MICROSCOPY RESEARCH AND TECHNIQUE 61:327–334 (2003)
In Vitro Fertilization and Polyspermy in the Pig: Factors
Affecting Fertilization Rates and Cytoskeletal
Reorganization of the Oocyte
HIROYUKI SUZUKI,1* YOSUKE SAITO,1 NORIKO KAGAWA,1
1
2
AND
XIANGZHONG YANG2
Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki 036-8561, Japan
Department of Animal Science U-40, University of Connecticut, Storrs, Connecticut 06269
KEY WORDS
IVF; spermatozoa; cumulus; cytoskeleton
ABSTRACT
Polyspermy is a common phenomenon in the pig. Extensive information has
become available from in vitro studies on not only the quality of oocytes but also the quality of
spermatozoa. However, little information is available on the relative penetration rates of fresh
and frozen spermatozoa from the same ejaculate from boars of different breeds. The present
results, based on a total of 15 boars of three different breeds, revealed that the inter-breed
variation in fertilization and polyspermic rates is larger than intra-breed variation. It was also
shown that the incidence of polyspermy as well as penetration rate was greatly decreased by
freezing and thawing, even if a higher number of sperm was coincubated with cumulus-free
oocytes for a longer period compared to fresh sperm of the same ejaculate. This study focuses
on the cytoskeletal organization of the oocyte with respect to the status of cumulus investment,
and monospermic and polyspermic fertilization. The status of cumulus cells correlated with the
density of transzonal cumulus-cell processes and with the maturation rate of oocytes and, to
some degrees, the incidence of polyspermy. Polyspermic zygotes formed multiple microtubule
domains in association with individual male pronuclei (PN), but in a high degree of polyspermy
(more than trispermy), the pronuclear apposition did not proceed. The effect of multiple PN of
paternal and maternal origin on the cytoskeletal reorganization is also discussed. Microsc. Res.
Tech. 61:327–334, 2003. © 2003 Wiley-Liss, Inc.
INTRODUCTION
Polyspermic fertilization occurs more frequently in
the pig than in the other species, even for in vivo
fertilization under diverse experimental conditions
(Hunter, 1967, 1990, 1991). Although techniques for
achieving in vitro maturation and in vitro fertilization
(IVF) of pig oocytes have been progressively improved
in recent years, a high incidence of polyspermy is still
one of the major problems in the pig IVF program
(Abeydeera, 2001; Nagai and Moor, 1990; Niwa, 1993;
Yoshida et al., 1990). Causes for this problem likely
include the quality of semen at fertilization (Kim et al.,
1996a; Pavlok, 2000; Sirard et al., 1993). Large variations among individual males have been noted in fertilization rates with fresh (Sirard et al., 1993) and
frozen sperm (Nagai et al., 1988). Further, because the
difficulty of cryopreservation of boar semen, little information is available on the relative penetration rates
of fresh and frozen boar sperm from a single ejaculate.
The objective of our Experiment 1 was to compare the
effects of boars of different breeds on the in vitro fertilizing capacity of fresh and frozen-thawed spermatozoa.
Causes for polyspermic fertilization may also depend
on the quality of the matured oocytes in the pig (Cran
and Cheng, 1986; Grupen et al., 1997; Wang et al.,
1997a). An intercellular communication between the
oocyte and cumulus cells are maintained during oocyte
growth and maturation, which is necessary for full
©
2003 WILEY-LISS, INC.
cytoplasmic maturation (Eppig, 1982; Familiari et al.,
1998; Mattioli et al., 1988) and stabilizes the distribution of cortical granules (Galeati et al., 1991). Our
previous studies showed that cytoskeletal alteration is
involved in the dynamic change of cumulus-oocyte cell
communication during oocyte maturation in the pig
(Suzuki et al., 2000). Cytoskeletal reorganization is
important during oocyte maturation and fertilization
in mammals (Kim et al., 1996b,c, 1997b; Long et al.,
1993; Maro et al., 1986; Navara et al., 1994; Schatten et
al., 1985; Sun et al., 2001; Wang et al., 2000). However,
little information is available on a relationship between the oocyte quality and cytoskeletal distribution.
Factors affecting in vitro maturation of the oocytes that
are associated with the incidence of polyspermy include
the effect of cumulus cells on the oocyte. In our Experiment 2, we analyzed (1) the relationship between the
status of the cumulus mass of cumulus-oocyte complex
(COC gradings) and cytoskeletal distribution of the
oocyte, (2) the relationship between COC gradings and
the rates of maturation and fertilization in vitro, and
*Correspondence to: Hiroyuki Suzuki, 3 Bunkyo-cho, Faculty of Agriculture
and Life Sciences, Hirosaki University, Hirosaki 036-8561, Japan.
E-mail: suzuki@cc.hirosaki-u.ac.jp
Received 20 February 2002; accepted in revised form 20 November 2002
DOI 10.1002/jemt.10345
Published online in Wiley InterScience (www.interscience.wiley.com).
328
H. SUZUKI ET AL.
(3) the cytoskeletal organization in monospermic and
polyspermic zygotes.
MATERIALS AND METHODS
In Vitro Maturation of Oocytes
COCs were aspirated from antral follicles (2–5 mm
in diameter) of ovaries collected from slaughtered prepubertal gilts. After being washed with Dulbecco’s
phosphate buffered saline (DPBS) containing 0.1%
polyvinyl alcohol, groups of 10 –15 oocytes were transferred to North Carolina State University 23 (NCSU23)
medium supplemented with 10% (v/v) porcine follicular
fluid, 10 i.u./ml eCG (Teikoku Hormone Mfg. Co. Ltd.,
Tokyo, Japan), and 10 i.u./ml hCG (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan). The oocytes were cultured for 24 hours, then incubated in NCSU23 without
hormonal supplements for a total period of 44 hours in
an atmosphere of 5% CO2 at 39°C.
Semen Preparation and Cryopreservation
Five boars each from the Large White, Landrace, and
Duroc breeds were randomly selected for study and
semen collection at Aomori Prefectural Experiment
Station of Animal Husbandry, Japan. The sperm-rich
fraction of the ejaculate was collected while filtering
out the gel component by an experienced operator using the glove-hand method. A portion of the fresh ejaculate semen was held in tightly capped tubes at 17°C
for 20 hours, and the other portion was frozen by the
pellet method and stored at ⫺196°C.
The method of semen cryopreservation was based on
the procedures reported by Pursel and Johnson (1975)
with minor modifications by Abeydeera and Day
(1997). After collection, semen was kept at 17°C for
2 hours and then was diluted (1:1) with Hulsenberg
VIII extender and centrifuged to remove the seminal
plasma. The sperm pellet was resuspended in BF5
extender at room temperature and cooled to 5°C over
90 minutes. Subsequently, the sperm suspension was
diluted (1:1) with BF5 containing 2% glycerol at 5°C
and frozen in 100-␮l aliquots on dry ice and stored in
liquid nitrogen. The final concentration of sperm was
5 ⫻ 108 cell/ml.
In Vitro Fertilization
Fresh semen was preincubated in modified Tris-buffered medium (Abeydeera and Day, 1997) supplemented with 1 mM caffeine and 0.1% BSA (IVF medium) for 90 minutes. After 44 hours incubation, the
COCs were washed and placed in 50-␮l droplets of IVF
medium. Sperm fraction (50 ␮l) was introduced to the
droplet containing oocytes to make a final concentration of 2 ⫻ 105 cells/ml (2,000 motile sperm/oocyte) for
6 hours of coculture. The oocytes were then transferred
to 100-␮l droplets of NCSU23 medium and cultured for
an additional 6 hours at 39°C in an atmosphere of 5%
CO2 in air.
With numerous preliminary experiments, the
swim-up procedure was found unsuitable for use with
frozen-thawed boar spermatozoa, because of a great
reduction of sperm motility during 90 minutes of incubation. In addition, when cumulus-enclosed oocytes
were coincubated for 6 hours with frozen-thawed spermatozoa, the total absence of fertilization was observed
in 14 out of 15 boars examined. Therefore, frozen-
thawed spermatozoa were coincubated with cumulusfree oocytes at a concentration of 5 ⫻ 106 cells/ml
(12,500 motile sperm/oocyte) for 12 hours without
swim-up.
Fluorescent Observations
To assess the nuclear configuration and the distribution of microtubules and microfilaments, the eggs were
processed as reported previously (Suzuki et al., 2000,
2001). Briefly, denuded oocytes were fixed in a microtubule stabilization buffer (Allworth and Albertini,
1993) at 37°C for 1 hour, washed extensively, and
blocked overnight at 5°C in the washing medium (calcium-free DPBS containing 2% BSA, 2% goat serum,
0.2% milk powder, 0.2% sodium azide, and 0.1% TritonX). The fixed samples were then exposed overnight to
anti-␤ tubulin primary antibody (1:200; Sigma Chemical Co., MO) at 5°C, and incubated with fluorescein
isothiocyanate (FITC)-conjugated secondary antibody
(1:200; Sigma) at 37°C for 2 hours. After rinsing, the
samples were stained with rhodamine- phalloidin (1:
1,000; Molecular Probes, Eugene, OR) for microfilaments for 1 hour, and then stained for DNA with
Hoechst 33342 (10 ␮g/ml) in mounting medium containing PBS and glycerol (1:1).
The samples were viewed under an Olympus microscope (BX-FLA, Olympus, Tokyo, Japan). A U-MNIBA
filter set (Olympus) was used for FITC, a U-MWIB set
(Olympus) was used for rhodamine, and a U-MWU set
(Olympus) for Hoechst. A cooled CCD video system
(ImagePoint, Photometrics Ltd., Tucson, AZ) was used
to obtain images on a computer and color adjustment
was performed by IPLab-Spectrum P software (Signal
Analytics Corporation, Vienna, VA).
Experimental Design
In Experiment 1, semen collections, from 5 boars
each of three different breeds, were repeated so that
there were duplicate samples. Each sample was divided into a portion that was used as fresh semen and
another fraction that was frozen and thawed. Thus, the
experiment consisted of a 3 ⫻ 5 ⫻ 2 ⫻ 2 factorial
arrangement.
In Experiment 2, COCs were graded in four categories described by Leibfried and First (1979). Grade
1 COCs are mostly covered with more than three cumulus layers; grade 2 COCs were covered with 2–3
layers; grade 3 COCs were partially covered with
clumped cumulus masses and the zona pellucida was
partially exposed; and grade 4 COCs were denuded and
the oocyte was only enclosed by zona pellucida. Before
and after maturation, oocytes for each COC grade were
processed for fluorescence staining to evaluate cytoskeletal distribution and oocyte maturation. Some
matured oocytes were inseminated with fresh sperm
from four (2 Landrace and 2 Large White) boars as
described above. At 6 and 12 hours after insemination,
oocytes were evaluated on the cytoskeletal distribution
with respect to the sperm penetration, polyspermy and
the PN formation by fluorescence staining.
Data in both experiments 1 and 2 were assessed by
analysis of variance with the help of the BMDP program (BMDP Statistical Software, Inc., Los Angeles,
CA). When appropriate, percentage data were arcsined
transformed. Differences between the means were de-
329
CYTOSKELETON IN POLYSPERMIC ZYGOTE
TABLE 1. Effects of the breeds and sperm condition on in vitro fertilization of pig oocytes
Sperm
Condition
Fresh
Frozen
Significance of fresh
vs. frozen data
within breed
Oocytes*
Breed
Motility
(%)1
Examined
(no.)
Penetrated
(%)
Polyspermic
(%)
Mean no. spermatozoa
in penetrated oocyte
Duroc
Landrace
L. White
Duroc
Landrace
L. White
77 ⫾ 2 (33 ⫾ 8)
84 ⫾ 2 (53 ⫾ 7)
81 ⫾ 1 (57 ⫾ 9)
33 ⫾ 2
35 ⫾ 4
40 ⫾ 3
366
499
691
401
438
461
48.7 ⫾ 10.3b
49.3 ⫾ 4.7b
79.4 ⫾ 5.0a
6.5 ⫾ 3.4c
0.9 ⫾ 0.7b
25.3 ⫾ 7.2a
21.5 ⫾ 7.8b
20.2 ⫾ 4.3b
54.1 ⫾ 4.7a
12.1 ⫾ 9.9
0
10.5 ⫾ 3.8
1.53 ⫾ 0.08b
1.23 ⫾ 0.04b
2.08 ⫾ 0.08a
1.10 ⫾ 0.05
1.00
1.16 ⫾ 0.03
P ⬍ 0.01
P ⬍ 0.01
P ⬍ 0.05 in Duroc
P ⬍ 0.01 in L. White
P ⬍ 0.01
1
Data in parentheses show sperm motility after 20 hours of sperm standing.
*Values with different superscripts within the sperm condition of the same column are significantly different (P ⬍ 0.01 between a and b; P ⬍ 0.05 between a and c).
termined using Tukey Studentized Range method.
Data were shown as the means ⫾ s.e.m. A value of P ⬍
0.05 was considered to be statistically significant.
RESULTS
Experiment 1. Effects of Boars on IVF
Average sperm motility decreased from 77– 84 to 33–
57% during semen holding at 17°C (Table 1). Proportion of motile sperm declined greatly after freezing and
thawing in all breeds examined (P ⬍ 0.01): overall
mean percentage of post-thawing motility was 36%,
ranged from 23 to 49% among boars.
As shown in Table 1, results of ANOVA showed that
the effect of breeds, as well as the effect of sperm
condition, was highly significant in terms of the sperm
penetration and polyspermy rates. In this study, 98%
of the penetrated oocytes showed PN formation. Spermatozoa from Large White boars penetrated at a significantly higher rate than those from Landrace and
Duroc boars irrespective of sperm condition. A similar
tendency was observed for polyspermic penetration in
the oocytes inseminated with fresh sperm (P ⬍ 0.01),
but not with frozen-thawed sperm. There was no penetration of frozen-thawed sperm from 6 out of 15 boars
examined (2 boars of Duroc and 4 boars of Landrace).
Similarly, the mean number of fresh spermatozoa penetrated per oocyte was significantly higher in Large
White than in the other two breeds. Of polyspermic
zygotes, about 80% were dispermic or trispermic and
the remainder were penetrated by 4 to 8 sperm when
using fresh sperm, while almost all polyspermic zygotes (84%) were dispermic when using frozen sperm.
The correlation coefficient for penetration rate on
polyspermy was significant (r ⫽ 0.68, P ⬍ 0.01), indicating that high penetration rates result in high
polyspermy rates, when fresh sperm were used.
Experiment 2. The Cytoskeletal Redistribution
of the Oocyte After Sperm Penetration
Effect of COC Gradings on Cytoskeletal Distribution. Representative pictures of the oocytes before
and after maturation are shown in Figure 1. More
profound differences were found in the distribution of
microfilaments rather than microtubules. At 0 hours of
incubation, almost all of the grade 1 oocytes (89%)
showed a dense and uniform distribution of the transzonal cumulus-cell processes (CCP, Fig. 1a). However,
about 40% of the grade 2 oocytes showed a partial loss
of CCP and about half of the oocytes at grade 3– 4 had
already lost a greater part of their CCP (Fig. 1b). At
44 hours of incubation, all grade-1 oocytes showed
strongly stained, thick microfilament bundles at the
cell cortex (Fig. 1c), whereas the oocytes of grades 2– 4
displayed a decreased density of the cortical microfilaments and uneven distribution of cytoplasmic microfilaments (Fig. 1d).
Effect of COC Gradings on Maturation and Fertilization Rates. Maturation and fertilization results
are summarized in Tables 2 and 3, respectively. At the
start of maturation, all of the oocytes assessed as grade
1 were at the GV stage, whereas significant fewer
grade 2– 4 oocytes were at the GV stage, with the
remainder (18 to 48%) at the prometaphase (Table 2).
After 44 hours of incubation, the grade 1–2 oocytes
showed significantly higher maturation rates. By contrast, lower-grade oocytes showed an increased incidence of degeneration during incubation.
Again, the sperm penetration rate was significantly
higher when using sperm from Large White than those
from Landrace, although no difference within the breed
was found regardless of the COC gradings. However,
there was a tendency that the proportion of oocytes
extruding the second polar body (PBII) decreased in
the grade-4 oocytes when compared to the better grade
oocytes. Polyspermy seemed to occur more frequently
as the penetration rate became higher, and as the COC
grade became lower (Table 3).
Cytoskeletal Organization in Monospermic and
Polyspermic Zygotes. Male PN formation was observed at 6 hours after insemination, where microtubules were developed to form the domain including
male PN (Fig. 2a). On the other hand, female PN was
incorporated in another microtubule-rich domain (Fig.
2b), which was concurrently developed. Microfilaments
became concentrated to the area between two microtubule-rich domains (Fig. 2c). At 12 hours after insemination, when the male and female PN had migrated to
a central region, one domain of microtubules included
female and male PN was found and microfilaments
were concentrated around the microtubule-rich domain. The microtubule-rich domains including female
and male PN, respectively, seemed to be unified around
the central cytoplasm during the PN apposition.
330
H. SUZUKI ET AL.
Fig. 1. Fluorescent micrographs of immature and matured pig
oocytes. Microtubules are green; microfilaments are red and nuclei/
chromosomes are blue; yellow shows the overlap of microtubules and
microfilaments. a: A GV-stage oocyte assessed at grade 1 showing a
dense and uniform distribution of cumulus cell processes consisting of
microfilaments within the zona pellucida (Z). Several cumulus cells
are attached (arrows). b: A GV-stage oocyte assessed at grade
3 showing partial loss of cumulus cell processes within the zona
pellucida (Z). Microtubules and microfilaments are not located coin-
cidentally. c: An MII-stage oocyte assessed at grade 1. Microfilaments
are strongly stained in the cortex and on the contact surfaces of the
oocyte and PBI (arrow). The MII spindle is located peripherally, with
its long axis perpendicular to the surface of the oocyte, and contains
condensed chromosomes at the equatorial plate. d: An MII-stage
oocyte assessed at grade 3 showing decreased density of cortical and
cytoplasmic microfilaments. The MII spindle near PBI (arrow) is not
radially oriented with its long axis. Bar ⫽ 50 ␮m.
In polyspermic zygotes, there were small multiple
domains of microtubules including the male PN derived from each penetrated spermatozoa at 6 –12 hours
after insemination (Figs. 3 and 4). In dispermic zygotes, the apposition of female and one male PN was
occasionally observed (Fig. 4). It seemed not to occur if
two male PN located distant from each other. In some
cases, the multiple microtubule domains became expanded to merge together over the ooplasm (Fig. 5).
However, the union of microtubule domains did not
proceed in a high degree of polyspermy (more than
3 spermatozoa penetrated).
DISCUSSION
In the pig, polyspermy is dependent on different
boars (Sirard et al., 1993; Wang et al., 1991) and on the
presence of a high concentration of capacitated spermatozoa during fertilization (Hunter, 1991; Kim et al.,
1997a). Xu et al. (1996) reported, based on fresh semen
from 3 boars of different breeds, that boars and sperm
concentration affect the rates of penetration,
polyspermy, and male PN formation. The present
study is based on a total of 15 boars and clearly showed
that the penetration and polyspermy rates were more
331
CYTOSKELETON IN POLYSPERMIC ZYGOTE
1
TABLE 2. Effect of investment morphology on meiotic maturation of pig oocytes
Hour of maturation*
0
Grade
of COC
n
1
2
3
4
44
% of GV
a
124
50
49
42
100
82.1 ⫾ 3.5b
71.0 ⫾ 4.5c
52.2 ⫾ 1.4d
n
% of MII
% of deg
138
194
175
129
93.1 ⫾ 1.1
79.5 ⫾ 6.2a,b
58.0 ⫾ 8.9b
33.0 ⫾ 6.9c
a
0
18.6 ⫾ 9.0
16.6 ⫾ 14.6
15.1 ⫾ 5.4
1
COC, cumulus-oocyte complex; GV, germinal vesicle stage; MII, the second meiotic metaphase; deg, degenerated.
*Values with different superscripts in the same column are significantly different (P ⬍ 0.05 between b and c; P ⬍ 0.01 between others).
TABLE 3. Effects of the breed and investment morphology on in vitro fertilization of pig oocytes1
Oocytes*
Breed
Landrace
L. White
Grade of
COC
Examined
(no.)
Penetrated
(%)
Extruded PBII
(%)
Polyspermic
(%)
Mean no. spermatozoa
in penetrated oocyte
1
2
3
4
1
2
3
4
118
103
131
60
177
126
105
99
30.4 ⫾ 2.7b
35.8 ⫾ 5.8b
30.4 ⫾ 6.5b
25.0 ⫾ 6.8b
93.6 ⫾ 3.6a
95.7 ⫾ 0.7a
90.3 ⫾ 7.7a
88.9 ⫾ 7.1a
88.8 ⫾ 6.5a
88.0 ⫾ 12.0
68.2 ⫾ 31.8
32.1 ⫾ 17.9b
87.9 ⫾ 4.7a
71.0 ⫾ 6.2
78.6 ⫾ 2.3
45.8 ⫾ 12.5
2.3 ⫾ 2.3d
23.9 ⫾ 3.9
35.6 ⫾ 17.4
14.3 ⫾ 14.3
38.1 ⫾ 7.9
55.9 ⫾ 0.3c
58.5 ⫾ 13.8c
48.6 ⫾ 9.7c
1.40 ⫾ 0.05b
1.57 ⫾ 0.05d
1.76 ⫾ 0.12
2.13 ⫾ 0.13a,c
1.68 ⫾ 0.10
2.00 ⫾ 0.13
1.96 ⫾ 0.19
1.85 ⫾ 0.02
1
COC, cumulus-oocyte complex; PBII, second polar body.
*Values with different superscripts in the same column are significantly different (P ⬍ 0.01 between a and b; P ⬍ 0.05 between c and d).
variable among the breeds rather than among boars
within the breed.
Due to a great reduction of sperm motility after
thawing, we realized different IVF conditions for fresh
and frozen-thawed sperm from the same ejaculate. By
estimating by post-thawing sperm motility, more than
6 times higher number of motile sperm were coincubated with cumulus-free oocytes for as long as
12 hours. Nevertheless, the incidence and degree of
polyspermy were consistently lower compared to insemination with fresh sperm. These results imply that
polyspermy may not solely be determined by the high
sperm:oocyte ratio. In the present study, all the oocytes
used for IVF were matured under the identical condition, and therefore the effect of the oocyte quality can
be eliminated from consideration. Thus, differences in
the penetration and polyspermy rates between fresh
and frozen-thawed sperm of the same ejaculate depend
on the changes in sperm characteristics during treatments.
One of the factors affecting the sperm characteristics
may be associated with the incubation conditions before IVF. In the present study, frozen-thawed sperm
were neither preincubated nor swim-upped. It is assumed, therefore, that the process of sperm capacitation may be delayed, thereby affecting sperm penetration. Morphological and functional alterations of the
acrosome during freezing and thawing might influence
the penetration rate and the incidence of polyspermy,
although we did not examine the morphological damage.
An alternating possibility may depend on the decreased velocity of motile sperm after thawing. Although the mechanism by which insemination with
fresh sperm increases the risk of polyspermy remains
unknown, it is likely that a functional block to
polyspermy is overridden on the grounds of excessive
numbers of capacitated spermatozoa reaching the egg
surface more or less simultaneously in vitro. By contrast, a relatively small number of capacitated spermatozoa with decreased velocity after freezing and thawing might positively affect fertilization condition to
avoid unphysiological distribution of excessive competent spermatozoa. Xu et al. (1996) described that the
optimal sperm:oocyte ratio for the maximal rate of
male PN formation is different among boars. It is possible, therefore, that the optimal sperm concentration
for successful IVF and development may depend on not
only different boars but also sperm condition (fresh vs.
frozen semen). Although biological significance of the
breed-specific difference is not clear, the focus should
not remain simply on boar effects within a single breed
on the fertilization parameters.
The present observations demonstrate that the status of cumulus cells when removed from follicles correlate with the density of transzonal CCP and that considerable variation in the distribution of microfilaments already exists among the porcine oocytes
assessed at different categories. In addition, the COC
gradings correlate well with the maturation of the pig
oocytes. However, once the oocytes could mature in
vitro, they seemed to be penetrated by sperm with
large variation among the breeds of sperm donor.
These observations suggest that the cytoskeletal architecture of the oocyte may alter irreversibly during the
intrafollicular development, affecting the ability of the
oocyte to become mature and fertilized, although gilts
of a similar age were immature. Further, the high
incidence of decreased cortical microfilaments could
account for the decreased rate of PBII expulsion in the
denuded (grade 4) oocytes, because microfilaments are
required for cytokinesis (Sun et al., 2001).
Polyspermic fertilization occurred more frequently in
the lower-grade oocytes, reaching 50 – 60% when in-
332
H. SUZUKI ET AL.
Figs. 2–5.
CYTOSKELETON IN POLYSPERMIC ZYGOTE
333
seminated with sperm from Large White boars. The
reason for this may be due to a delayed and incomplete
exocytosis of the cortical granules (Cran and Cheng,
1986; Wang et al., 1997b) or premature cortical granules release in poor-grade oocytes, causing a weaker
block to polyspermy. Some poor-grade oocytes had
shown GV breakdown at 0 hours of incubation, thus
aging of the oocytes could be another factor. These
observations suggest that cytoplasmic maturation may
not proceed in the grade 3– 4 oocytes so much as in the
higher-grade oocytes. The fact that the highest proportion of monospermic fertilization was derived from
grade-1 oocytes suggests that grade-1 oocytes were the
most synchronous group with respect to nuclear and
cytoplasmic maturation.
It has been known that the paternal centrosome is
active during fertilization in farm animals (Long et al.,
1993; Navara et al., 1994). Sperm aster enlarged during sperm head decondensation and extended throughout the cytoplasm during PN formation (Kim et al.,
1996c, 1997b). Recent reports describe the origin of the
active microtubule-organizing center as paternally derived and its role in PN migration and apposition (Kim
et al., 1996c, 1997b; Sun et al., 2001). In monospermic
zygotes, the male and female PN were included in two
different domains of microtubules, respectively. These
microtubule domains were anchored to the microfilament architecture. During polyspermy, multiple male
PN were found in separated microtubule domains, associated with individual penetrated sperm. A similar
situation was described by Szollosi and Hunter (1973)
and Kim et al. (1996c). From these observations, the
sperm aster may develop to a microtubule-rich domain
including the male PN as seen in the monospermic and
polyspermic zygotes. On the contrary, pronucleate parthenogenotes show one domain of microtubules in the
cytoplasm even if they are digynic (Suzuki et al.,
2002a,b). Furthermore, in pig oocytes of which PBI was
electrically fused back, the PBI’s chromatin was easily
incorporated into the microtubule networks of the oocytes, subsequently transforming into one “extra” nuclear-like structure (Suzuki et al., 2002a). From these
results, it is assumed that an interphase network of
microtubules, originated from the maternal centrosome and retained after the second meiotic division, is
distributed in the ooplasm and includes preferentially
the chromatin of the maternal origin, such as female
PN and also PBI chromatin.
In the monospermic zygote, paternal and maternal
microtubule domains unified during PN apposition. Interaction with microfilaments may be important for
merging of two microtubule domains. Multiple microtubule domains in the polyspermic zygote became
merged, but some delay or interruption was noted in
the oocyte showing a higher degree of polyspermy (usually over trispermy). These observations suggest that
the assemblies and interaction of microtubules and
microfilaments may be disturbed in the presence of
more than three male PN. The centration of the microtubule-rich domain as well as PN apposition may depend on the location and the number of male PN. A
recent report has shown that the correction of ploidy
can occur at the zygote stage before the first cell division, depending on the PN location (Han et al., 1999).
More recently, Xia et al. (2001) suggested that the
extra chromatin that does not affect the genome may
suffer from the phagocytic activity of lysosomes in the
ooplasm. The feasibility that antipolyspermic mechanism of the oocyte functions physiologically might depend on the location and the number of multiple male
PN in the polyspermic zygote.
In conclusion, there is a breed-specific variation in
fertilization and polyspermic rates and the freezing
and thawing treatment may affect the sperm characteristics. The status of cumulus investment correlates with
microfilament distribution during maturation, which
may affect the rates of maturation and polyspermy. Multiple male PN derived from polyspermy may exert different effects on cytoskeletal reorganization during PN formation and migration as compared to the monospermic
zygotes.
Fig. 2. Fluorescent micrographs of a monospermic zygote at
6 hours after insemination. a: Three components: microtubules are
green; microfilaments are red and nuclei/chromosomes are blue; yellow shows the overlap of microtubules and microfilaments. b,c: Microtubule and microfilament labeling, respectively. The first and second polar bodies are shown by small and large arrowheads, respectively. Female (large arrow) and male pronuclei (small arrow) are
included with different domains of microtubules, respectively (a,b).
Microfilaments are located between two microtubule domains (c).
Bar ⫽ 50 ␮m.
Fig. 3. Fluorescent micrographs of a dispermic zygote at 12 hours
after insemination. a: Three components: microtubules are green;
microfilaments are red and nuclei/chromosomes are blue; yellow
shows the overlap of microtubules and microfilaments. b,c: Microtubule and microfilament labeling, respectively. The first and second
polar bodies are shown by small and large arrowheads, respectively.
Female (large arrow in a) and two male pronuclei (small arrows in a)
are found in three domains of microtubules (asterisks in b). Microfilaments are concentrated around the microtubule domain (c).
Fig. 4. Fluorescent micrographs of another dispermic zygote at
12 hours after insemination. a: Three components: microtubules are
green; microfilaments are red and nuclei/chromosomes are blue; yellow shows the overlap of microtubules and microfilaments. b,c: Microtubule and microfilament labeling, respectively. The first polar
body is out of focus. The second polar body is shown by a large
arrowhead. Note the apposition of female (large arrow) and male
pronuclei (medium arrow) in a microtubule domain on the left side of
a. On the other side, a swollen sperm head (small arrow in a) is
associated with a microtubule aster (small arrow in b). Microfilaments
are located between two microtubule architectures (c).
Fig. 5. Fluorescent micrographs of a dispermic zygote at 12 hours
after insemination, showing pronuclear apposition. a: Three components: microtubules are green; microfilaments are red and nuclei/
chromosomes are blue; yellow shows the overlap of microtubules and
microfilaments. b,c: Microtubule and microfilament labeling, respectively. The first and second polar bodies are shown by small and large
arrowheads, respectively. The apposition of one female pronucleus
(large arrow) and two male pronuclei (small arrows) is almost completed (a), where the microtubule domain is merged to spread over the
ooplasm (b). Microfilaments are located concurrently a microtubule
domain (c).
ACKNOWLEDGMENTS
The authors thank the staff of the Gene Research
Center at Hirosaki University for use of the image
analyzing system and the staff of the Inakadate Meat
Inspection Office (Aomori, Japan) for supplying pig
ovaries. This study is a scientific contribution (#2/32) of
the Storrs Experiment Station at the University of
Connecticut.
334
H. SUZUKI ET AL.
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