). A lower rate of damage could, however, be
repaired and morphologically more or less normal mitotic
chromosomes could be formed. An intermediate level of
DNA damage could therefore unbalance mitosis-promoting
and checkpoint mechanisms in the cell. Alternatively, it is also
possible that some oocytes have a less strict surveillance
mechanism than others. In this case stimulation of mitosis
entry by cytoplasmic clocks and by the female pronucleus
could overcome the block of the cell cycle induced by damaged
sperm DNA in the male pronucleus. A higher than normal
level of M-phase promoting factor (MPF) should be present
in this case and could be responsible for male pronucleus
S-PCC and a meiotic-like structure of female pronucleus
chromosomes (Heald et al., 1993). In this regard, it would be
very interesting in the future to use this kind of model in
an attempt to correlate the embryo quality with adequate
functioning of the cell cycle checkpoint mechanisms. The
entry of mitosis with incompletely replicated and/or repaired
DNA could be one of the signs of defects in these checkpoint
mechanisms and one of the possible reasons for formation of
embryos of bad quality, which are so common in human IVF.
In conclusion, our results demonstrate that even if oocyte
activation can be obtained and zygotes can be formed, the
possibility of obtaining a pregnancy by fertilization through
ICSI with dead spermatozoa is extremely small. Even more
important, however, is the risk of introducing chromosomally
abnormal sperm cells. It has already been recommended by
several authors (Silber, 1995; Tournaye et al., 1996; Nijs et al.,
1996) to prefer TESE/MESA for the treatment of infertility
due to severe astheno- or necrozoospermia. Our data provide
experimental proof that this approach might indeed be the
more effective and safest way in these cases.
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Received on March 10, 1997; accepted on June 11, 1997
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