gene expression and development of mouse zygotes following droplet vitrification
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Gene expression and development of mouse zygotes following
droplet vitrification
A. Dhali, V.M. Anchamparuthy, S.P. Butler, R.E. Pearson, I.K. Mullarky,F.C. Gwazdauskas *
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0315, USA
Received 31 May 2007; received in revised form 22 August 2007; accepted 28 August 2007
www.theriojournal.com
Theriogenology 68 (2007) 1292–1298
Abstract
The concept of ultra-rapid vitrification has emerged in recent years; the accelerated cooling rate reduced injury attributed to
cryopreservation and improved post-freezing developmental competence of vitrified oocytes and embryos. The objectives of the
present study were to develop a simple and effective ultra-rapid vitrification method (droplet vitrification) and evaluate its effects on
post-thaw development and apoptosis-related gene expression in mouse zygotes. Presumptive zygotes were equilibrated for 3 min
in equilibration medium and washed 3 times in vitrification solution. A drop (5 mL) of vitrification solution containing 10–12
embryos was placed directly onto surface of liquid nitrogen, with additional liquid nitrogen poured over the drop. For thawing and
cryoprotectant removal, vitrified drops were put into dilution medium for 3 min, followed by M2 medium for 5 min. Although
cleavage rate did not differ significantly among the control (90.8 � 2.8%; mean � S.E.M.), toxicity control (83.5 � 3.2%), and
vitrified (86.2 � 3.1%) zygotes, rates of blastocyst and hatched blastocyst formation were lower (P < 0.01) in vitrified zygotes
(49.7 � 4.7% and 36.0 � 4.7%) and toxicity controls (47.3 � 4.6% and 40.3 � 4.6%) compared with controls (65.5 � 4.1% and
54.2 � 4.3%). Exposure of zygotes to vitrification solution, as well as the vitrification process, down-regulated the expression of
Bax, Bcl2, and p53 genes in blastocysts. Although droplet vitrification was efficient and easy, it altered the transcriptional activities
of Bax, Bcl2, and p53 genes in vitrified embryos, indicating a strong relationship between reduced developmental competence and
the altered transcriptional activities of these genes.
# 2007 Elsevier Inc. All rights reserved.
Keywords: Cryopreservation; Vitrification; Mouse; Zygotes; Apoptosis genes
1. Introduction
Cryopreservation of oocytes and embryos is an
integral part of assisted reproductive technology.
During the past decades, various methods of oocyte
and embryo cryopreservation have been standardized.
Among these methods, vitrification is now considered a
viable method to preserve oocytes and embryos [1].
* Corresponding author. Tel.: +1 540 231 4756;
fax: +1 540 231 5014.
E-mail address: guaz@vt.edu (F.C. Gwazdauskas).
0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2007.08.030
Vitrification is the process that induces a glass-like
solidification of living cells during freezing. The unique
advantage of the vitrification process is elimination of
ice crystal formation, the most dangerous cause of
cryoinjury. Vitrification can be induced when using high
concentration of cryoprotectants.
Efforts to induce ultra-rapid cooling during vitrifica-
tion used various methods, including dropping embryos
or oocytes containing solutions (droplet vitrification)
directly onto the surface of liquid nitrogen [2–5],
using electron microscope grids [6], open pulled straws
[7], cryoloops [8], or solid surface vitrification [9].
Ultra-rapid cooling during vitrification was successfully
A. Dhali et al. / Theriogenology 68 (2007) 1292–1298 1293
Fig. 1. Schematic presentation of the droplet vitrification method;
VS-1: vitrification solution 1 (1.8 M ethylene glycol (EG) and 20%
fetal bovine serum (FBS) in M2 media); VS-2: vitrification solution 2
(5.5 M EG, 20% FBS, and 0.5 M sucrose in M2 media).
achieved by reducing the volume of the vitrification
solution [10,11]. The open pulled straw method
achieved a very high cooling and warming rate during
vitrification and thawing [11]. Among the various
methods of ultra-rapid cooling, droplet vitrification was
easy and convenient, as it excluded the use of any
specialized device to introduce oocytes or embryos into
liquid nitrogen. Nevertheless, in the previous reports,
the major limitation of the droplet vitrification method
was either the use of a relatively large volume drop or
prolonged exposure of embryos or oocytes to cryopro-
tectants during the process.
The objective of this study was to use the murine
model to evaluate the effects of the modified droplet
vitrification method on embryo survival and develop-
ment, and to assess the expression of apoptosis-related
genes in blastocysts developed from the vitrified
zygotes (in an attempt to relate expression to devel-
opment). Apoptosis is a highly conserved and regulated
program that initiates cells death under a variety
conditions. Members of the Bcl2 gene family played
roles in regulating apoptosis; Bcl2 was anti-apoptotic
and promoted cell survival, whereas Bax was pro-
apoptotic and accelerated cell death [12]. Furthermore,
the tumor suppressor p53 was an important mediator of
responses to cellular stress [13,14].
2. Materials and methods
All chemicals and media were purchased from
Sigma–Aldrich Co. (St. Louis, MO, USA) unless
otherwise mentioned.
2.1. Embryo collection and culture
Female mice (strain ICR), aged 7–12 week, were
given 5 IU of eCG ip. After 48 h, females were given 5 IU
hCG ip and placed individually in cages with mature
males (strain ICR). After 18–20 h, the females were
sacrificed to harvest embryos. Presumptive zygotes were
harvested at the cumulus stage (day 0) and placed into M2
medium at 37 8C. The cumulus cells were removed from
embryos in 2 mL M2 medium containing hyaluronidase
(300 U/mL). Approximately 10–15 embryos were
cultured (until day 5) in each 20 mL droplet of potassium
simplex optimized medium (KSOM) plus amino acids
(AA; Chemicon International Inc., Temecula, CA, USA),
overlaid with light mineral oil (Chemicon International
Inc.). Developmental stages were recorded on each day
during the culture. A developmental score (0 = degen-
erate or 1-cell embryo; 1 = 2-cell embryo; 2 = 4- to 8-cell
embryo; 3 = morula; 4 = blastocyst; and 5 = hatched
blastocyst) was assigned to each embryo and an average
developmental score was calculated (total developmental
score/total number of embryos) for each treatment.
2.2. Vitrification and embryo recovery
Presumptive zygotes were vitrified using the modified
droplet vitrification method. The equilibration medium
(VS-1) consisted of 1.8 M ethylene glycol (EG;
Mallinckrodt Baker Inc., Phillipsburg, NJ, USA) and
20% fetal bovine serum (FBS; Gibco, Grand Island, NY,
USA) in M2 medium. The vitrification solution (VS-2)
consisted of 5.5 M EG, 20% FBS, and 0.5 M sucrose in
M2 medium. The schematic diagram of the vitrification
procedure is shown (Fig. 1). Briefly, 10–12 embryos were
A. Dhali et al. / Theriogenology 68 (2007) 1292–12981294
equilibrated in VS-1 for 3 min and subsequently washed
3 times in VS-2. The embryos were then aspirated, along
with VS-2, into a pipette. The pipette was held
horizontally until a drop (5 mL) containing embryos
was formed at the pipette tip. The pipette was then held at
a 458 approximately 5 cm above the surface of liquid
nitrogen (10 cm � 10 cm � 4 cm container) and the
drop was placed onto the surface of the liquid nitrogen by
shaking gently, and liquid nitrogen was poured over the
drop. The drop was vitrified and sank into liquid nitrogen
immediately. The time between the contact of embryos
with VS-2 and cooling did not exceed 45 s. For storage,
vitrified drops were placed in a pre-cooled 2 mL cryovial
(2 or 3 drops/vial) filled with liquid nitrogen with the help
of a pre-cooled forceps. The vial was quickly capped and
placed into a canister filled with liquid nitrogen; the top of
the canister was closed with a rubber cork and the canister
was placed in a liquid nitrogen container immediately.
During thawing, the contents of the cryovial were
quickly emptied into a small plastic container filled with
liquid nitrogen and the vitrified drops settled at the
bottom of the container. The vitrified drops were
transferred into dilution medium (0.3 M sucrose and
10% FBS in M2 medium) with the help of pre-cooled
forceps. Embryos were kept in dilution medium for 3 min
and then transferred into M2 medium and kept for 5 min.
The morphologically normal embryos were then subse-
quently washed in M2 and KSOM media and retained for
culture. An embryo was considered morphologically
normal in the absence of any signs of abnormal shape,
membrane damage, leakage of cellular content and
fragmentation, or degeneration of cytoplasm. The
temperature of all media was maintained at 37 8C.
During preliminary trials, developmental competence
of the vitrified embryos did not differ when warmed after
1 h or 2 weeks following the vitrification (data not
shown). Therefore, during the subsequent experiments,
vitrified embryos were warmed after 1 h following
vitrification. During the experiment, embryos were
cultured separately for control and toxicity control
groups. The embryos of control group were not exposed
to vitrification solution and liquid nitrogen. The embryos
of toxicity control group received all the treatments of
vitrified embryos, but they were not exposed to liquid
nitrogen. The experiment was performed in three
replicates of 25 embryos per treatment group.
2.3. Reverse transcription and quantitative real-
time polymerase chain reaction (PCR) analysis
The relative quantification of all gene transcripts was
carried out by real-time quantitative PCR (RT-PCR).
Three replicate PCR experiments were conducted for all
genes of interest, using blastocysts from the three
experimental pools. Blastocysts were collected at day 5
of culture and washed 3 times in Dulbeccos PBS
(Gibco) containing 0.1% polyvinyl alcohol (PVA) and
stored in pools of 25–30 embryos at �80 8C in a
minimum volume of media until further use. This
procedure inhibited RNase effectively [15].
Total RNA was isolated from blastocysts using
Absolutely RNA1 Microprep Kit (Stratagene, La Jolla,
CA, USA) following the manufacture’s protocol. The
RNA concentration of the samples was determined with
a ND-1000 spectrophotometer (Nanodrop Technolo-
gies, Wilmington, DE, USA). Purified RNA was
transcribed into cDNA immediately using the High-
Capacity cDNA Archive Kit (Applied Biosystems,
Foster City, CA, USA), following the manufacture’s
instructions.
Real-time PCR (RT-PCR) was performed in a 7300
Real-Time PCR System (Applied Biosystems) using a
SYBR1 Green detection protocol, as per the manufac-
ture’s instructions. Briefly, 3 ng of total cDNA, 0.2 mM of
each primer and 1� SYBR1 Green Mix (Applied
Biosystems) were used (total volume, 25 mL). When the
reaction was completed, melting curves were plotted to
confirm the product purity. Previously reported [16],
murine-specific primers were used to amplify b-actin
(sense 50-TTCGTTGCCGGTCCACA-30 and antisense
50-ACCAGCGCAGCGATATCG-30), Bax (sense 50-GG-
AGCAGCTTGGGAGCG-30 and antisense 50-AAA-
AGGCCCCTGTCTTCATGA-30), and Bcl2 (sense 50-ACTTCGCAGAGATGTCCAGTCA-30 and antisense
50-TGGCAAAGCGTCCCCTC-30). Whereas, to amplify
p53 (Gene bank accession number AY044188), murine-
specific primers (sense 50-AAAGGATGCCCATGCTA-
CAGAGGA-30 and antisense 50-AGTAGACTGGC-
CCTTCTTGGTCTT-30) were designed. The relative
abundance of gene transcripts was determined using a
2�DDCt method and b-actin was used as an endogenous
reference gene [17,18].
2.4. Statistical analysis
Data were analyzed using the SPSS 10.0.1 software
package (SPSS Inc., Chicago, IL, USA). Variations in
the occurrence of different developmental stages (2-cell
through hatched blastocyst) were analyzed among
treatments, each embryo was assigned a score of either
1 (embryo developed into the stage of interest) or 0
(embryo did not develop into the stage of interest) and
subjected to ANOVA, followed by multiple pairwise
mean comparisons using Student–Newman–Keuls
A. Dhali et al. / Theriogenology 68 (2007) 1292–1298 1295
Table 1
Mean (�S.E.M.) recovery, morphology and in vitro development of mouse zygotes in various treatments
Treatment
group
Exposure to
VS-1 (3 min)
Exposure to
VS-2 (45 s)
Vitrified Zygotes
recovered
Morphologically
normal zygotes
Cleavage
(%)
Morula
(%)
Blastocyst
(%)
Hatched
blastocyst (%)
Control � � � � 156 90.8 � 2.8 72.7 � 3.9 a 65.5 � 4.1 a 54.2 � 4.3 a
Toxicity
control
+ + � 146 146 83.5 � 3.2 54.9 � 4.5 b 47.3 � 4.6 b 40.3 � 4.6 b
Vitrified + + 162 157 145 86.2 � 3.1 61.3 � 4.5 b 49.7 � 4.7 b 36.0 � 4.7 b
VS-1: vitrification solution 1 (1.8 M ethylene glycol (EG) and 20% fetal bovine serum (FBS) in M2 media); VS-2: vitrification solution 2 (5.5 M EG,
20% FBS, and 0.5 M sucrose in M2 media). a, b: within a column, means without a common letter differed (P < 0.01).
Fig. 2. In vitro development (mean percentage � S.E.M.) of cleaved
mouse zygotes in a various groups; control: fresh embryos; toxicity
control: embryos exposed to the vitrification solutions without vitri-
fication; vitrified: embryos exposed to the vitrification solutions and
vitrified; A, B on error bar indicated values differed (P < 0.01); a, b on
error bar indicated values differed (P < 0.05).
(SNK) test. The model included treatments (fixed
effect) and day of experiment (random effect) as sources
of variation. Data on developmental score were
analyzed using ANOVA followed by multiple pairwise
mean comparisons using an SNK test. The model
included treatments (fixed effect) and day of experiment
(random effect) as sources of variation. Student’s t-test
was used to analyze the variations in different gene
transcripts assayed by quantitative RT-PCR.
3. Results
High recovery of the embryos and morphologically
normal embryos following warming was evident when
presumptive mouse zygotes were vitrified using the
droplet vitrification method (Table 1). Control and
toxicity control (exposure to cryoprotectants without
vitrification) recovery and development are also shown.
There were no significant difference in cleavage rates
among the vitrified, toxicity control and control
embryos. In contrast, the proportions of embryos that
developed into morula, blastocyst and hatched blas-
tocyst were higher (P < 0.01) in control zygotes.
However, there were no significant differences in
embryo survival and development between zygotes
exposed to vitrification solution and those that were
vitrified (Table 1); the results were similar when
developmental stages were calculated on the basis of
cleaved embryos (Fig. 2). Furthermore, the in vitro
developmental score (�S.E.M.) was higher (P < 0.01)
in control embryos (3.66 � 0.15), but the develop-
mental score did not differ significantly between
toxicity control embryos (2.91 � 0.17) and those that
were vitrified (3.06 � 0.17).
In order to assess the relationship between devel-
opment and apoptosis-related genes, the relative
expression pattern of the three apoptosis-related gene
(Bax, Bcl2, and p53) transcripts in control, toxicity
control, and vitrified embryos were evaluated (Fig. 3).
The expression of Bax was down-regulated (P < 0.05)
in blastocysts when zygotes were exposed to vitrifica-
tion solution alone, with further down-regulation
(P < 0.05) of Bax when zygotes were vitrified. The
expressions of Bcl2 and p53 were also down-regulated
(P < 0.05) in the toxicity control and vitrified embryos.
Although the expressions of Bcl2 and p53 were
comparatively lower in the vitrified embryos than the
embryos exposed to vitrification solution, the reduc-
tions were not statistically significant. Down-regulation
of Bcl2 in the treated embryos was more obvious than
Bax and p53.
4. Discussion
We demonstrated a simple, fast, and efficient droplet
vitrification method that can be used to cryopreserve
presumptive zygotes; zygotes retained a high develop-
mental competence following vitrification and thawing.
However, the expression patterns of Bax, Bcl2 and p53
A. Dhali et al. / Theriogenology 68 (2007) 1292–12981296
Fig. 3. Changes (�S.E.M.) in the relative expression of apoptosis-related genes (Bax, Bcl2 and p53) in mouse blastocysts in different groups.
Control: fresh embryos; toxicity control: embryos exposed to the vitrification solutions without vitrification; vitrified: embryos exposed to the
vitrification solutions and vitrified; b-actin was used as endogenous reference gene; a, b on error bar indicated values differed (P < 0.01).
were significantly altered when zygotes were vitrified or
exposed to vitrification solution without vitrification.
Vitrification is a potential alternative to traditional
slow-rate freezing for preserving oocytes and embryos
in various species. The unique advantage of vitrification
is elimination of mechanical injury caused by intra- or
extra-cellular ice crystal formation, and reduction of
chilling injury, by shortening the duration of exposure
of cell to the critical temperature zone. The concept of
ultra-rapid vitrification has emerged in recent years [2–
9]. An ultra-rapid cooling rate during vitrification can
practically be achieved either by minimizing the volume
of solution to be vitrified, by making direct contact
between vitrification solution and liquid nitrogen, or
through the combination of both. The major disadvan-
tages of the previous droplet vitrification protocols were
the relatively large volume of the vitrified drop and the
delay before the drop floating on the surface of liquid
nitrogen sank, which probably reduced the actual
cooling rate [1–5]. We have eliminated these dis-
advantages and tried to achieve a higher cooling rate by
reducing the volume of vitrified drop and by pouring
liquid nitrogen immediately over the drop once it was
placed onto the surface of the liquid nitrogen (which
helped the drop to quickly vitrify and sink). In addition,
we attempted to reduce cytotoxicity by shortening the
total duration of exposure of embryos to cryoprotectants
and by using an EG-based vitrification solution. It was
reported previously that EG had low cytotoxicity for
embryos [19,20]. Our method ensured a rapid thawing
rate of vitrified embryos by directly placing the vitrified
drop into sucrose solution at 37 8C. As expected, we
achieved high developmental competence of the
vitrified embryos. The blastocyst formation rate of
the vitrified embryos was similar to previous reports that
used a solid surface vitrification method or an open
pulled straw method to cryopreserve pronuclear mouse
embryos [21,22].
In the current study, we evaluated the effect of
droplet vitrification on the expression of three
apoptosis-related genes: Bax, Bcl2, and p53. Apoptosis
is a highly conserved and regulated program that
A. Dhali et al. / Theriogenology 68 (2007) 1292–1298 1297
initiates cells death under a variety of internal and
external controls. Among the different genes involved
in apoptosis, members of the Bcl2 gene family played
key roles in regulating apoptosis. At least 15
mammalian Bcl2 gene family members have been
identified that are involved in the process. Among these,
Bcl2 was anti-apoptotic and promoted cell survival,
whereas Bax was pro-apoptotic and accelerated cell
death [12]. The tumor suppressor p53 was an important
mediator of responses to cellular stress [13,14]. The
transcriptional activity of p53 protein was necessary for
cell death in some systems [23]. Although nuclear
translocation of the p53 protein was required for it to act
as a transcription factor for the Bax gene, apoptosis
appeared to have both transcriptionally dependent and
independent mechanisms [24]. Apoptosis is an under-
lying mechanism of oocyte degeneration and embryo
fragmentation [25]. Therefore, the decreased develop-
mental competence of embryos may be related to the
occurrence and regulation of embryonic apoptosis. In
our study, that the cleavage rate was not significantly
different between vitrified and control embryos indi-
cated that the embryos withstood the exposure to
vitrification solution or vitrification and initially
survived. Nonetheless, the developmental competence
of the vitrified or toxicity control embryos was
compromised, which was evident from the significantly
lower blastocyst formation rate. The expressions of all
three genes were down-regulated in the treated
embryos. The proportions of Bcl2 to Bax most likely
had changed in the vitrified and toxicity control
embryos, as Bcl2 displayed a greater decrease in the
treated embryos compared to controls. Interaction
between the Bcl2 family related proteins may play a
critical role in pre-implantation embryo development
[20]. The expression of Bcl2 was higher in normal
embryos than fragmented ones, and the expression of
Bax did not vary significantly among embryos of
varying quality. Therefore, the ratio of Bcl2 to Bax may
be used to predict the tendency of embryos towards
survival or apoptosis [20,25]. The activation of p53
stress-sensing pathway in the in vitro generated
embryos could be a major cause of the loss of
developmental competence [26]. The results of the
present study clearly indicated a strong relationship
between the compromised developmental competence
and altered transcriptional activities of Bax, Bcl2, and
p53 genes in the treated embryos.
In conclusion, the modified droplet vitrification
protocol was an efficient and easy ultra-rapid cooling
method for cryopreserving zygotes that resulted in high
recovery and survival of vitrified mouse zygotes.
Nevertheless, there were altered transcriptional activ-
ities of Bax, Bcl2, and p53 genes in embryos that were
exposed to vitrification solution and vitrified.
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