Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes
Reproductive Biology and Endocrinology
Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes
Bongkoch Turathum 0
Kulnasan Saikhun 1
Parisatcha Sangsuwan 1
Yindee Kitiyanant 0 1
0 Department of Anatomy, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400 , Thailand
1 Institute of Molecular Biosciences, Mahidol University , Nakhon Pathom 73170 , Thailand
Background: Cryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. Methods: Immature oocytes (germinal vesicles) isolated from ovaries of normal bitches (> 6 months of age) were either vitrified in open pulled straw (OPS) using 20% ethylene glycol (EG) and 20% dimethyl sulfoxide (DMSO) as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were investigated for nuclear maturation following in vitro maturation (IVM), ultrastructural changes using transmission electron microscopy (TEM) and gene expression using RT-PCR. Fresh immature oocytes were used as the control group. Results: The rate of resumption of meiosis in vitrified-warmed oocytes (53.4%) was significantly (P < 0.05) lower than those of control (93.8%) and exposure (91.4%) groups. However, there were no statistically significant differences among groups in the rates of GV oocytes reaching the maturation stage (metaphase II, MII). The ultrastructural alterations revealed by TEM showed that cortical granules, mitochondria, lipid droplets and smooth endoplasmic reticulum (SER) were affected by vitrification procedures. RT-PCR analysis for gene expression revealed no differences in HSP70, Dnmt1, SOD1 and BAX genes among groups, whereas Bcl2 was strongly expressed in vitrified-warmed group when compared to the control. Conclusion: Immature canine oocytes were successfully cryopreserved, resumed meiosis and developed to the MII stage. The information obtained in this study is crucial for the development of an effective method to cryopreserve canine oocytes for establishment of genetic banks of endangered canid species.
A major obstacle for the development of assisted
reproductive technologies in canines is the low percentage of
oocytes reaching the maturation stage (i.e., metaphase
II, MII) following IVM. In contrast to most of other
mammals that oocytes are at the MII stage when
ovulated, canine oocytes released from ovaries are at the
prophase I stage of the first meiotic division and they
subsequently completed nuclear maturation within
6072 h in the oviduct . Several studies have been made
to improve the rates of oocyte maturation in vitro,
however, little progress has been achieved and usually
less than 20% of canine oocytes complete nuclear
maturation [2,3]. Although the low efficiency of IVM of
bitch oocytes remaining unresolved, the development of
oocyte cryopreservation is important for establishing
genetic banks as well as for developing applications for
conservation of endangered canid species . The first
successful IVF producing live offspring from
cryopreserved mouse oocytes frozen and stored in liquid
nitrogen was reported in 1977 . Subsequently, successful
cryopreservation of oocytes has been achieved in other
mammalian species [5,6] including human [7,8].
However, there is no report on the cryopreservation of
Previous studies reported success in cryopreservation
of bovine oocytes but mature (MII stage) oocytes were
susceptible to cooling damage resulting in disruption of
meiotic spindle and chromosome . Ultrastructural
studies on vitrified bovine oocytes have revealed that
intercellular communication between the cumulus cells
and oocyte might have been interrupted and that the
zona pellucida might have been modified by premature
cortical granule release . Ultrastructural alterations
of the cytoskeleton, mitochondria, cortical granules and
nucleoli have also been observed in bovine oocytes
[11,12]. Structural changes of vitrified oocytes have also
been observed in porcine  and human oocytes .
Immature oocytes in which organization of the meiotic
spindle did not develop may be an alternative source for
genetic banks. Therefore, the aim of this study was to
investigate the effects of vitrification on nuclear
maturation, ultrastructural changes and gene expression on
vitrified-warmed immature canine oocytes.
Chemicals and media
All chemicals in this study were purchased from Sigma
Chemical Company (Sigma, St. Louise, MO, USA),
unless indicated otherwise. Media was prepared once a
week, filtered (0.2 μm, Sartorius, Minisart, CA, USA)
and kept in sterile bottles. Synthetic oviductal fluid
(SOF) cultured media was incubated at 38.5°C under 5%
CO2 in air at least 4 h before use.
Collection of oocytes
Ovaries were obtained from normal bitches of various
breeds at various ages (> 6 months old) by
ovariohysterectomy at the veterinary clinic of the Veterinary Public
Health Division, Bangkok Metropolitan Administration.
Ovaries were placed in 0.9% NaCl (containing 100 IU/
ml penicillin) and transported to the laboratory (at
2532°C) within 2-4 h after removal. Ovaries were washed
three times in 0.9% NaCl containing 100 IU/ml
penicillin. To collect cumulus-oocyte complexes (COCs),
ovaries were sliced repeatedly in Petri dishes containing
TCM 199 (Invitrogen, Carlsbad, CA, USA)
supplemented with 25 mM HEPES, 0.1% polyvinylalcohol, 0.1 mM
glutamine, 2.5 mM sodium pyruvate and 1%
penicillinstreptomycin. Cumulus-oocyte complexes were washed
and graded under a stereomicroscope (200×) using
criteria based on the uniformity of ooplasm and cumulus
cell complement, as previously described . Grade 1
oocytes were surrounded with more than five layers of
compact cumulus cells and had homogeneous dark
cytoplasm. Grade 2 oocytes were surrounded by three to
five layers of compact cumulus cells and had
homogeneous dark cytoplasm. Grade 3 oocytes were partially
surrounded by cumulus cells and lacked homogeneous
cytoplasm. Grade 4 oocytes were denuded (without
surrounding cumulus cells) and lacked homogeneous
cytoplasm. Cumulus-oocyte complexes with more than three
layers of cumulus cells with dark pigment oocyte
cytoplasm (grade 1 and grade 2) were selected for the
Vitrification and warming of immature oocytes
The procedures for cryopreservation of oocytes were
performed at room temperature. Immature oocytes
(germinal vesicle stage) were exposed to the holding
medium (HM) (TCM 199 supplemented with 20 mM
Hepes and 20% FBS) for 5 min and then equilibrated in
HM+4% EG for 5 min. They were exposed to HM+10%
EG+10% DMSO for 1 min and then placed in 2-3 μl
drop of the vitrification solution (HM+20% EG+20%
DMSO+0.5 mM sucrose) for 30 sec. The COCs were
then loaded in OPS straws (five oocytes per straw),
plunged directly into the liquid nitrogen and stored for
7 days. Warming was done by immersing the tip of OPS
straw into 1 ml of HM+0.3 mM sucrose at 37°C for
1 min. The oocytes were directly expelled into the
medium after the vitrified medium became liquid. The
oocytes were transferred into HM+0.15 M sucrose for
further rehydration. They were then washed and
incubated in holding medium for 5 min before further
Morphological assessment of vitrified-warmed oocytes
Vitrified-warmed oocytes were assessed for viability
according to their morphology. The COCs surrounded
with compacted cumulus cells, of symmetrical shape
and showing no signs of lysis were classified as of
normal morphology (Figure 1A). Contrarily, COCs with
damaged cumulus cells, ruptured zona pellucida or
leakage of cytoplasm were classified as abnormal oocytes
(Figure 1B). Only vitrified-warmed oocytes with normal
morphology were subjected to in vitro maturation.
In vitro maturation
The COCs were cultured in SOF supplemented with 40
ng/ml epidermal growth factor (EGF) . Twenty COCs
were cultured for 48 h in a four-well Petri dishes
containing 500 μl SOF under mineral oil in each well. The
dishes were held in an incubator at 38.5°C in a
humidified atmosphere of 5% CO2 in air.
Experiment I: Nuclear maturation of vitrified-warmed
Cumulus-oocyte complexes were divided into 3 groups:
1) control, 2) exposure; oocytes were exposed to
vitrification media without loading into straws and storage in
liquid nitrogen, and 3) vitrified-warmed; oocytes were
Figure 1 Morphology of vitrified-warmed oocytes after warming (200×). A) Normal oocyte with compacted cumulus cells, symmetrical
shape and no signs of lysis. B) Abnormal oocyte with damaged cumulus cells, ruptured zona pellucida and leakage of cytoplasm.
vitrified, loaded into straws, stored in liquid nitrogen for
7 days and warmed at 38.5°C. Cumulus-oocyte
complexes were cultured in SOF for 48 h at 38.5°C.
Following 48 h culture, they were denuded, fixed and stained
with Hoechst 33342. They were then evaluated for the
meiotic stage under a fluorescence microscope.
Assessment of nuclear maturation
After culturing for 48 h, COCs were denuded by
exposure to 1% hyaluronidase for 5 min and gentle pipetting
to remove cumulus cells. To analyze meiotic maturation,
the denuded oocytes were fixed and permeabilized in
Dulbecco’s phosphate buffer saline (PBS) containing
3.7% (w/v) paraformaldehyde and 0.1% (v/v) Triton
X100 for 20 min at room temperature. They were washed
three times in PBS and then transferred to small drops
of PBS supplemented with 90% (v/v) glycerol and
1.9 μM Hoechst 33342, placed on glass slides. The
oocytes were overlaid with a coverslip supported by four
droplets of vaseline/paraffin. They were examined using
a fluorescence microscope with a 355-nm wave length
excitation filter. The meiotic stage of IVM oocytes was
classified as previously described : germinal vesicle
(GV), germinal vesicle break down (GVBD), metaphase I
(MI) and metaphase II (MII) (Figure 2).
Experiment II: Ultrastructural changes of vitrified-warmed
Based on the results of experiment I that there were no
significant differences in rates of nuclear maturation
among groups the control and vitrified-warmed groups
were included in this study. COCs at 0 h of IVM
collected from both groups were fixed in 2.5%
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for
120 min at 4°C and washed three times in the same
buffer for 10 min at 4°C. Samples were post-fixed with 1%
osmium tetraoxide in 0.1 M sodium cacodylate buffer
for 1 h at 4°C. They were gradually dehydrated for
10 min twice in each concentration in series of ethanol
(35, 50, 70, 80, 90, 95 and 100%) and treated twice for
10 min with propylene oxide, infiltrated with 1:1
propylene oxide/resin in embedding capsules (Electron
Microscopy Sciences, Washington, PA, USA) overnight, and
finally embedded in fresh resin. Thin sections (60-80
nm) were cut with an ultramicrotome and collected on
glass slides or 200-mesh thin bar copper grids. Thin
sections were stained with saturated uranyl acetate in 80%
methanol and lead citrate. These sections were observed
and photographed with a transmission electron
microscope at 80 KV.
Experiment III: Gene expression of vitrified-warmed canine
Based on the results of experiment I that there were no
significant differences in rates of nuclear maturation
among groups the control and vitrified-warmed groups
were included in this study. They were collected at 0, 24
and 48 h of IVM to investigate gene expression using
RT-PCR. All transcripts of COCs were performed by
PCR using the Array Pure Nano-scale Purification Kit
(Epicentre, WI, USA). The examined genes were
apoptosis related BAX and Bcl2, DNA methyltransferase
(Dnmt1), heat shock protein 70 (HSP70), superoxide
dismutase1 (SOD1) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH). The amplified PCR products
were subjected to electrophoresis for visualization on a
1.5% agarose gel follow by UV analysis using the
BioDoc-It System (UVP, Upland, CA, USA). The primers
used for RT-PCR analysis are shown in Table 1.
All oocytes were randomly distributed within each
experimental group and each experiment was repeated
at least three times. Data were arc-sine transformed and
then subjected to ANOVA; when necessary, differences
Figure 2 Nuclear status of canine oocytes. Stained with Hoechst 33342: (A) GV; (B) GVBD; (C) MI; and (D) MII (400×).
were located with Duncan’s New Multiple Range Test.
Differences of P < 0.05 were considered significant.
Experiment 1: The effect of vitrification on nuclear
maturation of vitrified-warmed canine oocytes
A total of 284 oocytes were vitrified. After warming,
95.7 and 4.3% were classified as normal and abnormal
oocytes, respectively. A total of 292, 209, and 200
oocytes of control, exposed and vitrified-warmed groups
were cultured and evaluated for nuclear maturation
(Table 2). The percentage of vitrified-warmed oocytes
arrested at GV stage (46.6%) was significantly (P < 0.05)
higher than those of control (6.2%) and exposure (8.7%)
groups. The rate of meiotic resumption (GVBD-MII) of
vitrified-warmed oocytes (53.4%) was significantly (P <
0.05) lower than those of control (93.8%) and exposure
(91.4%) groups. However, there were no statistically
significant differences in rates of GV oocytes reached the
Experiment 2: Ultrastructure of control, exposed and
vitrified-warmed canine oocytes
Transmission electron microscopy (TEM) analysis was
performed to investigate the ultrastructural changes of
canine oocytes caused by vitrification. A semi-thin
Table 1 Primers used for RT-PCR analysis
section of normal COC showed a large germinal vesicle
oocyte with a central nucleus and surrounded with
densely compacted cumulus cells (Figure 3A). Figure 3B
showed the typical ultrastructure of a normal oocyte at
low magnification, which is characterized by numerous
mitochondria uniformly scattered within pericortical
cytoplasm and a small alignment of cortical granule
beneath the plasma membrane. Lipid droplets were of
typical round shape (Figure 3C). The smooth
endoplasmic reticulum (SER) was concentrated in the perinuclear
region and scattered throughout the cytoplasm but in
rather low quantities. It was partially or totally
surrounded by lipid droplets (Figure 3D). Mitochondria
were characterized by a few cristae rarely crossing an
electron dense matrix (Figure 3E). They were typically
round or elliptical in shape and were distributed around
lipid droplets (Figure 3C and 3E). The cortical granules
were already present but in highly variable quantities,
round in shape and more electron dense. They were
either dispersed or grouped in small lines of 3 to 5
granules next to the membrane (Figure 3F). Ultrastructural
alterations of oocytes were observed following
vitrification. There was an increased mitochondrial density but
a decreased cortical granule density at the cortical zone
and an irregular plasma membrane (Figure 4B). Lipid
droplets became smaller and less electron-lucent. The
lipid droplet membranes were broken and mitochondria
infiltrated inside lipid droplets (Figure 4C). The smooth
endoplasmic reticulum was normal in size but decreased
in electron density as compared to the control group
(Figure 4D). The mitochondria were elongated and their
surfaces became coarse, vague, broken and decreased in
electron density (Figure 4E). Furthermore, broken
cortical granules were also observed (Figure 4F).
Experiment 3: Gene expression of control and
vitrifiedwarmed canine oocytes
In order to assess the effect of vitrification on the
expression of stress (HSP70, Dnmt1 and SOD1) and
apoptosis-related genes (Bcl2 and BAX), the expression
pattern of these selected genes in control and
vitrifiedwarmed oocytes were analyzed (Figure 5). The
expression pattern of stress genes (HSP70, Dnnt1 and SOD1)
did not differ between control and vitrified-warmed
oocytes. HSP70 was strongly expressed at 0 h and
decreased over time, whereas Dnmt1 and SOD1 were
constantly expressed at 0, 24 and 48 h of cultivation.
For apoptosis-related genes (Bcl2 and BAX), Bcl2 was
constantly expressed in both groups but strongly
expressed in vitrified-warmed group when compared to
the control. BAX was absent at 0, 24 and 48 h of
Successful cryopreservation of canine oocytes is a
stepping stone towards applying assisted reproduction
technology (ART) for conservation of endangered
canids. To our knowledge, this is the first report
conducted on vitrification of immature canine oocytes.
Although the rate of meiotic resumption of
vitrifiedwarmed oocytes was significantly lower than those of
the control and exposure groups there was no
statistically significant difference in rates of meiotic
progression to the MII stage.
Various types of cryoprotectants (i.e., EG, DMSO,
glycerol, propylene glycol, polyethylene glycol and
1,2propanediol) have been used in different combinations
for the vitrification of mammalian oocytes and
embryos . EG has been found to be less toxic than
glycerol and propylene glycol to mouse embryos .
In addition, it was shown to allow much higher
survival rates of bovine embryos . DMSO has been
found to be an effective cryoprotectant for vitrification
of mouse and hamster oocytes [19,20]. In consequence,
Table 2 Mean (± SD) nuclear status of control, exposed and vitrified-warmed canine oocytes after in vitro culture for
Within a column, percentages without a common letter differed (P < 0.05).
Figure 3 Transmission electron micrographs of control canine geminal vesicle (GV) oocytes. A) Semi-thin section of a COC with a central
nucleus (N). The cumulus cells densely compact around the oocyte. B) The cortical zone of oocyte, contains a small alignment of cortical
granules (CG) beneath the plasma membrane and numerous mitochondria (M). C) The normal shape of lipid droplets (L) and mitochondria (M)
scatter in the pericortical cytoplasm of the oocyte. D) Smooth endoplasmic reticulum (SER) are scattered in pericortical cytoplasm. E) Higher
magnification of mitochondria showing outer and inner membranes (cristae) with matrix inside. F) Higher magnification of cortical granule (CG)
demonstrates the round shape with strong electron dense and smooth endoplasmic reticulum (SER). C = cytoplasm, ZP = zona pellucida, N =
nucleus, Cu = cumulus cells, CG = cortical granule, L = lipid droplet, SER = smooth endoplasmic reticulum.
EG and DMSO were chosen as cryoprotectants in this
present study to avoid ice formation based on the
evidence of previous studies in which cryoprotection was
useful for the successful vitrification of immature
buffalo oocytes . The vitrification technique used high
cryoprotectant concentrations, which have been
described as toxic to cells . Contrarily, an
appropriate phase composition of cryoprotectant mitigates the
toxic and osmotic consequences of highly concentrated
cryoprotectants . Thus, a mixture of
cryoprotectants can decrease individual specific toxicity. In the
present study, we vitrified canine oocytes in OPS using
mixture of 20% EG and 20% DMSO as previously
Vitrification of immature bovine and equine oocytes
using OPS resulting in subsequent cleavage and
development to the blastocyst stage has been reported
[25,26]. The OPS method used for cryopreservation of
oocytes offers many advantages over other methods. It
is simple, inexpensive, achieves a great increase in the
speed of cooling by reducing the volume to be vitrified
and by thinning the isolating layer between the cooling
agent (LN2) and the vitrification solution. A further
advantage of using very small volumes of vitrified
Figure 4 Transmission electron micrographs of vitrified-warmed canine germinal vesicle (GV) oocytes. A) Semi-thin section of a COC
with a central nucleus (N). The cumulus cells densely compact around the oocyte. B) The cortical zone increases mitochondria (M) numerical
density and decrease in cortical granule (CG) numerical density. C) Lipid vesicle turns into smaller droplets with broken membrane and replaced
by mitochondria (asterisks). D) Smooth endoplasmic reticulum vesicles (SER) are scattered in pericortical cytoplasm. E) Mitochondria (M) are
elongated and of decreased electron density. F) Showing broken CG (asterisk). C = cytoplasm, ZP = zona pellucida, N = nucleus, Cu = cumulus
cells, CG = cortical granule, L = lipid droplet, SER = smooth endoplasmic reticulum vesicle.
drops was a reduction of the amount of damage to the
zona pellucida which occurred during cooling and
Our results demonstrated that vitrified-warmed
immature canine oocytes in OPS can successfully resume
meiosis and develop to the MII stage following IVM.
However, the efficiency of oocyte cryopreservation
methods is still unsatisfactory. Vitrification procedure reduced
oocyte competence to resume meiosis. The assessment of
cryo-damaged processes and organelles is fundamental in
the evaluation and refinement of current and future
cryopreservation protocols. The ultrastructural alterations of
vitrified-warmed oocytes revealed by TEM indicated that
vitrification procedures affect the pericortical distribution
and morphofunctional integrity of cortical granule,
mitochondria, lipid droplet and SER.
Morphological examination demonstrated that cortical
granules were reduced in numerical density and were
damaged in vitrified-warmed oocytes, similar to results in
previous studies [12,28,29]. Wessel et al.  reported
that cortical granules are Golgi-derived,
membranebound spherical or slightly ovoid organelles formed
during the early stages of oocyte and at maturation. Cortical
granules are believed to establish the block to polyspermy
Figure 5 RT-PCR analysis. GAPDH, HSP70, SOD1, Dmnt1, Bcl-2 and
BAX cDNA expression in control and vitrified-warmed immature
oocytes following cultured 0, 24 and 48 h.
by preventing penetration of additional spermatozoa.
Fuku et al.  showed that the numbers of cortical
granules along the oolemma were substantially reduced,
and fusion of cortical granules with the plasma
membrane followed by exocytosis of granule core or intact
cortical granule into the perivitelline space was seen in
all vitrified bovine IVM oocytes. Ghetler et al.  found
that the cryopreservation procedure resulted in the loss
of cortical granules from the cortical area and in
appearance of vesicles within the cytoplasm of both immature
and mature human cryopreserved oocytes, which might
indicate structural damage occurring from the freezing
and warming process.
It has been suggested that the shape and intracellular
distribution of mitochondria were related to the level of
cell metabolism, proliferation and differentiation, and
that these organelles generate the essentials required in
a crucial period of the cell cycle . In addition, the
present study demonstrated that mitochondria
corresponding to the cortical zone were shown to increase in
numerical density, to elongate, develop a coarse surface,
broken and unclear, of decreased electron density.
Similar findings were reported in the exposure of porcine
oocytes to vitrification solutions . Roberto et al. 
reported mitochondria of frozen-thawed human oocytes
with decreased matrix electron density or with ruptures
of the outer and inner membranes. Mitochondria were
the most abundant organelles in mammalian oocytes
and their dysfunction or abnormalities would critically
determine oocyte and embryo developmental
competence. Structural changes to lipid droplets in the present
study were in agreement with previous reports in bovine
and porcine oocytes [13,31,32]. Xiang et al. 
suggested that the increased small lipid droplets came from
broken larger lipid droplets and exist in the form of
smaller drops during vitrification of porcine oocytes.
Isachenko et al. also reported that lipid droplets in porcine
oocytes changed morphologically during cooling; they
changed into a spherical form with lucent streaks .
The present study showed the expression of stress
genes (HSP70), Dnnt1 and SOD1 were similar in control
and vitrified-warmed groups. The identical expression
patterns of HSP70, Dnnt1 and SOD1 in the control and
vitrified groups, indicating that the vitrification protocol
did not alter the expression pattern of these stress
genes. It is well known that members of Bcl2 gene
family play a major role in regulation of apoptosis. In
that regard, Bcl2 is anti-apoptosis and promotes cell
survival, whereas BAX is pro-apoptosis and promotes cell
death . Apoptosis is an underlying process in oocyte
degeneration and embryo fragmentation . This
experiment found that Bcl2 was strongly expressed in
the vitrified-warmed group when compared to the
control, whereas BAX wasn’t expressed in both groups. Our
results were similar to report in mouse embryos that the
expression of BAX gene did not differ from control
when morulae were vitrified . Contrarily, Dhali et al.
 showed that Bcl2 displayed a greater decrease in
the vitrified mouse embryos compared to control and
expression of BAX gene was down-regulated in the
treated group. The expression of Bcl2 was higher in normal
than fragmented ones, and the expression did not vary
significantly among embryos of varying quality .
Moreover, the expression of Bcl-2 was comparatively
lower in the vitrified embryos than the normal embryos
. Therefore, the differential pattern of gene
expression observed in the present study may be used to
predict the quality and developmental ability of vitrified
immature canine oocytes.
In conclusion, the present study demonstrated that it
is possible to cryopreserve immature canine oocyte by
vitrification using EG in combination with DMSO and
modified OPS protocol. Our results showed that
vitrified-warmed immature canine oocytes can resume
meiosis and develop to the MII stage. Further studies are
required to investigate the fertility and developmental
ability of MII oocytes following fertilization.
We thank Professor LM Lewin for critical reading and editing the manuscript.
This study was supported by Mahidol Research Fund, Mahidol University.
BT participated in all aspects of the experiment and writing the manuscript.
PS contributed to the RT-PCR analysis. KS and YK designed the experiment
and contributed to the analysis and discussion of data. All authors read and
approved the final manuscript.
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