BIOLOGY

OF

REPRODUCTION

736-742

45,

(1991)

In Vitro-Matured/In Vitro-Fertilized Bovine Oocytes Can into Morulae/Blastocysts in Chemically Defined, Protein-Free Culture Media1 TANU

Department

of Veterinary

PINYOPUMMINTR2

Science,

and

Universiiy

BARRY

D.

Develop

BAVISTER

of Wisconsin-Madison,

Madison,

Wisconsin

53706

ABSTRACT of bovine

Development

culture

media:

20 amino

hamster

acids; and

experiment

in four

conditioned

(1) 199

cultured

in HECM;

When

the

culture

inated

oocytes).

four

the

10%

second

experiment,

the

in a 2 X 2 X 2 factorial

mentation

did

the

not

cleavage

first

enhance and

from

NM/IVF

oocytes

using

oviduct

cell

stimulation can

of morula

develop

in

vitro

or serum;

was

no

greater

to the

morula

In many

serum

laboratories, obtained oocytes.

are

bovine

morulae

and

from in vitro-matured/in When media supplemented

used,

most

embryos

cease

blastocysts

stage

are

formation.

or at the 8-16-cell stage [1-3]. This block to development at the 8-16-cell stage can be overcome by culturing embryos in a medium containing cumulus cells or oviduct cell monolayers [4]. Alternatively, cell-free medium, typically tissue culture medium (TCM)-199 plus serum, in which oviductal tissue has been cultured for 24-48 h (conditioned medium), is capable of supporting early bovine embryo development equally as well as oviduct cell monolayers [1, 5]. However, with both procedures, the proportion of embryos that cleave to the blastocyst stage is quite variable (25-40% [2, 6-9]) and needs to be increased to maximize the usefulness of IVM/IVF embryos for research and commercial purposes. Moreover, although a few IVM/IVF bovine embryos have developed to term after transfer to re-

‘This

March research

‘Correspondence.

26,

FAX

(608)

by USDA-CRGO

grant

no.

The

and/or

analysis

induced

modifications.

we

cells

with

no

embryo dium

that

to the

chemically

Glucose

and/or

embryo

culture of components

nally,

the

tissue

conditioning

in a 2

X

effects 2

X

(HECM),

phosphate hamster of serum

(2)

common

(1)

8-cell

examined and

TCM-199

me-

embryos

TCM-199,

a more

somatic

cells.

components blocks

embryos

supplementation experiment.

2-cell

for

in the

hamster

simple

of

responsible

IVM/

conditions

media:

(P1), were

on HECM 2 factorial

of bovine

culture

be

defined, Therefore,

culture

and

ovi-

would

a chemically embryos.

for

2- and

were

hypothesis

a relatively

[13-16],

cell-

in the serum-

of hamster

designed media,

oviduct

present

defined

development stage

medium

for

is that

“conventional”

medium

and

obscure isocomponents,

hypothesis This

to use bovine

cells,

may

of development

both

in supporting

somatic

medium

possible to culture

of two

blastocyst

complex

of serum,

medium.

under

supports

cells

substances

a study

culture

embryonic growth embryotoxic subboth. However, de-

complex medium of any embryotrophic

complex

use

TCM-199

inhibitory

conducted

with

of

use of a conventional + bovine serum) will

An alternative

if it were medium

have

at

derived

advantage

of oviduct

of conditioned

detoxify

IVF embryos and

736

embryos

through

presence

metabolites in the and identification

supported serum-free

89.37240-4562.

actual

difficult.

supple-

inhibition

development.

embryo development (oviduct cells +

supplemented

262-7420.

bovine

role

of the

In were

serum with

termination

duct

1991. supported

without

media,

(13.8%).

TCM-199

could provide or remove medium, or

their lation

8, 1991. was

protein-free

blastocyst

oviduct

+

199

and

effect,

that

P, alone.

of insem-

BCS

TCM.

HECM

a biphasic show

to

or with

cell(M&B)

(42-51%

or TCM-199

data

oviduct

compared

or

first

IVM/IVF

stages

oviduct cell monolayers stimulatory components, stances from the culture

These Received

on

HECM

for

velopment July

conditioning

defined,

be very

cipient cattle [e.g., 1, 10-12], these have been highly selected, and the viability of the great majority of embryos is not known. At present, the actual role of oviduct cells in the coculture or conditioned medium has not been elucidated. The

Accepted

(9.7%)

These

needed

are

P when

(29.7%)

BCS

exhibited

and

development

HECM

of either

bovine protocol

before

10%

cell

in chemically

factors

vitro-fertilized only with

development

with

supplementation

INTRODUCTION routinely (IVM/IVF)

in M&B

two

The

of bovine

(BCS),

serum

in

containing

cells.

of somatic

morula/blastocyst

and

glucose

in nonsupplemented

blastocyst

serum

of

oviduct

calf

to the

difference

conditioning

Serum and

however,

presence

examined

medium

the development

bovine

developed

supplemented

cell

Oviduct

unheated

significant

and/or

development. compaction

10%

was

for culture

(2)

and

oocytes

defined, protein-free

designed

HECM

oocytes

than

supplementation

experiment.

medium

in the

depressed

in TCM-199

significantly

embryo

+

inseminated

there

development

bovine

contributions

was

of serum

complex (Pt) using

TCM-199

of the

compared,

was

effects

examined

45%

development

(21.6%)

a more

phosphate

TCM-199,

WF,

were

blastocyst

medium

and/or

(IVM/IVF)

vitro-fertilized

a relatively simple, chemically

(TCM)-199,

After

blastocyst

in vitro-matured/in

(HECM),

HECM,

BCS.

conditions

However,

cell-conditioned

medium

conditions: +

when

from

medium

effects of glucose

different

TCM.

derived

culture

tissue culture

studied

embryos

embryos

embryo

[13,

present and/or were

of to de17, 181.

study. oviduct examined

Fi-

IN VITRO

MATERIALS

AND

DEVELOPMENT

OF

BOVINE

METHODS

For experiment the

Culture

Media used in this study were as follows: TALP HEPES, used for IVF and oocyte washing, respectively, as described

Parrish

by

et al. [191.

dium was made to volume with water osmosis and filtration through a 4-bowl

[201.

Corporation, and tested HECM

was

New with

purified Milli-Q

as described

Uwere TCM-

weekly Gibco, the me-

by reverse system (Mu-

Bedford, MA). Water the hamster sperm

prepared

and

Medium

used for NM and embryo culture, was prepared from a powdered medium (cat. no. 400-1100EB; Grand Island, NY). Bicarbonate was added, and 199,

lipore weekly

was prepared motility assay

by Schini

and

Bay-

ister [131; this medium is chemically defined (protein-free), contains no glucose or phosphate (P1), and is supplemented with 20 amino acids. To reduce the possibility of intrinsic inhibitory

effects

leted from ster 1-cell

HECM embryos

mented

of the

in some

calf serum acid-free

culture

medium,

groups

treatment

(BCS; Hyclone (cat. no. 5711,

with

10%

containing 50 ig/ml

0.25 mM gentamicin.

oviducts of the

The

sodium

TCM-199 containing antibiotics/antimy-

WF

pyruvate,

in saline

were

containing

ferred HEPES

and

obtained

and five

PSA.

sediment was ml of medium

allowed times.

and

status,

Then

toward [1]. The

the

abattoir,

and

were trimmed washed three

oviducts

sediment to After

introduced TCM-199

was

were

the infundibulum extruded tissue

redispersed

free times scraped

with a was trans-

in 10 ml

settle. This washing the final wash, 0.25 into a 50-mi supplemented

flask with

re-

transported

TALPwas TALP-

step was reml of tissue containing 10% BCS

10 and

PSA. The tissue suspension was cultured under 5% CO., in air with high humidity at 39#{176}C for 48 h; during this time, viable oviduct cell cultures grew as free-floating spheres of epithelial cells, with outwardly directed, actively beating cilia.

of either HECM to subsequent

collected

and

or TCM-199 treatments.

as the

period, of viability,

Only

from

conditioned

medium

the conditioned media were a 0.2-pm millipore filter and

Maturation

five corwas

one assigned or TCM-199 h. At the end

oviduct cell as described

viable

sterilized stored

10 con-

(without serum) Each tissue pellet

of this second 48-h incubation were re-examined for signs

Cow ovaries mediately post to the laboratory

g for

X

used

then resuspended and allowed to condition medium (HECM, HECM + 10% BCS, TCM-199, + 10% BCS) during culture for another 48

cultures

cultures above. was

passage at -20#{176}C.

used;

through

by

(NM)

from random breeds were collected immortem at a local abattoir and transported in saline (25-28#{176}C) containing PSA. The

ovaries were pooled regardless of stages of the estrous cycle of donors. Fluid was aspirated from small antral follicles (2-6 mm) using an 18-g needle connected to a 10-mi syringe, then pooled and allowed to settle in 50-mi conical

completed natant was

with aluminum foil to protect exposure to light. The aspiration

within 4-6 discarded

1-2-mI

amounts

power oocytes

(20-30X) with evenly

in

fluid

14 randomly of maturation

h after ovary collection. and the sediment was sterile

petri

a fine-tip

selected medium.

Becton,

Dickinson

5%

in air

In Vitro The

of a low-

only cumulus-intact were selected from

“Unopette”

(Becton,

Dickin-

oocytes was allocated to each For NM, oocvtes were cultured

and with

Fertilization OCCs

aswas

The superdivided into use

By

the

Rutherford, NJ). The oocvte-cumulus comwere washed two times in 10 ml TL-HEPES with 10% BCS and PSA; then a group of 10-

24 h in 50-pA drops of medium affin oil in sterile 60 x 15-mm CO2

dishes.

stereo-microscope, granulated cytoplasm

with

son and Co., plexes (OCCs) supplemented at a local

endocrine

isthmus slide

the

TALP

BSA,

to a 12-ml conical test tube containing 10 ml medium and allowed to settle. The supernatant

discarded, HEPES peated

was

for cell growth,

at 250

For experiment 2, after centrifugation, pellet was washed separately in

test tubes wrapped pirated fluid from

Media

donors’

from the microscope

medium 6 mg/mI

on ice to the laboratory. The oviducts from surrounding connective tissue and gently glass

bovine

BSA was fatty from Sigma

incubation

centrifuged

was

ditioned medium. the oviduct tissue changes responding

the 48-h

was

supernatant

follicular

of Conditioned

Bovine gardless

de-

100 p.g/ml streptomycin, and B; PSA), 50 ng/ml epidermal (5 g/ml LH, 0.5 g/ml FSH,

1713-estradiol).

Preparation

was

unheated

Labs, Logan, UT). lot, no. 29F-9315)

cotic (100 LU/mI penicillin, 0.25 g/ml amphotericin growth factor, and hormones I pg/ml

pyruvate

1, after

suspension

the

In Vitro

because of its inhibitory effect on ham[211. TCM-199 and HECM were supple-

Chemical Co. (St. Louis, MO). The oocyte maturation medium was 10% BCS, 0.25 mM sodium pyruvate,

and

tissue

mm;

Media

prepared

737

EMBRYOS

Co.).

saturated

overlaid with petri dishes The

culture

humidity

drop for

10 ml of par(Falcon 1007,

conditions

were

at 39#{176}C.

(IVF)

contained

in

each

drop

of

maturation

me-

dium were removed, washed three times in TL-HEPES containing 10% BCS and PSA, then introduced into a SO-p.1 drop of IVF medium under 10 ml paraffin oil. For each experiment, 2 straws of frozen semen containing 5 X 10 sperm/ straw (American Breeders Service, DeForest, WI) were thawed in a 35#{176}C water bath for 1 mm, then subjected to swim-up separation in Sperm-U medium [19] in a 39#{176}C water bath The 106/ml.

for final

1 h to sperm Sperm

increase the concentration capacitation

proportion of motile sperm. used in 1W was 0.6-2.0 was

enhanced

by

including

X

2

PINYOPUMMINTR

738 TABLE

1.

of glucose

Effect

and/or

P, on the development

AND

of bovine

IVM/$VF

BAVISTER embryos. Stage

inseminated Treatment

HECM HECM HECM HECM

No.

oocytes

+

P,

+ glucose

2-7-Cell

8-16-Cell

4.1#{176} 5.6#{176}

296b 253b

21.2

14.3#{176}

18.9

9.9#{176}

3#{216} 3.2#{176}

0b 274b

1-Cell

6” 7 7 7

Morula

12.5c

*Results

p.g/ml

shown

in Tables

heparin

1 and

sulfate

2 were

in the

conditions for IVF were at 39#{176}C for 18 h.

In Vitro The

Embryo oocvtes

obtained

from

the same

5%

IVF medium CO2

ignated

in air with

Incubation

high

treatments.

IVF drop

The

fixed and examined mation). Experiment I.

or P, on bovine For supporting

This

stripped

of cumulus

to more

10%

and

BCS,

seminated medium,

(9-10)

of each

allocated to the remaining fertilization

preliminary

group

randomly oocytes

of prewere

(pronuclear study

was

forto

development were embryo development,

complex

media:

conditioned

oocvtes were nonsupplemented:

TCM-199 randomly HECM,

+

TCM-199

10%

The

BCS.

allocated to (1) HECM + glucose,

+

in-

simple HECM

2

199

+

97h 11,1h

,43h

5.7’

media

BCS;

Embryo under

culture

in both in air

Data

At the for

end

of each

IVF + 8 days

uated

with

Nomarski

development of nuclei

supporting

ference

2.

Development

of bo vine

IVM/IVF No. of inseminated oocytes

Treatment

emb ryos

examined

in four

diffe rent

HECM

136

6”

TCM-199

153

7

+ TCM-199

10%

BCS +

10%

BCS

129

60

154

7

condi-

saturated

1 and

2 was

10 ml

paraffin

humidity

undisturbed

for

experiment

(=24

done oil

at 39#{176}C. Em-

8 days

(range

188-

The design

data

culture),

optics

for the

h of NM

all embryos

with

the

morphological

fluorescent,

+

18

were

the counted)

stages

of

number were

DNA-specific

dye

were

analyzed

ANOVA

followed

comparison

by use by

of a randomized

protected

block

least-significant

dif-

of means.

media of embryo

development

(%)“ Compact

1-Cell

2-7-Cell

4.1#{176}

29.6c

6.4#{176}

28.9c

325b

7.6#{176}

“Results shown in Tables 1 and 2 were obtained from the same experiment. ““Stage of embryo development was evaluated on Day 8 after the IVF (percentage “““One replicate lost due to contamination. ““Numbers in same column with different superscripts differ (p < 0.05).

8-16-Cell 212d 184d0

18.6” 28.9”

h

eval-

[221.

Stage No. of replicates

by

reached. In some experiments, each embryo (all embryos

in 33342

were

as above,

experiments

of embryo

by staining

development

in (1) or TCM-

Analysis

Hoechst

embryo

incuba-

TCM-199,

media

overlaid

with

incubated

counted

bovine

48-h placed

h).

+ glucose + P; (2) TCM-199 + 10% BCS; or oviduct cell-conditioned TCM-199 + 10% BCS. Experiment 2. Effects of media (HECM vs. TCM-199) ± serum supplementation ± oviduct cell conditioning on P,, HECM

(3)

same

of medium

CO2

were

192

the

BCS,

ab-

growth

for condition-

second

randomly

10%

de-

cell

removed the

were +

oviduct

This in the

cells.

drops 5%

(2)

the

was during

HECM

or

conditioning

for

oocytes

oviduct

experiment.

cell

incubation)

HECM,

in 50-p.l

2 factorial

X

used

inseminated

by

oocytes).

of oviduct

48-h

10%

tioned

2

X

serum

unconditioned

also studied. HECM was

TCM-199,

(first The

inseminated

study

in protein-free

bryos

deter-

total

of serum;

tion.

and then to des-

from

permitted

ing

NM/lw embryos could develop in chemically defined medium, HECM, by serum. Effects of glucose and!

embryo bovine

compared

Cond.

21.2’ 29.2’ 26.9’ 26.5’

14.7#{176}

a complete

sign cultures

were

majority

to verify

mine whether bovine a relatively simple, without supplementation

calculated

using

humidity

(ED)

inseminated oocvtes was then assigned embryo culture media;

TCM-199

Blastocyst

experiment.

sence

Development in each

[19].

by passing them through a fine-tip Unopette washed four times in medium corresponding

TABLE

morula

9.3#{176}

226d

Stage of embryo development was evaluated on Day 8 after the IVF (percentage #{176}“One replicate lost due to contamination. #{176}Numbers in same column with different superscripts differ (p < 0.05).

+

(%)*

P

+

cells were

development

Compact

replicates

136 163 166 166

+ glucose

of embryo

of

7.0’ 12.8#{176}’

calculated from

Morula

morula

Blastocyst

14.3

21.2 l3.4J

13.8’

0.8”

11.5’

29.7”

11.0”

18.1’

21.6”

19.2’

total inseminated

oocytes).

9.7’

IN

VITRO

DEVELOPMENT

OF

RESULTS

The

possibility

simple,

of growing

chemically

glucose morula no

were stage

embryo the

(all

the

on bovine stage (p

both

Tables ferent

2 and

bovine

ulae tween cytes, the

able

When

cell

no

beyond was

ditioning.

the (Table

necessary. past

of inseminated supplementation However,

the

critical

difference

cell

deper

pressed

supplementation that

failed +

serum

serum

TABLE

3.

The

to the

without

and

Mean

cell

number

of

embryos

con-

No. of embryos

Treatment

serum supplementation, to the relatively simple reaching the blastocyst without

serum

conditioning

development

to the

defined or

contribution

media,

without from

by reducing

present

four

in

different

levels

conventional

any that cells

of inhibitory culture

No. of blastocysts

148,6d

154

45,9”

33

152.8#{176}

same

column

with

different

superscripts

differ

(p

such

“Mean no. of nuclei/blastocyst

111.1C

of variance.


or

presence

containing with

1, p

27-32)

treatments

though

first.

across

differences

(range

(HECM)

examined

noted

(Table

significant

embryos

medium

P, were

development

bovine

defined

and/or

739

EMBRYOS

hatching (14/37 [39%] and hatched, respectively) compared 20 [14%]). Furthermore, these

I

Experiment

BOVINE

typically as

TCM-199

740

AND

PINYOPtJMMINTR

TABLE

4.

Effect

of serum

supplementation

and/or

No.

oviduct

10% HECM HECM

BCS

+

10%

+

BCS

TCM-199 TCM-199

10%

+

Cond. Cond.

BCS

TCM-199 TCM-199

“Stage

10%

+

of embryo

wNumbers

in

BCS

development

same

supplemented

column

with

1W embryos

was with

serum.

developing

in this study were similar vestigators using somatic

results

also

clearly

entiation of morulae In a preliminary and

glucose

Glucose bovine

on

175

6

3.4#{176}

177

6

176

6

176

6

the

and

bovine

187b

4.4#{176} 37.8”

8 after the differ (p