BIOLOGY OF REPRODUCTION 39,157-167 (1988)
Interspecies Differences in the Stability of Mammalian Sperm Nuclei Assessed in Vivo by
Sperm Microinjection and in Vitro by Flow Cytometry'
SALLY D. PERREAULT,2.3 RANDY R. BARBEE,3 KENNETH H. ELSTEIN,4
ROBERT M. ZUCKER,4 and CAROL L. KEEFER5
Reproductive Toxicology Branch3
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 2 7711
Northrop Services, Inc.
Research Triangle Park, North Carolina 2 7709
and
Department of Physiology and Pharmacology5
College of Veterinary Medicine
University o f Georgia
Athens, Georgia 30605
ABSTRACT
To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm
nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 1 5 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact
over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and
hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm
nuclei were pretreated with dithiothreitrol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same sh species of sperm nuclei were induced
to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear
size were monitored by phase-contrast microscopy and flow cytometry. A s occurred in the oocyte, human
sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after
a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be
related t o the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn,
be determined by the type(s) of sperm nuclear protamine(s)present.
INTRODUCTION
The nuclei of mammalian spermatozoa are genetically inactivated and structurally stabilized by association of sperm DNA with protamines, highly basic
proteins that replace somatic histones during spermi-
Accepted February 19, 1988.
Received September 14, 1987.
'The research described in this article has been reviewed by the
Health Effects Research Laboratory, U. S. Environmental Protection
Agency and approved for publication. Approval does not signify that
the contents necessarily reflect the views and policies of the Agency
nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
Reprint requests.
'
157
ogenesis (reviewed by Bellve, 1979; Poccia, 1986). As
sperm pass through the epididimides, protamine
sulfhydryls are oxidized to disulfides, which further
stabilize the sperm nuclei (Calvin and Bedford, 1971;
Marushige and Marushige, 1975; Meistrich et al.,
1976). Disruption of these bridges is a prerequisite
for decondensation of the fertilizing sperm nucleus
(Perreault et al., 1984). Decondensation, in turn, is a
prelude to protamine replacement by histones and
subsequent reactivation of the sperm genome in the
oocyte (reviewed by Poccia, 1986; Zirkin et al.,
1988).
Decondensation of sperm nuclei also can be
induced in vitro by treatment with a disulfide reducing
agent such as dithiothreitol (DTT), usually in com-
158
PERREAULT ET AL.
bination with a charged detergent such as sodium
dodecyl sulfate (SDS) (Calvin and Bedford, 1971;
reviewed by Wolgemuth, 1983). Interspecies variations have been reported in the relative resistance of
sperm nuclei to this treatment (Bedford et al., 1973;
Mahi and Yangimachi, 1975), suggestive of differences in the extent or arrangement of protamine
disulfide bonding. Indeed, the protamine present in
the sperm of eutherian mammals is known for its
interspecies variability in amino acid content, a
consequence of which is different numbers and
arrangements of cysteine residues and potential
disulfide bridges (Calvin, 1976; McKay et al., 1985;
Hecht, 1988). Thus, protamine composition may
account for differences in the stability of sperm
nuclei exposed to DTT/SDS in vitro.
We showed recently that the extent of sperm
protamine disulfide bonding in hamster sperm nuclei
is a limiting factor in the time course of sperm
nuclear decondensation in the oocyte (Perreault et
al., 1987). For example, following microinjection
into hamster oocytes, condensed hamster spermatid
nuclei, which have relatively few disulfide bonds,
decondense faster than cauda epididymal sperm
nuclei, which are rich in disulfide bonds. In other
words, the greater the extent of protamine disulfide
bonding, the more time is required for oocyte reducing
factors to initiate decondensation.
The present study was designed to determine
whether sperm nuclei from heterologous species and
with varying protamine contents would decondense
at variable rates following microinjection into hamster oocytes, and, if so, whether these differences
would correlate with those observed in vitro with
DTT/SDS treatment. Six sperm species (human,
mouse, chinchilla, hamster, rat, and bull) were
selected because they contain different types of
protamines (Calvin, 1976; McKay et al., 1985, 1986;
Corzett et al., 1987, Hecht, 1988, and Balhorn, personal communication), and most of these have
been reported to exhibit differences in susceptibility
to DTT/SDS-induced decondensation in vitro (Bedford
et al., 1973; Mahi and Yanagimachi, 1975). Hamster
oocytes were selected because they are known to
support decondensation of a variety of sperm nuclei
(Yanagimachi, 1984). We found that the time course
of sperm nuclear decondensation in the oocyte varies
by species in a manner similar to that found in vitro,
and that the timing of decondensation also correlates
with the type of protamines present in each species of
sperm nuclei.
MATERIALS A N D METHODS
lsolation of Sperm Nuclei
Sperm nuclei were isolated as described in Perreault
and Zirkin (1982) from the cauda epididymidis of
adult Syrian hamsters and Sprague-Dawley rats.
Briefly, a suspension of cauda epididymal sperm was
sonicated in tris( hydroxymethy1)aminomethane (Tris)
buffer (50 mM, pH 7.7, Sigma Chemical Co., St.
Louis, MO), centrifuged through a two-step sucrose
gradient to collect nuclei free of tails, and washed by
centrifugation (2500 X g, 10 min, three times)
in Tris buffer. Suspensions of cauda epididymal
sperm from the chinchilla and mouse were sonicated
briefly (10 s) to dislodge the tails and washed three
times in Tris buffer. Straws of frozen bull semen
(kindly provided by Atlantic Breeders, Inc., Lancaster,
PA) were thawed at 35°C for 30 s, and the contents
were added to 5 ml defined medium (Brackett and
Oliphant, 1975). An aliquot was examined for
motility (>30%) by phase-contrast microscopy, and
the sperm were washed, as above, to remove seminal
fluid and cryoprotectant, sonicated briefly, and
washed again. Human semen was collected by masturbation, allowed to liquify at room temperature for
30 min, washed in Tris buffer to remove seminal
fluid, sonicated briefly, and washed again. The human
semen, provided by three healthy volunteers, was
normal in quality (as defined in Perreault and Rogers,
1982).
Isolated sperm nuclei were suspended in Tris buffer
at a concentration of approximately 100 million
sperm/ml and stored at 4°C for use within 48 h or
frozen (-60°C) in small aliquots without cryoprotectant and thawed just before use. Preliminary
experiments indicated that this treatment did not
alter the subsequent behavior of the nuclei either in
vitro or in vivo, as described below.
For some experiments, isolated bull and rat sperm
nuclei were treated sequentially with 5 mM DTT
(Sigma) and 10 mM iodoacetamide (Sigma), as
described (Perreault et al., 1984), to reduce disulfide
bonds and block the resulting free sulfhydryls.
Sonication-resistant spermatid nuclei were also
prepared from rat testes, as described in detail (Perreault et al., 1984). Free sulfhydryls in these nuclei
were similarly blocked with iodoacetamide treatment.
Sperm Microinjection
Superovulated hamster oocytes were injected with
a single sperm or spermatid nucleus (except as noted
SPERM NUCLEAR DECONDENSATION
in Results) and cultured at 37"C, as described previously (Perreault and Zirkin, 1982; Perreault et al.,
1987). Oocytes were fixed and stained at various
times after injection (15-60 min), and the sperm
nucleus was scored as condensed or decondensed (partially or fully, as illustrated in Perreault et al.,
1987). Oocytes were also examined 3-6 h after
injection to determine whether male pronucleus
formation had occurred.
For each species, the time required for 50% of the
injected sperm nuclei to decondense (TDS0) was
calculated, along with the 95% confidence interval,
by using a computer-based implementation of Finney's
maximum likelihood probit technique (Lieberman,
1983). Lack of overlap of the 95% confidence intervals was considered statistical evidence that the TDsos
were significantly different. Species differences in the
proportions of oocytes containing decondensed
sperm or male pronuclei at a given time were also
determined with Fisher's exact test. Differences were
considered significant at p< 0.05.
In Vitro Decondensation
in DTT/SDS
An aliquot (0.1 ml) of sperm nuclei was diluted to
1 ml with Tris buffer (50 mM, pH 8.0). To this were
added 0.25 ml 3% SDS (Sigma) and 0.14 ml 50 mM
DTT dissolved just before use in Tris buffer (pH 8.0).
This provided final concentrations of approximately
0.5% SDS and 5 mM DTT, similar to the original
protocol of Calvin and Bedford (197 1). The sample
was immediately divided in half so that decondensation of the sperm nuclei could be monitored by both
flow cytometry (see below) and light microscopy.
For the latter, small aliquots were glutaraldehydefixed at various times and examined with phasecontrast microscopy at 5OOX . Sperm nuclear lengths
were determined to the nearest micron with the aid
of an ocular micrometer.
Flow Cytometry
To evaluate size changes in a large number of
sperm nuclei, DTT/SDS-treated sperm nuclei were
monitored by flow cytometry. Before and at each
selected interval after the addition of DTT (as described above), approximately 10,000 nuclei were
analyzed in an Ortho 50H flow cytometer at a rate of
about 500 counts/s. The machine was configured to
measure the forward light scatter (an indicator of
size) from a 0.8 mW helium neon laser (wavelength,
159
630 nm) by using a 1.5 mm blocker bar. The assay
was carried out at the ambient temperature in the
flow cytometry lab of 27-28°C. Samples from three
or more species were tested on each occasion, each
sample was tested on at least two occasions, and
hamster sperm nuclei were always included as an internal standard.
RESULTS
Timing of Sperm. Nuclear Decondensation
in Hamster Oocytes
Of the six species of sperm nuclei tested, four
(human, mouse, chinchilla and hamster) decondensed
within the first hour, and did so at different rates
(Fig. 1). Human sperm decondensed ahead of the
other species with a TDso (calculated time to decondensation of 50% of the sperm nuclei) of only 9 min
(95% confidence limits, C. L. = 6-12 min). Mouse
sperm decondensed more slowly than human (TDSo=
23 min, C. L. = 10-33 min). Although the C. L.s of
these two species overlap slightly, human sperm were
significantly more likely to be decondensed at 15
min, as determined by Fisher's exact test. Chinchilla
and hamster sperm nuclei decondensed at similar
rates, with TDSOsof 45 rnin (C. L. = 41-51 min) and
44 rnin (C. L. = 42-46 min), respectively. These rates
were significantly slower than those of human and
mouse sperm nuclei.
Very different results were obtained with bull and
rat sperm nuclei; only 8% of the bull and none of rat
sperm nuclei showed any signs of decondensation at
60 min after injection (Fig. 2). However, pretreatment of these sperm with DTT to reduce protamine
disulfide bonds prior to microinjection enhanced
significantly the likelihood of their decondensing
within this time frame (Fig. 2). Most of the DTTtreated rat sperm nuclei decondensed only partially,
with the central shaft remaining intact. However,
condensed spermatid nuclei, isolated from the rat
testis prior to disulfide bond formation, decondensed
fully in most oocytes (Fig. 2).
Representative micrographs of the six species of
sperm nuclei in the ooplasm are shown in Figure 3. In
one experiment, each oocyte was injected with a
mouse and a hamster sperm nucleus. In these oocytes,
only the mouse sperm nucleus was decondensed at 30
rnin (Fig. 3C), whereas at 60 min, both nuclei were
swollen (Fig. 3D). Thus the difference between the
decondensation rates of mouse and hamster sperm
160
PERREAULT ET AL.
100,
c
g
I
-1
0 ?I5
0
15
30
TIME (MINUTES)
45
60
75
FIG, 1. Time
of decondensation of sperm nuclei
into hamster oocytes. Each point is the percentage ofoocytescontaining
a decondensed sperm nucleus and is based on 21-1 11 oocytesper point.
Each of the species of sperm nuclei was tested in 2-10 separate experdecondense in the
iments, H~~~~ sperm required only 15-30 min
ooplasm. Mouse sperm decondensed consistently by 30 min, and
chinchilla and hamster sperm required 45-60 min. Statistical analysis
indicates that human and mouse sperm nuclei decondensed at significantly faster rates tha? chinchilla and hamster sperm nuclei (see text).
nuclei remained when oocyte factors were kept
absolutely constant and could not, therefore, be
attributed to subtle interoocyte variations in sperm
decondensing activity.
Oocytes were also examined 3 or more hours after
microinjection to determine whether the sperm nuclei
transformed into male pronuclei. The species of
sperm nuclei that decondensed within the first hour
(human, chinchilla, and mouse) were highly likely to
form male pronuclei (Table l ) , as is the case with
homologous hamster sperm nuclei (Perreault et al.,
1987). In contrast, sperm nuclei that did not decondense within the first hour were significantly less
likely to form pronuclei (Table 1).For example, only
35% of injected bull sperm nuclei formed male
pronuclei by 3 h, and this proportion remained
essentially unchanged even after 6 h. Rat sperm
nuclei were even less likely to transform into pronuclei (Table 1); those that did so by 6 h appeared
retarded developmentally when compared with their
female counterparts. Many bull and rat sperm nuclei
decondensed partially in activated oocytes, i.e.
oocytes containing a female pronucleus (Table 1, Fig.
4A), but were not transformed into male pronuclei.
27/28
I
CTL
24/26
DTT
BULL
RAT
FIG. 2. Decondensation of untreated (CTL) and dithiothreitoltreated (DTT) bull and rut sperm nuclei and condensed rat spermatid
(TID) nuclei 60 min after microinjection into hamster oocytes. The
number of oocytes containing a decondensed s p e r d t o t a l number of
injected oocytes is shown above each bar. AlthoLgh untreated bull and
rat sperm nuclei rarely decondensed, DTT-treated bull and rat spermatid nuclei consistently decondensed. DTT-treated rat sperm nuclei were
more likely than untreated to decondense at least partially (typically
at the base of the nucleus) but did not decondense fully, as did the rat
..
..
spermatld
Interestingly, the DTT-treated rat sperm nuclei that
decondensed partially in the first hour (typically in
the basal region) often formed pronucleus-like
structures that remained associated with the condensed nuclear shaft (Fig. 4B). On the other hand,
DTT-treated bull sperm nuclei and rat spermatid
nuclei, which decondensed fully within the first hour
after injection, subsequently formed morphologically
normal pronuclei (Table 1, Fig. 4C).
The microinjection procedure failed to activate a
small proportion (<5%) of oocytes that remained
arrested at Metaphase I1 of meiosis when examined 3
hours after injection. Untreated rat and bull sperm
nuclei were usually decondensed fully in these
oocytes. Thus the hamster ooplasm apparently was
capable of supporting decondensation of rat and bull
sperm nuclei when activation of the oocyte was
prevented or delayed.
Timing o f Sperm Nuclear
Decondensation in Vitro
The same six species of sperm nuclei were incubated in DTT/SDS under carefully controlled conditions to compare rates of thiol-induced decondensation in vitro. The kinetics of decondensation were
monitored by flow cytometry before and at various
SPERM NUCLEAR DECONDENSATION
161
FIG. 3. Phase-contrast micrographs of sperm nuclei at various times after microinjection into hamster oocytes. A , ) Human sperm nucleus decondensed at 15 rnin. B . ) Chinchilla sperm nucleus decondensed a t 45 min. C.) Mouse sperm nucleus (left) decondensed, and hamster sperm nucleus
(right) condensed in the same oocyte fixed 30 min after injection. D) Mouse (left) and hamster (right) sperm nuclei decondensed in the same oocyte
fixed 60 min after injection. E . ) Bull sperm nucleus partially decondensed at its base at 60 min. F . ) Rat sperm nucleus intact at 60 min. Acetolacmoid stain, X 1400.
162
PERREAULT ET AL.
TABLE 1. Fate of heterologous sperm nuclei 3-6 h after microinjection into hamster oocytes.*
% Sperm nuclei in each condition
Species
Human
(3-6 h)
Mouse
(3-4 h)
Chinchilla
(4-5 h)
Bull
(3 h)
Bull
(6 h)
DTT-treated bull
(3 h)
Rat
(3 h)
Rat
(6 h)
DTT-treated Rat
(3-6 h)
Rat spermatid
(3 h)
Condensed
Partly
decondensed
Fully
decondensed
Male
pronucleus
Number of
oocytes
98a
57
93a
103
100a
40
9
46
10
35b
a9
11
44
4
4lb
54
0
0
0
1ooa
36
11
80
9
OC
3s
7
65
0
28d
43
9
19
7
6Se
43
0
0
5
9sa
20
*These oocytes were activated by the injection and contained a female pronucleus. Data is the sum of 3 or more experiments conducted on different days with each type of sperm nucleus. Percentages of male pronuclei with different superscripts differ by Fisher's exact test (p<O.O5).
times after the addition of DTT/SDS. Aliquots were
removed at these times and fixed for simultaneous
assessment of morphology by phase-contrast microscopy. As was observed with microinjected sperm
nuclei, decondensation rates varied by species.
Figure 5 shows representative examples of the
relative change in sperm nuclear length with time in
DTT/SDS, determined by microscopy. Again, human
sperm nuclei decondensed the fastest, swelling within
10 min to about three times their original length.
Furthermore, and in contrast to the other species,
human sperm did not swell uniformly; rather, a
subpopulation (<20%) swelled ahead of the rest.
Chinchilla sperm nuclei decondensed about 5 min
after human sperm nuclei. Mouse and hamster sperm
nuclei decondensed at slightly slower rates, swelling
rapidly between 5 and 15-20 min. Mouse sperm
nuclei increased less in relative length and more in
relative width compared with hamster sperm nuclei.
In marked contrast, rat and bull sperm nuclei showed
little, if any, change in morphology during the first
15 min, and swelled very gradually thereafter. In all
cases, initial size increases occurred while the nuclei
remained phase-dark, but as the nuclei continued to
swell they became translucent and finally transparent, as illustrated with micrographs of rat sperm
nuclei (Fig. 6).
Results of the flow cytometric analysis of the same
samples are shown in Figure 7. For each sample, the
mean forward red scatter signal at each time point is
plotted, and is expressed as a percentage of the initial
signal. In all samples, this signal first increased in
accordance with or slightly ahead of the size increase
of the phase-dark nuclei, and then declined steadily as
the nuclei became more transparent, thereby scattering
less light. This sequence of events occurred most
rapidly with human sperm nuclei, followed in close
order by chinchilla, mouse, and hamster sperm nuclei.
Bull and rat sperm nuclei also showed this biphasic
response, but over a broader time span,
The timing of decondensation in vitro was repeatable within 5 rnin from sample to sample and day to
day, as long as the temperature was controlled. In
addition, species differences in decondensation rates
relative to hamster sperm nuclei were always consistent. Temperature dependance was demonstrated
further with hamster sperm nuclei. For example, a
sample of hamster sperm nuclei incubated in DTT/
SDS at 4°C failed to decondense within a l-h observation period. The same sample required 30 min to
swell grossly at 22"C, 20 min at 28°C (as in Fig. 5 ) ,
and less than 15 min at 3 7" C.
SPERM NUCLEAR DECONDENSATION
163
FIG. 4. Fate of rat sperm or spermatid nuclei 3-6 h after microinjection into hamster oocytes. A , ) Rat sperm nucleus (arrow) partially decondensed in an activated oocyte with a female pronucleus and two polar bodies (top). B . ) Dithiothreitol-treated rat sperm nucleus (bottom) in oocyte
with female pronucleus (top). Remnants of the anterior end of the sperm nucleus (arrow) remain associated with a pronucleus-like structure,
apparently derived from the portion of the sperm nucleus that had decondensed. C . ) Morphologically normal male pronucleus (arrow) derived from a
rat spermatid nucleus. The female pronucleus (top) is identified by its proximitry to the second polar body nucleus at left. Acetolacmoid stain,
X 1800 (A, B); X 1000 (C).
DISCUSSION
Using two experimental approaches, we have found
that the stability of mammalian sperm nuclei differs
markedly among species. In the first approach, sperm
nuclei were microinjected into hamster oocytes and
the time course of sperm nuclear decondensation was
determined. Exposed to a common ooplasm, sperm
nuclei from heterologous species underwent decondensation at very different rates. With these rates as a
basis, the species fell into two categories: those that
decondensed within the first hour after injection
(human in 15-30 min, mouse in 30 min, and chin-
chilla and hamster in 45-60 min), and those that did
not (bull and rat).
In the second approach, sperm nuclear decondensation of the same six species of sperm nuclei was
monitored in vitro after exposure to disulfide reducing
agent and detergent (DTT/SDS). Species differences
similar to those observed in the microinjected oocytes
were found; again, human sperm nuclei swelled the
fastest in vitro, followed by chinchilla, mouse, and
hamster, while bull and rat sperm nuclei were considerably slower. These in vitro results confirm previous
reports (Bedford et al., 1973 ;Mahi and Yanagimachi,
164
PERREAULT ET AL.
340
-
o
300-
o
Em-
A
o
I
Human
Chinchilla
Mouse
Hamster
Rat
Bull
I-
Ym-
/*/ /
!!i
--5
fJlI
'
0
5
10
/
15
20
25
A-AlAPA
30
TIME (MINUTES)
35
40
45
50
I
55
FIG. 5. Timing of dithiothrietol/sodium dodecyl sulfate-induced
sperm nuclear decondensation in vitro in representative samples as
monitored by phase-contrast microscopy. Sperm nuclear length was
measured to the nearest micron and expressed as percentage of untreated length. Human sperm nuclei decondensed the fastest, followed at
about 5-min intervals by chinchilla, mouse, and hamster. Rat and bull
sperm nuclei were considerably slower t o decondense. Data are from
fived aliquots of the same samples analyzed by flow cytometry in
Figure 7. All species of sperm nuclei swelled uniformly except human.
For the latter, data shown are for the modal population (80%)of sperm
nuclei.
1975) that interspecies differences in sperm nuclear
stability exist, and suggest that these differences may
be related to variations in the extent and efficiency of
sperm nuclear disulfide bonding.
The similarity between the in vitro and microinjection results constitutes indirect evidence that a major
factor limiting decondensation of the sperm nuclei in
the ooplasm is the reduction of sufficient protamine
disulfide bonds. This argument is strengthened by the
observation that bull sperm nuclei underwent decondensation in the hamster ooplasm when their disulfide bonds were prereduced in vitro, as did rat spermatid nuclei, which lack disulfide bonds altogether.
Thus, once sperm protamine disulfide bonds have
been broken, hamster oocytes can induce decondensation of all of these heterologous sperm nuclei.
However, these results do not rule out the existence
of additional factors that might also limit sperm
nuclear decondensation in vivo, such as the presence
of disulfide bonds among other nuclear proteins, or
the activity of species-specific oocyte proteinasessuch as those reported in rat oocytes (Betzalel et al.,
1986), which may be involved in protamine degradation during or subsequent to sperm nuclear decondensation. As we have discussed in depth elsewhere,
the specific oocyte factors responsible for decondensing the sperm nucleus are largely unknown, and
although reduction of disulfide bonds is required, it is
not sufficient for sperm decondensation (Zirkin et al.,
1988).
The relationship between sperm nuclear disulfide
bond content and the timing of sperm nuclear decondensation in the oocyte has been established with
homologous sperm nuclei (Perreault et al., 1987). For
example, hamster sperm nuclei with diminished
numbers of disulfide bonds decondense faster than
their disulfide-rich counterparts. In the present study,
interspecies differences in decondensation rates may
be related to the number and/or arrangement of
nuclear disulfide bonds, which, in turn, is determined
FIG. 6. Phase-contrast micrographs of rat sperm nuclei fixed at various times after the addition of dithiothrietol/sodium dodecyl sulfate. A , ) Zero
minutes, intact nucleus; B . ) 15 min, slightly enlarged phase-dark nucleus; C.) 30 min, moderately swollen translucent nucleus; D.)40 min, grossly
swollen transparent nucleus. X 1600.
SPERM NUCLEAR DECONDENSATION
o
x
D
d./
Human
Chinchilla
Mouse
Hamster
u
5 loo
‘.
-5
0
5
10
15
20
I
I
25
30
TIME (MINUTES)
35
40
45
50
55
FIG. 7. Pattern of dithiothreitol sodium dodecyl sulfate (DTT-SDS)induced sperm nuclear decondensation in vitro in representative samples
as monitored by flow cytometry. The data are presented as the percentage of change in the mean forward red scatter signal at various times
after the addition of DTT/SDS. Each species of sperm nuclei produced
a unique biphasic pattern with the signal increasing initially and then
decreasing until it was lost. The time course of this pattern was shortest
for human sperm nuclei, followed by chinchilla, mouse, hamster, and,
after a lag, by rat and bull sperm nuclei.
by the type of protamine(s) present in the sperm
nucleus. For example, bull and rat sperm nuclei
contain only Type I protamine (Calvin, 1976). A
characteristic feature of bull sperm protamine (and
presumably all Type I protamines) is that each
cysteinyl sulfhydryl is oxidized to form an intra-or
intermolecular disulfide bridge (Balhorn, 1982) ; thus,
Type I protamine is maximally cross-linked and
would be expected to be very stable. On the other
hand, Syrian hamster, chinchilla, mouse, and human
sperm nuclei contain Type I1 protamine, as well
as-and in varying proportions with-Type I protamine (Calvin, 1976; Balhorn et al., 1984; McKay
et al., 1985, 1986; Corzett et al., 1987; Hecht, 1988;
Balhorn, personal communication). Type I1 protamine contains less cysteine and more histidine, and
might be expected to be less efficiently cross-linked
on the basis of Balhorn’s model (Balhorn, 1982).
Furthermore, human sperm contain two variants of
Protamine I1 that are the least similar to Type I
protamine of all mammalian protamines examined to
date (McKay et al., 1986). Based on these interspecies
differences in protamine composition, and the
differences in sperm nuclear stability reported here,
we propose that species with both types of protamine
are less efficiently cross-linked by disulfide bonds,
165
and therefore more readily decondensed both in vitro
and in vivo (in hamster oocytes) compared with
species containing only Type I protamine. Further
evidence in support of this contention has come from
preliminary experiments in which sperm nuclei from
the Chinese hamster (recently reported by Corzett et
al., 1987, to contain only Type I protamine) also fail
to decondense within the first hour after microinjection into hamster oocytes (unpublished observation).
The pronounced instability and heterogeneity of
human sperm nuclei observed in vitro in the present
study has been reported previously (reviewed by
Huret, 1986). For example, a subpopulation of
human sperm decondenses in SDS alone, without
supplemental disulfide reducing agent (Bedford et al.,
1973; Kvist et al., 1980; Blazak and Overstreet,
1982). This property has been attributed to the
presence of free sulfhydryls in human sperm protamine that are reversibly blocked by zinc and may
trigger an intrinsic mechanism of sperm nuclear
decondensation (Kvist et al., 1980; Bjorndahl and
Kvist, 1985). The present results indicate that human
sperm nuclei are also relatively unstable in the oocyte,
in comparison with the other species tested, but
whether the intrinsic mechanism proposed by Kvist et
al. (1980) contributes to this instability in the ooplasm
remains untested.
Interspecies comparisons of DTT/SDS-induced
decondensation were facilitated by the use of flow
cytometry to quantify changes in nuclear size over
time. This method allowed us to assess many thousands of sperm nuclei within a few seconds and gave
us a decondensation “fingerprint” for each species.
Furthermore, we observed changes in the light scatter
signal that occurred before changes in nuclear size, as
visualized by light microscopy. We think that this
early increase in light scatter may correlate with
changes in internal structure, possibly related to
disulfide bond reduction, that precede chromatin
dispersion. The light scatter signal continued to
increase as the opaque sperm nuclei began EO swell
and then declined as the nuclei became translucent
and eventually transparent. This biphasic pattern in
the forward red scatter signal suggests that sperm
decondensation may be a two-step process, with
reduction of at least some sperm nuclear disulfide
bonds occurring early when the sperm nucleus is
intact or only slightly enlarged, followed by dramatic
expansion of the nucleus as the detergent (or corresponding oocyte factors) gains access to the sperm
DNA. In vivo, changes in sperm nuclear stainability
166
PERREAULT ET AL.
indicative of disulfide bond reduction have been
reported to occur during fertilization, shortly after
sperm-egg fusion but before the sperm nucleus
decondenses (Miller and Masui, 1982).
The occurrence and extent of sperm nuclear
decondensation in the microinjected sperm nuclei
influenced the likelihood of male pronucleus formation. Those species of sperm nuclei that decondensed
within 60 min of injection also formed morphologically normal pronuclei by 3 h. This was expected
in light of similar experiments with homologous
sperm nuclei (Perreault et al., 1987). In contrast, rat
and bull sperm nuclei, which showed little if any
evidence of decondensation by 60 min, were unlikely
to form male pronuclei by 3-6 h. This failure was
apparently due to changes in the ooplasm that are
known to occur with oocyte activation and to result
in the arrest of sperm decondensation. I t has been
known for some time that sperm decondensing
activity is maximal in mature, Metaphase I1 oocytes,
and that this activity declines after fertilization to
become diminished or absent in pronuclear eggs (Usui
and Yanagimachi, 1976; Komar, 1982). Furthermore,
this change appears to be due to a loss in the oocyte’s
ability to reduce sperm nuclear disulfide bonds
(Perreault et al., 1984; Zirkin et al., 1985), possibly
regulated by modulation of oocyte glutathione levels
(Perreault et al., 1988). Thus failure of the rat and
bull sperm to form male pronuclei was apparently secondary to their failure to decondense within the
limited window of time prior to female pronucleus
formation. This contention is supported by two
observations: first, bull and rat sperm nuclei did
decondense in unactivated oocytes, given sufficient
time ( 3 h as opposed to 60 rnin), and second, DTTtreated bull sperm nuclei and rat spermatid nuclei, which decondensed within 60 min of injection
(i.e. prior to female pronucleus formation), also
formed morphologically normal pronuclei.
A practical consideration arising from the above
observations relates to the use of sperm microinjection to assess heterologous sperm chromosomes in
hamster oocytes (Libbus et al., 1987) as an alternative to in vitro fertilization of zona-free hamster
oocytes (reviewed by Yanagimachi, 1984). While
sperm microinjection bypasses the need for sperm
capacitation and acrosome reaction, making it particularly useful for species that are difficult to capacitate in vitro, the failure of rat and bull sperm nuclei
to decondense in hamster oocytes after microinjec-
tion would preclude their forming chromosomes.
However, pretreatment of the nuclei with disulfide
reducing agent or use of spermatid nuclei may resolve
this problem. Using the latter approach, it would
appear to be important to confirm that the nuclei
decondense fully, since partially decondensed sperm
nuclei may form pronucleus-like structures with less
than the full DNA complement (as with the DTTtreated rat sperm illustrated in Fig. 4B), resulting in
abnormal karyotypes.
The biological significance of interspecies variations in sperm nuclear stability remains to be determined. Recent evidence indicates that sperm DNA is
susceptible to the adverse effects of alkylating agents
while the spermatozoa are in epididymal transit
(Trader et al., 1987). This raises the possibility that
the DNA of less stable sperm species, including
humans, might be relatively more accessible to chemicals and hence more vulnerable to their damaging
effects. If so, the implications for interspecies extrapolation in risk assessment are noteworthy. Furthermore, abnormalities in sperm chromatin sturcture,
such as those induced by drug or toxicant exposures
during spermiogenesis (reviewed by Wyrobek et al.,
1983), may be associated with changes in sperm
nuclear stability, which, in turn, could perturb the
decondensation process during fertilization, resulting
in adverse effects on development.
ACKNOWLEDGMENTS
The authors thank Ms. Judy Duncan for her excellent technical
assistance, Ms. Julia Davis for her photographic expertise, Dr. Robert
Clarke for the gift of chinchilla sperm, Dr. Benjamin Brackett for the
gift of bull semen, and Dr. John Laskey for statistical advice.
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