Cryopreservation of kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols

ELSEVIER

CRYOPRESERVATION

OF KUNMING

ULTRARAPID

MOUSE OOCYTES USING SLOW COOLING,

COOLING AND VITRIFICATION

PROTOCOLS

Shang, SC. Yang and R.J. Zou

H.S. Men, J.C. Chen, W.Z JI ‘E.Y.

Kunming Institute of Zoology, Kunmin g Cell Bank, The Chinese Academy of Sciences Kunming,

Yunnan

650223, P.R. China

Received for publication: Accepted:

Augus t 2 3 , 19 9 4 May 6,

I996

ABSTRACT The cryopreservation protocols,

including

of oocytes has been only marginally

slow cooling, rapid cooling and vitriication.

that oocytes from a single mouse strain would freeze successfully Unfertilized randomly

Kunming assigned

mouse

oocytes

obtained

to be cryopreserved

14 h after

after slow cooling,

successful

with any of the current

We wished to test the hypothesis by 1 of the 3 mentioned PMSG/hCG

ultra rapid cooling

cryopreservation.

capability

Survival

broken membranes

were compared

of oocytes

or zona pellucida.

cell embryo after IVF Survival

appearance

with that of oocytes that had not undergone

was indicated Functional

were

and vitrification.

Oocytes were thawed by straws being placed into 37 ‘C water, and their morphological and in vitro fertilization

protocols.

administration

by the absence

integrity

of darkened

was evaluated

ooplasm

by the formation

or by of a 2-

rate of slow cooled oocytes did not differ from that seen in vitrified

oocytes (55.1 vs 65.9%) but was significantly

lower in the rapidly cooled oocytes (24.2%; PiO.01).

The results of IVF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P>O.O5). It appears using

the slow

permeating

cooling

method

and nonpermeating

that Kunmin g mouse oocytes can be successfully

with

1,2-propanediol

and vitrification,

which

cryopreserved contains

both

cryoprotectants.

0 1997 by Elsevler Science lnc Key words:

Kunmin g mouse oocytes, slow cooling, ultrarapid cooling, vitrification,

cryoprotectants

INTRODUCTION Various freezing techniques for the rryopreservation of embryos have been developed since the pioneering work of Whittingham et al(32). Among these, slow cooling, ultrarapid cooling and Acknowledgments This work was supported by funds from the National Nature Sciences Foundation, 39170109,

fund No.

and from the Applied and Basic Research Fund ofYunnan

‘Correspondence

and reprint requests.

Thenogenology 47:1423-l 431, 1997 0 1997 by Elsevier Science Inc.

FAX: (0871) 5151823 to Dr. Weizhi Ji.

0093-69 1X/97/$1 7 00 PII SOO93-691X(97)00133-7

1424

Theriogenology

vitrification

and have all worked

Unfortunately,

recent attempts

such protocols successful (16,1,30)

well for the cryopreservation

to cryopreserve

are not easily transferable

cryopreservation

to oocytes.

Although

of various

species.

have indicated

that

there have been several reports on

of mouse (2, 3, 5, 12, 18, 22, 25), rabbit (11, cow (13, 28) and human

oocytes, the survival and fertilization

Currently,

of embryos

oocytes with these methods

rates have varied.

most of the data on the cryopreservation

of mouse oocytes have been obtained &om

the cooling of oocytee from different strains of mice. There are few reports on the cryopreservation freezing

have indicated

and oocytes is affected by genotype

that the post-thaw

(12, 311, thus oocytes

viability

from different

of mouse embryos

strains

behave

differently

methods.

during

Previous

of

mouse oocytes from the same strain of mouse using various

cooling

studies

and warming.

Therefore, in our present study we used oocytes from the K unming mouse to test 3 selected hezing methods

(slow cooling, ultrarapid

cryopreservation

cooling, and vitrification)

in order to assess the efficacy of each

technique while avoiding the influence of strain differences MATEXIALS

AND METHODS

source of oocytes Female Kunmin g mice (6 to 8 wk old) were induced to superovulate of 10 IU PMSG and, 48 h later,

10 IU hCG. Intact cumulus

oviducts into HTF (human tubal fluid) medium containing hCG injection. washed

After the cumulus

3 times

containing

in HTF medium

by intraperitoneal were released

0.1% hysluronidase

cells had been detached and incubated

masses

from the oocytes,

for approximately

injection

from excised

(25) 14 to 15 h after the oocytes

were

20 min in HTF medium

20% fetal calf serum (FCS, Sigma, St. Louis, MO, USA) at 37 ‘C under 5% CO2 in air

before cryopreaervation. Freezing and Thawing Oocytes vitritlcation

were frozen by slow cooling

with Hepes-buffered Hepes-buffered

HTF with 20% heat-inactivated

for 5 min each

propauediol

A; 291, ultrarapid

cooling

(Group

B; 25), and

serum (HS), and for Group C with

Table 1) oocytes

freezer

were equilibrated

at room temperature

to 0.5 M, 1.0 M, 1.5 M, 1.2-propanediol

plus 0.2 M sucrose.

programmable

human

saline (HBl; Table 2).

Group A (slow cooling; exposure

(Group

(Group C; 17,181 protocols (‘lhble 1). Freezing solutions for Groups A and B were made

(Planner

first cooled to -7 “C at 2 oC/min.

and finally

with serial

to 1.5 M 1.2-

Groups of 15 to 20 oocytes were frozen in 0.25~ml straws with a KYRO

10, Perkasie,

After manual

PA, USA). After equilibration,

seeding,

oocytes were

the oocytes were subsequently

cooled at

Theriogenology

1425

0 3 “C/min to -30 “C and finally plunged into liquid nitrogen. released into 1.0 M l.‘&propanediol 1.2-propanediol

Oocytes were thawed at 37 “C! and

plus 0.2 M sucrose, followed by subsequent

dilutions in 0.5 M

plus 0.2 M sucrose and in 0.2 M sucrose alone. The oocytes were finally washed in

Hepes-HTF with 5 mg/ml BSA. Table 1. The freezing and thawing protocols for unfertilized Kunming mouse oocytes Group A (room temperature) Freezing: 0.5 M PDa‘C+hold

1.0 M PD-1.5

M PD-1.5

1 min and manual seeding-hold

Thawing:

37 ‘C water bath-l.0

sucrose AH-HTP/20%HSdH-HT’F/5

M PD/O.2 M sucrose+cool2

5 mix+coolO.3

‘Ctmin to -7

OCAnin to -30 ‘C-LN2

M PD/0.2 M sucrose-O.5

M PD/0.2 M sucmse+0.2

M

mg/ml BSA

Group B (room temperature) Freezing: 0.25 M sucrose-+0.5 Thawing:

M sucrose~0.5

37 “C water batlaO.

M sucrose/3.5 M DMSO-LN2

M sucrosedO.

M sucrose+H-HTP/20%HS+H

HTP/5 mg/ml BSA Group C (cold room, 4 ‘0 Freezing: VSlb-LNz Thawing:

0 ‘C water bath+506

VSl,

lOmin+25%

VSl,

l&nm+washed

4 times

in PBl

(roomtemperature ) a. 1,2-propanediol. b: VSl: 20.5% (w/v) dimethyl sulfoxide, 15.5% (w/v) acetamide, 10% (w/v) propylene glycol, 6% (w/v) Polyethylene glycol (Mr 8000). Group B (ultrarapid

cooling;

exposure to 0.25 M sucrose,

Table 1) oocytes

underwent

3-&p

equilibration

with serial

0.5 M sucrose and 0.5 M sucrose plus 3.5 M DMSO for 5 min each

before being plunged into liquid nitrogen. Thawing was achieved by agitatig

the straws in a 37 ‘C

water bath. Oocytes were diluted in solutions of decreasing sucrose concentrations

and Hepes-HTP

containing 20% human serum. Finally, oocytes were washed in Hepes-HTP with 5 mg/ml BSA. Group C (vitrification; 0 25-ml straws Immediately 25% VSl

Table 1) oocytes were transferred

and equilibrated

into 90% VSl and then loaded into

for 5 min at 4 ‘C before being plunged into liquid nitrogen.

after thawing in a 37 “C water bath, the oocytes were placed into 50% VSl and then

for 10 min each at 4 ‘C. F’inally, the thawed

oocytes

were washed in PBl at room

temperature The toxicity of freezing

media also influences

the survival

and fertilizing

undergoing freezing. We tested solution toxicity by exposing oocytes (Groups

A

ability of oocytes Band C:) to freezing

Theriogenology

1426

media in a stepwise fashion, as listed for each group, without freezing them. We also compared the potential

of frozen-thawed

oocytes to be fertilized

in vitro and subsequent

development

to 2-cell

stage embryos by fertilizing oocytes in vitro without treatment (control). Morphological Observation The frozen-thawed onto an inverted

oocytes were transferred

microscope

for morphological

into fresh medium after dilution and mounted observation.

pellucida /or without a dark ooplasm were classified /or with a broken zona or membranes

Oocytes

with an unbroken

zona

as living, while oocytes with a dark ooplasm

were classified as dead. The living oocytes were subsequently

used for in vitro fertilization. Table 2. Composition of HTP, PBI, PBS and Hepes-buffered

saline in the vitrification

solution

Final concentration (mM) Component NaCl

PBl

HTF 101.60

Vitrification

136.9

136.90

KC1

4.69

2.68

2.68

Mgso4.7Hzo

0.20

__

__ 0.88

KHzPO4

0.37

1.47

MgCl2.6H20

-_

0.49

0.25

NazHP04

-_

8.04

__

CaClZ NaHC03

2.04 25

0.90

0.07

_-

__

Glucose

2.78

5.56

Hepes

-_

__

Na pyruvate Na lactate BSA

0.33

0.33

__

.4.35

__

-_ 100 &Ill

Streptomycin SO4

50 ug/ml

Phenol red

5.56 20.00

21.40

Penicillin

0.001% (WVVOl)

HTF’=human tubal fluid; PBl=moditied

solution

4 mg/ml

0.75 mg/ml

100 ulml

__

__

__

0.001%

(wvvol)

Dulbecco’s phosphate-buffered

0.001% (wt/vol) saline.

Theriogenology

1427

In Vitro Fertilization Spermatozoa

were obtained from the cauda epididymidis

males and suspended in HTF medium containing 5% COZ in air. After incubation morphologically

and the unfrozen coincubation,

oocytea (control) incubated

was adjusted

for 1 h at 37 “C under to lo8 sperm/ml

(Groups A to C), the freezing medium-exposed

were introduced

the oocytes were transferred

and were further

10% BSA, then incubated

the sperm concentration

normal cryopreserved

of 12 to 15 wk old Kunming mouse

into the sperm suspension.

into HTF medium containing

for an additional

The

(Groups A Cl

Following

6 h of

0.3% BSA, 1.0 mM EDTA

24 h, after which the 2-cell stage embryos

were

counted. RESULTS Oocytes underwent

shrinkage due to osmotic dehydration during equilibration.

protocol, the process of shrinkage

In slow cooling

was relatively slow and caused little damage to oocytes, and the

oocytes regained their initial spherical shape when transferred into Hp-HTF (Hepes-HTF) In contrast, a high concentration caused drastic shrinkage

of cryoprotectant

in the vitrification

and even the breakdown

of oocytes.

observed that about 10% of the vitriIication medium-exposed

medium

solution of Rall and Fahy (18)

During the dilution

step, we also

oocytes exhibited the characteristic

of

a dark ooplasm. In ultrarapid cooling, most of the oocytes were killed during the process of freezing and thawing. The results of the present study are detailed in Table 3. Similar survival rates of oocytes were observed between Groups A and C (P>O.O5). Fertilization embryos to the total number of oocytes inseminated rates were compared solution-exposed the frozen-thawed

at 2 levels:

groups; 2) frozen-thawed protocols.

dikference compared

Group B was significantly

in each Group. In this study the fertilization groups

versus

the corresponding

groups versus controls. The fertilization

groups and the corresponding

(P>O.O5) in the 3 employed significant

1) frozen-thawed

rates were defined as the ratio of 2-cell

freezing solution-exposed

The fertilization

freezing

rates between

groups were very similar

rates of Groups

A and C showed

with those of the control group. However, the fertilization

no

rate in

lower than that in Groups A, C and the controls (P
Previous findings have demonstrated ment differ physiologically different freezing techniques. a demonstrably

fertile

that oocytes and embryos at various stages of develop-

and morphologically

(7, 8, 10) and that different

cell types require

Unfertilized oocytes have been diEcult to cryopreserve

state. The ability of unfertilized

oocytes

to withstand

while retaining freezing

affected by the genetic strain (24, 31) and by species (4, 10, 12, 14, 15). The variations reflect differences in cell size, membrane permeability et al. (32) obtained a 48% fertilization by slow thawing.

and intracellular

rate of frozen-thawed

composition.

may be probably

Whittingham

mouse oocytes by slow cooling followed

Surrey and Quinn (25) compared the results of cryopreservation

of oocytes from

Theriogenology

1428 Table 3. Survival,

fertilization

and development

in vitro of mouse oocytes

cryopreserved

with

different freezing methods Oocytes used

Freezing methods

oocyte

(n)

Fertilized oocytes

survival

(%)

n

355 (55.1)

258 (72.1)

258

484

418 (86.4)

303 (72.5)

303

643

148 (24.2)

85 (58.6*)

85

509

475 (93.3)

291 (61.3*)

291

316

210 (65.9)

155 (73.9)

155

339

266 (78.5)

A: slow cooling; a: slow-cooling-solution-exposure; solution-exposure;

C: vitrification;

hybrid

mice by employing

method. They achieved satisfactory study to cryopreserve

192 (72.2)

192

405 (77.5)

405

B: ultrarapid cooling; b: ultrarapid-coobng-

c: vitri&ation-solution-exposure;

*: Significant difference with the control (P
present

embryos

647

512

C57BU6XC3H

2-cel.l stage

(8)

D: control.

t-test).

the ultrarapid

cooling

method

result5 in both groups. The 2 protocols

Kunming

mouse oocytes:

we obtained

and the slow cooling were also used in this

55.1% survival

and 72.1%

fertilization rate using the slow cooling method but only 24.2% survival and 58.6% fertilization employing

ultrarapid

oocytes may respond

cooling

method.

differently

This observation

to cryopreeervation

suggests

that Kunming

mouse

Partial dehydration

formation

of the cytoplasm

is essential for the survival of cryopreserved

cryopreservation

solution, which was originally

developed by Pall and

has been succe55fidly

of various species (11, 19, 26-28). However, vitritication

(4). High rates of survival

and fertilization

of mouse

employed

our study also. The result was comparable (data not shown) demonstrated cryoprotectants

to that of Nakagata.

Nevertheless,

that oocytes exposed to vitrification

(propyleneglycol

survival. This suggested that vitrification

the

in embryo

of oocytes needs further

oocytes

were achieved

Nakagata (12) using the medium of F&dl and Fahy (18). We used a similar vitrification

permeating

of

embryos

a glassy state not only within but also outside the cell and thus prevents

of ice crystal5 during cooling. Vitrification

investigation

incidence

observations).

and oocytes before cooling. The vitrification Fahy (18), induces

strain

since they require a longer time (6 h) than

oocytes from other strains of mice (4 h) to be fertilized in vitro without a significant polyspermy (our unpublished

rate

by

procedure in

our previous

work

medium which contains only

and glyceml) resulted in very poor post-thaw

solutions which contain both permeating

oocyte

cryopmtectants

Theriogenology

1429

(DMSO, glycerol, acetamide,

and propyleneglycol)

glycol) provide better protection oocytes. Vitrification

solutions which contain only permeating

can cause membrane membrane

function

equilibration

Ledezma

breakage

cryoprotectant

due to oocyte swelling

due to the large amount

cryoprotectanta

(polyethylene

to the survival

of

at high concentrations

(22), or they may lead to a disturbance

of water flow through

the membranes

in

(24) during

and dilution procedures.

Chemical organisms.

and nonpermeating

to oocytes during freezing and thus contribute

toxicity

Published and Wright

significantly

of solutes is always

a serious

consideration

results indicate that propanediol

improved

stage. The vitrification at room temperature

(6) reported

that the use of propanediol

the survival

and development

instead

of cryopreserved

solution used for the cryopreservation due to its high concentration

reduced by lowering the temperature

when cryopreserving

living

is less toxic than DMSO (20). Hernandezor DMSO

of embryos is highly toxic at 37 “C or

of cryoprotectants,

during equilibration

of glycerol

mouse oocytes to the 2-cell

but its toxicity

is greatly

and dilution, and by reducing the length

of exposure before plunging into liquid nitrogen (2). We examined the toxicity of freezing solutions in the 3 freezing protocols under study by exposing oocytes stepwise to Beezing solutions used in each of the protocols and fertilizing the ultrarapid the survival

them in vitro without freezing. The lowest fertilization

protocol, probably due to the hardening rate of slow-cooled

control, the in vitro fertilization the process of freezing

oocytes

was significantly

capability

of slow-cooled

and thawing.

This probably

and nonpermeating

as the cryoprotectant

affected by

that slow cooling as the vitrification

of oocytes from Kunming mouse strain

oocytes from K unming strain mice can be successfully

cooling method with propanediol permeating

lower than that in its corresponding oocytes was not significantly

indicates

protocol is an acceptable method for the cryopreservation In summary,

rate was with

of the zona pellucida by DMSO (9). Altbough

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cryopreserved

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