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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