Effect of human leukemia inhibitory factor on in vitro development of IVF-derived bovine morulae and blastocysts

EFFECT OF HUMAN LEUKEMIA INHIBITORY FACTOR ON IN VITRO DEVELOPMENT OF IVF-DERIVED BOVINE MORULAE AND BLASTOCYSTS Y.M. Han’, ES. Lee2, T. Mogoe2, K.K. Lee’ and Y. Fukui

2a

1 Korea Research

Institute of Bioscience & Biotechnology,KIST 52 Gun-Dong Yusong-Gu Taejon 305-333,Korea 2 Laboratmy of Animal Genetics and Reproduction,Obiiiro Universityof Agriculture

and Veterinary

Medicine, Obii

080, Japan

Received for publication : January I 7, I gg5 Accepted : March 23, 2995

ABSTRACT This study was conducted to investigate whether human leukemia inhibitory factor (hLIF) improves the subsequent development of W-derived bovine morulae To obtain IVF-derived bovine morulae, ova were matured and and blastocysts. fertilized in vitro and cultured in 0.5 I& of synthetic oviduct fluid (SOF) medium supplemented with 10% human serum (HS) for 5 d at 39C under a gas atmosphere of 5% COz, 5% 02, 99% Nz. Morulae and early blastocysts at Day 5 of culture were cultured in 0.5 mt?of SOF medium with or without 5000 U/me recombinant hLIF for 2 or 3 d (2 groups). To investigate the effect of addition of hLIF on the subsequent development of morulae, SOF medium was supplemented with 8 mp/d BSA instead of HS. To test whether hLIF affects the subsequent development of IVF-derived bovine blastccysts, only good blastocysts that developed from SOF medium with or without hLJF at Days 7 and 8 of culture were frozen by a conventinal slow freezing method and again cultured in SOF medium with or without the addition of hLIF for 3 d after thawing (4 groups). Survival of frozen-thawed bovine embryos was evaluated for re-expansion and hatching of blastocysts during 3 d of culture. There was no significant difference in the developmental rate of Day 5 embryos to blastocysts between those cultured with (47.8%) and without (47.6%) addition of hLIF. However, the addition of hLlF before freezing significantly increased the hatching rate of IVF-derived bovine morulae (P
0093-691 X/95/$1 0.00 SSDI 0093-691 X(95)00222-T

508

INTRODUCTION Leukemia inhibitory factor (LB?), a cytokine, is a glycoprotein that suppresses the proliferation of the murine-derived Ml

myeloid leukemia cell line (Ml)

inducing irreversible differentiation of the cells to macrophage cells (8-10).

by It is

expressed in the uterine endometrial glands specifically on the fourth day of pregnancy (1).

It is known that LlF plays a specific role in preimplantation mouse

development, inhibiting the formation of the primitive ectoderm while permitting differentiation of the primitive endodenn (191.

It has been reported that LIF

improved the in vitro development of murine (141, ovine (3) and bovine (5,ll) embryos to expanding or hatched blastocysts.

Lavranos and Seamark (14) also

reported that hLIF-treated mouse embryos showed a higher implantation rate than the untreated controls. The in vitro development of 2-cell ovine embryos cultured for 6 d was also improved (higher final cell number) when the medium was supplemented with hLIF (4).

This result indicates that LIF may affect the subsequent development of

early-stage embryos.

Recently, Fukui and Matsuyama (7) reported that the addition

of hLIF at morula and early blastocyst stages resulted in significantly better development than at the early stages (l-,

4- and 8-cell) when cuhured in SOF

medium supplemented with BSA or polyvinyl alcohol (PVA), suggesting that the effect of hLIF addition is critical to embryonic stages.

However, there

has been

no report on the significance of the addition of LlF at the mid to expanded blastocyst stage of IVF-derived bovine embryos. In this study, we tried to determine the exact stage in the embryogenesis when the activity of hLIF is most critical for developing bovine embryos, with special focus on the morula and blastocyst stages.

The effect of LIF addition on

the survival and the subsequent development of blastocysts after freezing and thawing was also evaluated

MATERIALS

AND

METHODS

In Vitro Maturation The

ovaries were collected fmm Holstein cows

and heifers at a local

slaughterhouse and were transported to the laboratory in 0.9% saline at 30 to 3SC

509

Theriogenology withii

1 h.

The immature

oocytes were obtained from follicles with the diameter

2 to 5 mm using an l&gauge

needle connected

times with 3 I& of aspiration

medium

with L-glutamine

and without

to a 5-d

(10 mM-Hepes

sodium bicarbonate)

salts

and L-glutamine, [FEE1 and

cytoplasm

and surrounded

maturation

medium

(Nunclon,

(US

according

to the criteria of Moor and

medium.

They

centrifuging

were

washed

follicles

with granulosa

of about

(171, and then twice

at 400X g for 10 min.

co-cultured

USA)

by Leibfried and First

Trounson

by

and Pituitary

of Agriculture,

antral

more

The

fetal bovine

evenly

granulated

into 0.5 I& of

Program,

Granulosa

once with

the

aspiration

cells were by the

the aspiration medium

of granulosa

cumulus-oocyte

cells (2 X lo6 cells/&)

multidish

oocytes were selected

(15).

washed

with

USA) and 5

a 4-well

in

10 mm in diameter

The concentration

a haemocytometer.

with Eagle’s

(Sigma Chemical Co, St Louis, MO

Only normal cumulus-enclosed

defiied

determined

with

cells were transferred

(US National Hormone

from 5 to lo-mm

3

salts

with 0.3% (W/V)

10% [v/v1 heat-inactivated

1 &me estradiol

Department

with Eagle’s

medium (TCM-199

10 to 20 oocytes

by compact cumulus

Roskilde, Denmark).

collected

with

NaHCOs),

containing

USA), 10 &meLH-B-5 ,wI/& FSH-B-1

method

supplemented

25 mM

After washing

TCM-199 supplemented

BSA and 2 mM NaHCo3, and once with maturation serum

syringe.

of

by

a

cells was then

complexes

(COC)

were

for 22 to 24 h at 39”c, 5% Co2

in air. In Vitro Fertilization Frozen

straws

of semen from 3 Holstein

for 1 min, and the pooled spermatozoa in 1 I& of capacitation

medium

bulls were thawed

were separated

for 1 h at 39C,

capacitation

medium

supplemented

with 5 mM HEPES, 1 n-&l Na-pyruvate,

(w/v) BSA (6). pooled

in

centrifugation

a

After swim-up,

conical

dissolved During

tube

and

of

modified

5% CQ

Tyrode’s

in air

the

semen

12.5 mM glucose

suspension

was medium.

swim-up,

washed

3 times

medium

containing

and 0.6% twice

was by

The sperm pellet solution

(200

medium was added to sperm solution.

cumulus-expanding

with the washing

The

medium

medium

washed

in the same medium, and an equal volume of heparin in the capacitation

procedure (18).

calcium-free

the upper portion of the capacitation

at 500X g for 10 min with the capacitation

was resuspended ra;/d)

consisted

at 399c in water

using the swim-up

medium

oocytes

cultured

for 22 to 24 h were

(6).

Thereafter,

3 Lrl! of the washing

5 to 7 oocytes was introduced

into a 43-2

drop of fertilization

510

Theriogenology

medium under light mineral oi! (E.R. Squibb & Sons Inc., Princeton, ahquot

of 4 Z

of spermatozoa

final sperm concentration in air, cumulus

suspension

NJ, USA).

was then added to the 46-d

: 2.0 X lo”/&.

After 30 h of incubation

An

drops (a

at 399=, 5% CO2

cells were completely removed from oocytes by gentle pipetting.

In Vitro Culture The cleaved embryos d in 0.5 rdZ of synthetic human

serum

(HS).

embryos

fluid (SOF) medium

The SOF medium

(21) and supplemented acids solution

(2 to 4 cells) 30 h after insemination oviduct

were cultured

amino

Inc., Grand-island,

was designated

whether

at Day 5 of culture

supplemented

with

0.8%

a gas atmosphere

examined

in the morning and Thawing

The IVF-derived good blastocysts

of hLIF affects

were incubated

(w/v)

BSA

(Sigma)

to blastocysts. instead

Ltd., Australia

and early blastocysts

of HS

or

without

Blastocysts

with or without hLIF treatment

bovine blastocysts

were classified by embryo quality

that were

slow freezing

were

with 15% FBS (16).

Briefly,

for 10 min in freezing medium (10% glycerol and 50%

at room temperature per straw.

Yano Science, Japan).

min later, cooling was initiated

(12), and

The embryos

saline (PBS) supplemented

and then loaded into 0.25-d

straws,

with

a

The straws were sealed with straw powder and

placed at -5°C in the cooling chamber of a programmable : Fujiya

with

of Bovine Embryos

in phosphate-buffered

of 5 embryos

Morulae and early

of Days 7 and 8 of culture.

were equilibrated

in PBS)

maximum

of

: Batch No. H113, 5000 U/d)

(Grades A and B) were selected for freezing.

washed

the development

at 39’c in 0.5 I&? of SOF medium

and then frozen by a modified method of conventional FBS

The

of 5% C& 5% 02, 90% Nz.

of 5% CGZ, 5% 02, 90% NZ (2 groups).

from morulae

blastocysts

NY, USA).

as Day 0.

the addition

hLIF (AMRAD Corporation

developed

separately

and 1% (w/v) Eagle’s essential

bovine morulae and early blastocysts

blastocysts

Freezing

according

1

IVF-derived

under

et al.

at 39C under a gas atmosphere

It was investigated

recombinant

for 5

with 10% (v/v) to Tervit

(Gibco BRL, Life Technologies

The day of in vitro fertilization Experiment

was formulated

with 1 mM glutamine

were cultured

supplemented

After 2 min at -5’c at 0.5’c/min

freezing machine

seeding was induced,

and interrupted

(ET-UM and, 10

at -3O’c before plunging

Theriogenology and storing

511

the straws

into liquid nitrogen.

each straw in 37’c water a petri dish.

Embryos

for 20 sec.

supplemented

fresh PBS containing blastocysts

and hatched

blastocysts

was recorded.

The blastocysts

on PBS

were washed

the number

The embryos

into

of viable

were considered

blastocysts

development

of W-derived

after freezing and thawing

was examined.

that developed from SOF medium with or without hLIF addition

or triplicates in Experiment

was further cultured

(Experiment

at

1) were frozen on the day of observation.

of frozen blastocysts 1 were pooled.

that developed in the culture with hLIF

After thawing,

one half of the blastocysts

for 3 d in SOF medium with hLIF (5000 U/mQ addition, while

the other half in SOF medium without hLlF (2 groups). without hLIF addition in Experiment

Blastocysts

that developed

1 were examined in a similar way (2 groups).

Analysis

The statistical

significance

and for detailed comparison thawing;

was based

them

stages with good morphology.

of hLIF on the subsequent

to hatching

Days 7 and 8 of culture

Statistical

by equilibrating

The embryos

After 3 d of culture,

to later developmental

The effect of addition

addition

by agitating

2

bovine blastocysts

Duplicates

manner

The diluent

and 0.5% BSA.

30% (v/v) FBS.

viable if they progressed Experiment

in a stepwise

for 5 min each time.

with 0.25 M sucrose

was conducted

The content of each straw was expelled into

were rehydrated

in 6, 3 and 0% glycerol

Thawing

Duncan’s

of the results

was analyzed by the Chi-square

among the survival

and hatching

test

rates of 4 groups after

new multiple range test was used after the arcsin transformation.

RESULTS In Experiment

1, the Day-5

embryos

were cultured

in 0.5 ml of SOF medium

with or without 5600 U/d

It

addition

was

shown

W-derived

that

bovine

developmental

rates

the morulae

of hLIF

did

and early blastocysts

of the W-derived

bovine

for an additional

recombinant

not

affect

the

early blastocysts proportion to the

from the same experiment

of expanded

SOF

medium

blastocysts

development

to blastocysts morulae

(Table

cultured

addition of hLIF before freezing were 41.9 and 40.7%, respectively;

2 or 3 d

hLIF (2 groups).

with

11.

of The

or without

likewise, those of

were 81.1 and 86.8%, respectively.

The

was also similar whether or not hLIF was added

for the culture

of Day-5

embryos.

Thus,

there

was

no

512

Theriogenology

significant difference in the developmental rates of Day-5 bovine embryos to the blastocyst stage due to the addition of hLlP. Table 1.

Effect of human leukemia inhibitory factor ChLIF) on the development of Day-5 embryos to blastccysts

Group Embryo No. embryos No. embrvos develouedto BL J3x Cultured stase Morula hLrF(+) Fslv blastocysts

Development % of expanded blastccystsb

rate(%la

303

78

49

127 (41.9)

88.6

53

16

27

43 (81.1)

62.8

Sub-total

356

94

76

170 (47.8)

44.7

Morula

302

76

47

123 (49.7)

88.2

58

23

23

355

99

70

hLIF(-1 Early blastocysts Sub-total

46 C38.8)

56.0

169 (47.6)

41.4

Exeperimentswere contemporaneously repeated12 times in each group. BL : blastccysts, EX : expandedblastocysts. (+),(-I Embryoswere cultumclin SOF mediumwith or withouthLIF CXXXI VW!), respectively. 8No. of blastocysts and expandedblastocysts/No.embryos culturedx 100. bNo. of expandedblastocysts/No.of blastocystsand expandedblastccysts x 100. In Experiment 2. when the frozen-thawed blastocysts treated with h.LIP before fmezing were cultured for 3 d after thawing, the survival and hatching rates were 61.9 CZW2) and 26.6% (12/42) for the embryos cultured with hLIF, and 67.5 (27/40) and 25.0% (10/&I) for those cultured without liLIF, respectively (Table 2).

In the

group of blastocysts cultured without hLIP before freezing, the survival and hatching rates of the embryos cultured with or without hLIF after thawing were 58.7 (27/46) and 6.5% (3/46), and 62.2 &WI5) and 6.7% (3/45), respectively.

There was no

significant difference in the survival rates of frozen-thawed blastocysts treated with and without hLIF before freezing.

However, the addition of hLIF before freezing

significantly increased the hatching rate of IW-derived

bovine morulae (P
whereas addition of hLIF after thawing did not increase the subsequent development of the blastocysts.

Theriogenology Table 2.

513

Effect of hLIF addition on subsequent development of IVF-derived bovine blastocysts after freezing and thawing Survival

Hatching

rate(%?

rate(%Jb

16

61.9

28.6’

10

13

67.5

25.0’

31

22

29

64.6

26.8’

46

24

3

19

58.7

6.5*

(-)

45

25

3

17

62.2

6.7*

Sub-total

91

49

6

36

60.4

6.6*

Additionof

Additionof

No.of

hLIF before freezing

hLIF after thawing

embryos cultured

No.embryosdevelopedto EX

HI3

Beg

(+)

42

14

12

(-)

40

17

Sub-total

82

(+)

(+)

(-)

Experiments were contemporaneously repeated 5 times in each group. EX : expanded blastocysts, HB : hatched blastocysts, Deg : degenerated embryos. %o. of expanded and hatched blastocysts/No. of embryos cultured x 100. bNo. of hatched blastocysts/No. of embryos cultured x 100. c~dColumnswith different superscripts differ P
DISCUSSION In this study, it was shown that hLIF did not affect the in vitro development of IVF-derived bovine morulae to blastocysta before freezing (Table 1). is different from those of other investigators.

This result

Fukui and Matsuyama (7) reported

that hLIF improved the development of BP-derived

bovine morulae and early

blastocysts to expanded blastocysts when SOF medium was supplemented with BSA or WA.

Fry et al. (5) showed that in the in vivo produced bovine morulae,

significantly more embryos developed to the expanded blastocyst stage using SOF + BSA supplemented with hLIF after 48 h in culture. In the present study, the bovine embryos martmed and fertilized in vitro were cultured in SOF medium supplemented with 10% HS to produce IVF-derived morulae.

Lack of the hLIF effect on the

development of IVF-derived morulae and early blastocysts to the blastocyst stage may be due to HS used for the culture of 2-cell embryos, since it is known that hLIF has no effect on development of early embryos when SOF medium is

514

Theriogenology

supplemented

with HS (5,7).

Murine bovine

LIF

morulae

knLI.F)

significantly

to blastocysts

there was no significant control group of cultured increased number

(11). ovine

the number

freezing

improved

blastcysts

after

activity

the

expression domestic

hatching

rate LIF.

Therefore,

bovine may

the

morulae

it is important

early embryogenesis

and

early

be due to different to determine

in different

the

species

the

study,

hatching

rate.

embryos

LIF plays its role.

development

hLIF

morula than

improved the hatching

addition

However,

the

at Day-5

it remains

of

and

bovine

Fukui and Matsuyama early

blastocyst

at the early

stages

As shown in Table 2, the present

embryos

4-

also

at which

increased

stage

the

of bovine

resulted

in significantly

and El-cells).

But, they

of m-derived

bovine

study indicates

may act upon the hatching

ovine embryos.

(7) reported that the addition of

stages

(l-,

Bhatt et blastocyst

that the addition

rate of Day-6

to be determined

the effect of hLIF on the development stage

on Day 4 of pregnancy,

in vivo may be to regulate

On this respect, Fry et al, (3) reported

In our

morula

medium

study, addition of hLlF before

difference

that mLIF is expressed

and implantation.

examine

The

to the culture

up to 4-fold, and decreased

of IVF-derived

thawing.

that one of LIF’s functions

of hLIF to SOFM significantly

at

of hLIF

In the present

of LIF during

Based on the result

hLIF

with that of the

animals.

al. (4) suggested growth

(Day 71, although

rate compared

ovine blastocysts (2).

and human

and binding

the addition

embryos and

blastocysts

of IVF-derived

that mLIF has little effect on the development

whereas

of hatching

freezing

of murine

in the hatching

It was reported

of degenerating

the development

(Day 6) and expanded

difference

embryos,

improved

better did not

blastocysts.

that hLIF added at Day 5 of the

process

to improve

the development

of

bovine embryos. It has been shown resulting

in high

recipient

ewes

was

similar

whereas

implantation

receiving to that

ewes receiving

reduced pregnancy bovine morulae hLIF needs

that LIF plays a role in the implantation frequency

an embryo

of ewes

(13,14,20).

cultured

receiving

The

in medium

an embryo

process

pregnancy

containing

immediately

in mice,

rate

of the

hLIF for 48 h after

collection,

an embryo cultured for 48 h in medium without hLIF had a

rate (3).

and blastocysts,

To test the effect of hLIF on viability the transfer

to be carried out in the future.

of the embryos

treated

of IVF-derived with or without

Thus, LIF may have potential

use in

515

Theriogenology animal breeding

to improve the rate of successful

of IVF-derived

bovine

survival

embryos

rates are currently

In conculusion,

the addition

rate of IVP-derived

addition

of hLJF after freezing

results

bovine

indicate

or nuclear

after manipulation

transplantation

where

low.

hatching

IW-derived

embryo transfer

by DNA injection

of hLF

before freezing

significantly

bovine morulae and early blastocysts and thawing

blastocysts

to the

the

(P
had no effect on the development

hatching

blastocyst

that hLIF has an effect on JYF-derived

Day-5 morula and early blastocyst

improved

stage.

The

bovine embryos

of

present

only at the

stages.

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