Zygote 20 (November), pp. 371–378. C ?Cambridge University Press 2011
doi:10.1017/S0967199411000451First Published Online 27 July 2011
Cat fertilization by mouse sperm injection
Yong-Xun Jin2, Xiang-Shun Cui2, Xian-Feng Yu3, Sung-Hyun Lee2, Qing-Ling Wang2,
Wei-Wei Gao2, Yong-Nan Xu2, Shao-Chen Sun2, IL-Keun Kong4and Nam-Hyung Kim1
Chungbuk National University, Cheongju, Chungbuk, South Korea; Jilin University, Changchun, Jilin Province, China;
and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, GyeongNam, South Korea
Date submitted: 10.06.2011. Date accepted: 13.06.2011.
Interspecies intracytoplasmic sperm injection has been carried out to understand species-specific
differences in oocyte environments and sperm components during fertilization. While sperm aster
organization during cat fertilization requires a paternally derived centriole, mouse and hamster
fertilization occur within the maternal centrosomal components. To address the questions of where
sperm, we studied the fertilization processes of cat oocytes following the injection of cat, mouse,
or hamster sperm. Male and female pronuclear formations were not different in the cat oocytes at
6 h following cat, mouse or hamster sperm injection. Microtubule asters were seen in all oocytes
following intracytoplasmic injection of cat, mouse or hamster sperm. Immunocytochemical staining
with a histone H3-m2K9 antibody revealed that mouse sperm chromatin is incorporated normally with
cat egg chromatin, and that the cat eggs fertilized with mouse sperm enter metaphase and become
normal 2-cell stage embryos. These results suggest that sperm aster formation is maternally dependent,
and that fertilization processes and cleavage occur in a non-species specific manner in cat oocytes.
Keywords: Centrosome, ICSI, Interspecies, Mouse embryo, Sperm injection
Fertilization is a series of processes that occur
as the egg and the sperm create a new life.
Fertilization begins with sperm entry into mature
oocytes, and a union of male and female genomes
occurs inside the egg, followed by progression to
mitotic metaphase, and then to 2-cell cleavage. In
most animals, including humans, cats, pigs and sea
urchin, after the sperm enters the cytoplasm of
an oocyte, a radial microtubule array, the so-called
‘sperm aster’, is organized from the sperm centrosome
1All correspondence to: Nam-Hyung Kim. Department of
Animal Sciences, Chungbuk National University, Cheongju,
Chungbuk 361–763, South Korea. Tel: +82 43 261 2546. Fax:
+82 43 272 8853. E-mail: firstname.lastname@example.org
2Department of Animal Sciences, Chungbuk National
University, Cheongju, Chungbuk 361–763, South Korea.
3Laboratory Animal Center, Jilin University, Changchun,
Jilin Province, 130062, China.
4Division of Applied Life Science, Institute of Agriculture
and Life Science, Gyeongsang National University, Jinju,
GyeongNam, South Korea.
(Schatten, 1994). Sperm aster formation seems to be
essential for pronuclear movement resulting in the
union of the male and female genomes (Schatten, 1994;
Simerly et al., 1995). In contrast, in rodents (mouse
and hamster), the sperm centriole is not introduced
into oocytes during fertilization. Acentriolar mouse
zygotes accomplish early embryonic cleavage using
a microtubular organization center (MTOC). The
differences between rodents and other animals with
respect to centrosome introduction from sperm during
fertilization are evolutionarily puzzling.
Interspecies intracytoplasmic sperm injection has
been carried out to understand species-specific differ-
ences in oocyte environments and sperm components
during fertilization. In rabbit oocytes following human
sperm injection, astral microtubules radiated from
the sperm neck and enlarged as the sperm head
underwent pronuclear decondensation (Terada et al.,
2000). Beaujean et al. (2004) showed, using interspecies
sperm injection with mouse sperm and sheep eggs,
that the sperm demethylation process in the mouse
is not dependent on the oocyte environment. In pig
oocytes, normal pronuclear formation, DNA synthesis
Jin et al.
and metaphase entry were observed following mouse
sperm injection (Kim et al., 2003). Human sperm
injection into mouse and rabbit oocytes has been used
widely for studies related to assisted human repro-
duction (Yanagimachi, 2005; Yamazaki et al., 2007).
Successful 2-cell division of mouse oocytes following
the injection of human and acrosome less hamster
has been observed (Morozumi & Yanagomachi, 2005).
However, it is not clearly understood which of the
processes of normal metaphase entry and cleavage are
maintained during interspecies fertilization by sperm
To address whether sperm aster assembly is medi-
ated by the maternal components and whether inter-
species sperm are able to achieve complete fertilization
in cat oocytes following cat, hamster or mouse sperm
injection. In this study, we saw normal pronuclear
formation, sperm aster assembly and metaphase entry
in cat oocytes after mouse sperm injection. In order
to confirm the incorporation of mouse and hamster
sperm in the entry to mitotic metaphase and 2-cell
stage division, we used immunocytochemical staining
male and female components.
Materials and methods
Generation of oocytes
For collection of cat oocytes, 1–8-year-old female
cats were induced to superovulate with 400 IU of
pregnant mare serum gonadotropin (PMSG, Daesung
Microbiological Labs, Co., Ltd., Seoul, Korea), fol-
lowed by 100 IU of human chorionic gonadotropin
(hCG; Daesung Microbiological Labs) 96 to 100 h
later. Animals were treated according to the guidelines
of the Chungbuk National University Institutional
Animal Care and Use Committee. Expanded cumulus
cell oocyte complexes (COCs) were collected 24 to 27 h
after hCG injection from ovaries by slicing the ovarian
cortex in feline-optimized culture medium (FOCM;
Herrick et al., 2007) supplemented with HEPES
(FOCMH; Sigma-Aldrich Co.) and 5–10 U/ml heparin
(Yin et al., 2006), and washing twice in FOCMH
without heparin. Unfertilized metaphase II eggs (MII)
were collected (day 0) after 6–9 h of in vitro maturation
and the cumulus cells were removed. Oocytes with
visible polar bodies and superior morphology were
centrifuged at high speed and used for injection.
Preparation of spermatozoa
Preparation of spermatozoa was performed using
the methods of Kim et al. (1998). Frozen–thawed
semen was induced by capacitation and stained with
MitoTracker fluorochrome (Molecular Probes) before
injection. For ICSI with ejaculated cat spermatozoa,
we used alternatively frozen and thawed semen
from three proven breeder males (one male/replicate).
Motile ejaculated spermatozoa were selected by swim-
up processing in complete FOCMH medium. Mouse
and hamster sperm mass was taken from the cauda
epididymis and placed at the bottom of a 1.5 ml tube
containing 200 ?l of M2 medium for 30 min at 37◦C to
allow spermatozoa to swim-up through the medium.
Intracytoplasmic sperm injection (ICSI)
ICSI was carried out according to the methods of
Kimura and Yanagimachi (1995) with some modifi-
cations. An aliquot (10 ?l) of sperm suspension was
mixed thoroughly with FOCMH containing 10–12%
(wt/vol) polyvinylpyrrolidone (Sigma). A microdrop
of suspended spermatozoa was placed on a dish,
and the dish was placed on a Nikon Differential
Interference Contrast inverted microscope equipped
with Nikon micromanipulators. High quality sper-
matozoa were selected and each sperm was injected
using a piezo-micromanipulator (MB-U; Prim Tech)
after immobilization by touching the sperm tail with
the injection pipette and aspiration into the injection
pipette. Using the holding pipette (i.d. 120 mm),
metaphase II oocytes were held by negative pressure
with the polar body at the 6 or 12 o’clock position. The
injection pipette (i.d. = 7 mm) was pushed through
the zona pellucida and into the cytoplasm of the
oocyte at the 3 o’clock position. After a minimal
amount of cytoplasm had been aspirated into the
injection pipette to ensure plasmalemma breakage,
the immobilized spermatozoon was deposited into
the oocyte. After injection, oocytes were cultured as
After injection, oocytes were cultured in FOCM
supplemented with 4.0 mg/ml BSA at 38.7◦C in 6%
CO2 in room air under mineral oil. Some groups of
oocytes were activated by 5 min exposure to 7% (v/v)
ethanol or 5 ?M ionomycin in IVC medium at 3 h post-
ICSI (Kim et al., 1996; Nakamura et al., 2001). Oocytes
were then fixed and stained at 3 h, 6 h, 9 h and 15 h
post-ICSI. A total of three replicates were performed.
Analysis of DNA synthesis
Cat oocytes following sperm injection were pulse-
labelled in vitro with 5-bromo-deoxyuridine (BrdU) in
order to evaluate antibody accessibility. The zygotes
were incubated in IVC medium supplemented with
100 mM BrdU (Sigma) for 1 h at 38.7◦C in 6% CO2
Cat fertilization by mouse sperm injection
in room air. After incubation, the zygotes were rinsed
in PBS (phosphate-buffered saline), fixed for 15 min
in 4% paraformaldehyde in PBS, and permeabilized
with 0.2% Triton X-100 in PBS for 15 min at room
The following antibodies were used in this study:
mouse anti-?-tubulin (Sigma), rabbit anti-dimethyl
lysine 9 in histone H3 (H3K9; Santa Cruz), and BrdU
(Invitrogen and Sigma).
To determine the expression and distribution of pro-
teins, cat embryos were fixed with 4% formaldehyde
for 20 min. In the case of ?-tubulin, embryos were
incubated for 5 min in 0.1 ?M taxol medium, and
incubated with Buffer M (50 mM KCl, 0.5 mM MgCl2,
0.1 mM EDTA, 1 mM ethylene glycol bis, and 1
mM ?-mercaptoethanol) for 5 min, before being fixed
in –20◦C methanol for 10 min. Embryos were then
permeabilized with 0.2% Triton X-100 for 10 min and
incubated with primary antibodies for 1 h followed
by incubation with fluorescein isothicyanate (FITC)-
or Alexa-labelled secondary antibodies. Propidium
iodide (PI) or Hoechst 33343 was used to stain the
nuclei. Slides were examined using laser-scanning
confocal microscopy performed using a Leica DM
IRB equipped with a krypton–argon ion laser for the
simultaneous excitation of fluorescence for proteins
The general linear models (GLM) procedure in the
SAS program (SAS User’s guide, 1985, SAS, Inc., Cary,
NC, USA) was used to analyse the data from all
experiments. Significant differences were determined
using Tukey’s multiple range test (Steel and Torrie,
1980) and p-values < 0.05 were considered significant.
Male and female pronuclear formations were ex-
amined in cat oocytes at 6 h following ICSI of a cat,
mouse, or hamster spermatozoon. The rate of male and
female pronuclear formation in cat oocytes following
mouse or hamster sperm ICSI was not different from
that in cat oocytes with cat sperm ICSI (Fig. 1).
No DNA synthesis had occurred in cat oocytes at 4
h following cat, mouse or hamster sperm injection
(Fig. 2). However, DNA synthesis was observed in cat
oocytes at 6 h following injection of cat (5/6), mouse
(4/4) or hamster (4/5) sperm, and at 8 h following
Figure 1 Percentage of pronuclear formation (1PN, 2PBs),
metaphase entry and 2-cell division in cat oocytes following
means ± SEM of four independent experiments (p < 0.05).
injection of cat (3/3) or mouse (5/5) sperm (Fig. 2). The
microtubule organization and chromatin configuration
in cat eggs after ICSI with mouse or hamster sperm
are shown in Fig. 3 (microtubules, green; DNA, blue;
sperm tail, red). At 3 h and 6 h post-ICSI, the sperm
aster, a radial microtubule array extending from the
sperm centrosome, was seen in cat oocytes after cat
(6/23, 26%), mouse (7/20, 35%) or hamster ICSI (2/13,
15%). No microtubules were observed to be organized
around the female pronucleus (Fig. 3).
Mitotic metaphase and the 2-cell division stage were
observed in cat oocytes at 14–15 h and 15–16 h after the
injection of cat, mouse or hamster sperm (Figs. 1, 4 and
5, respectively). Immunocytochemical staining with a
histone H3-m2K9 antibody was used to confirm male
and female components. A previous study had shown
that male chromatin was rapidly demethylated in
oocytes, but not female chromatin (Oswald et al., 2000),
suggesting the existence of discrimination among male
and female chromatin. Fig. 4 shows that throughout
metaphase, chromatin was stained with histone H3-K9
in the parthenotes. In contrast, in mitotic metaphase
in oocytes, only half of the chromatin was stained
with H3-K9 following cat or mouse sperm injection
(Fig. 4). Similarly, half of the chromatin was stained
in cat oocytes at the time of 2-cell division after cat
or mouse sperm injection (Fig. 5). The incidence of
normal mitotic metaphase (57%) and 2-cell division
(35%) in cat oocytes following cat sperm injection was
not different from that (normal metaphase, 51%; 2-cell
division, 33%) following mouse sperm injection. A few
cat oocytes progressed to normal mitotic metaphase
(12%, p < 0.005) or 2-cell division (4%, p < 0.001)
following hamster sperm injection (Fig. 1). However,
many deformed 2-cell stage and arrested 1-cell stage
embryos were observed among cat oocytes subjected
to hamster sperm injection (Figs. 1 and 5).
Jin et al.
Figure 2 Representative laser-scanning confocal microscopic images of DNA synthesis in cat oocytes following injection of cat,
mouse or hamster sperm (×630). Blue staining, chromatin; red staining, sperm tail; green staining, DNA synthesis; ♂, male
pronucleus; ♀, female pronucleus; T, tail. (The sperm tail is detected by MitoTracker.) (A) At 4 h following ICSI (cat, mouse or
hamster sperm), DNA synthesis had not been initiated in any pronuclei. (B) At 6 h following ICSI, DNA synthesis had initiated
in both male and female pronuclei. (C) At 8 h following ICSI, DNA synthesis was complete in fully developed pronuclei. PB,
Previous studies have shown that interspecies ICSI
in pig oocytes (Kim et al., 1999, 2003) and in mouse
oocytes (Kimura et al., 1998; Fulka et al., 2008) can
result in the successful production of male and female
pronuclei. Similarly in our study, the incidence of male
and female pronuclei was not different in cat oocytes
subjected to cat sperm injection than in cat oocytes
receiving interspecies sperm injection. To address the
question of whether pronuclei from an interspecies
sperm component are functional in cat oocytes, we
observed DNA synthesis in cat oocytes following cat,
mouse or hamster sperm injection. DNA replication
in all groups had a similar onset 6 h after ICSI, and
completely normal male and female pronuclei were
observed at 8 h after ICSI. The similarity of pronuclear
formation and onset time of DNA replication observed
in cat oocytes following cat, mouse or hamster sperm
injection suggests that the formation of functional
pronuclei is a non-species specific process.
zygote has a large sperm aster after ICSI with
ejaculated sperm (Murakami et al., 2005; Comizzoli
et al., 2006). Sperm asters are observed during
fertilization in most animals, with the exception of
Cat fertilization by mouse sperm injection
Figure 3 Laser-scanning confocal microscopic images of microtubules and chromatin in cat oocytes following intracytoplasmic
sperm injection (ICSI) of cat, mouse or hamster sperm (×630). Microtubules (green), sperm tail (red) and chromatin (blue) are
shown. (A) Cat oocytes following cat sperm ICSI. At 3 h post-ICSI, a midbody structure (arrow) connecting the decondensing
female chromosomes was observed. The sperm nucleus (arrowhead) was decondensed, and a radial array of microtubules
(the sperm aster, arrow) was organized from the sperm centrosome. Microtubules were not organized around the female
pronucleus. (B) Mouse sperm injection group at 3 h post-ICSI. At this time, the male and female pronuclei had decondensed,
and the sperm aster was organized from the sperm centrosome. (C) Mouse sperm injection group at 6 h post-ICSI. At this time,
the sperm aster had enlarged toward the male and female pronuclei. (D) Hamster sperm injection group. At 6 h post-ICSI,
a small sperm aster was seen at the base of the hamster sperm head, the male and female pronuclei had decondensed and
they had done so more fully than the cat or mouse sperm injection groups. PB, polar body; ♀, Female pronucleus; ♂, male
pronucleus; T, tail.
Jin et al.
Figure 4 Laser-scanning confocal microscopic images of microtubules, dimethyl histone H3K9 and chromatin assembly are
shown in normal and abnormal pronuclear and mitotic metaphase formations in activated oocytes, or after interspecies sperm
injection of cat oocytes (×630). Blue staining, chromatin; red staining, methyl-H3K9 or sperm tail; green staining, microtubules;
♂, male chromatin; ♀, female chromatin; T, tail. Images were taken 14–15 h after activation of oocytes or injection of sperm.
(A) Metaphase chromatin was stained with histone H3-m2K9 in pathenogenic cat oocytes. (B, C) Metaphase chromatin from
cat oocytes following cat or mouse sperm injection was seen in half of the chromatin stained with H3-m2K9. (D) Metaphase
chromatin from cat oocytes following mouse sperm injection shows two metaphase chromatin strands, one was stained with
H3-m2K9 and the other was not. (E, F) Abnormal chromatin and deformation of cytoplasmic embryos in cat oocytes following
hamster sperm injection. PB, polar body.
rodents. In rodents, fertilization is accomplished by
means of a maternally inherited centrosome. In mice,
the paternal centrosome seems to be degenerated
during spermiogenesis, and sperm asters are not
observed at the base of the incorporated sperm
head (Schatten, 1994). Interestingly, in the absence
of functional sperm centrosomal components, such
as parthenotes, the maternally derived microtubules
are organized in the cytoplasm as is seen in mouse
fertilization (Kim et al., 1996; Terada et al., 2000). In
the starfish (Saiki & Hamaguchi, 1992, 1998), in the
absence of sperm components, introduction of the
maternal centrosome (polar body centrosome), which
is involved in the meiotic progress of oocytes, is able
to serve as an aster even during cleavage, suggesting
an involvement of maternal components in sperm
aster formation. Unexpectedly, we have demonstrated
the existence of sperm aster formation in cat oocytes
following mouse or hamster sperm injection. The
organization of microtubule asters by mouse sperm in
cat oocytes suggests that mouse sperm components do
not completely lose their ability to form a functional
centrosome during spermiogenesis, as opposed to the
strictly maternal inheritance pattern in mice that has
been suggested based on previous studies (Schatten,
In our study, we have demonstrated, for the
first time, the complete incorporation of interspecies
sperm components in cat oocytes. Previously, Kim
et al. (2003) had demonstrated the ability of pig
oocytes injected with mouse sperm to enter into
metaphase. In this study, by staining H3K9, which
stains female chromatin only, we have demonstrated
theincorporation ofmale chromatin during metaphase
entry and 2-cell division. This result suggests that
these fertilization processes occur in a non-species
specific manner. In the present study, we also found a
high incidence of deformation of cat oocytes following
hamster sperm injection. Similarly, Kimura et al. (1998)
previously reported that mouse oocytes injected with
acrosome-intact rabbit and hamster spermatozoa were
deformed and did not develop into 2-cell embryos.
Cat fertilization by mouse sperm injection
Figure 5 Laser-scanning confocal microscopic images of microtubules, dimethylation histone H3K9 and chromatin assembly
are shown at the 2-cell division stage in activated oocytes and after sperm injection of cat oocytes (×630). Images were taken
15–16 h after the activation of oocytes or the injection of cat, mouse or hamster sperm, respectively (A–D). Blue staining,
chromatin; red staining, methyl-H3K9; green staining, microtubules; ♂, male chromatin; ♀, female chromatin. (A) Two-cell
division-stage chromatin was stained with histone H3-m2K9 in pathenogenic oocytes. (B, C) Two-celled cat embryos from cat
or mouse sperm injection showed half of their chromatin stained with H3-K9. (D) Two-celled cat embryos from hamster sperm
injection. One blastomere showed staining with H3-K9 and the other did not.
to injection, the oocytes did not deform (Yamauchi
et al., 2002; Morozumi & Yanagimachi, 2005). The
observation of deformation in cat oocytes, which
is similar to that seen in mouse oocytes following
hamster sperm injection, suggests that the contents
of acrosome may have detrimental effects on the cat
fertilization following hamster ICSI.
In summary, we have demonstrated that there is a
similar incidence of pronuclear formation and DNA
synthesis in cat oocytes following cat, mouse or
hamster sperm injection. This finding suggests the
existence of non-species specific processes in func-
tional male pronuclear formation during fertilization.
The observation of sperm aster formation by mouse
sperm in cat oocytes supports a maternal role in
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Jin et al.
functional microtubule assembly in the cat. Successful
incorporation of mouse sperm and progression of the
zygote into metaphase and 2-cell cleavage suggests
that complete fertilization occurs in a non-species
specific manner in the cat oocyte.
This work was supported by a grant from the Next-
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