Role of the H-2 Complex in Preimplantation Mouse Embryo ...

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McLaren and. Bowman. (1973) have shown that throughout the preimplantation period. C3H mice have fewer cells per embryo than mice of the outbred. Q strain ...
BIOLOGY

OF

REPRODUCTION

26,

591-596

(1982)

Role of the H-2 Complex Mouse SIMON

B.

GOLDBARD,

in Preimplantation

Embryo

KATHRYN

Department

M.

of

Development VERBANAC

Biochemistry State

Iowa

Ames,

and

and

CAROL

M.

WARNER’

Biophysics

University

Iowa

50011

ABSTRACT

The H-2 complex was was shown to be associated demonstrated that gene(s)

rate

of subsequent

found to influence early mouse embryo development. Slow development with the H.2k haplotype. By the use of congenic strains, it was clearly in the H-2 complex affect the time of the first cleavage division and the

preimplantation

development. This paper hypothesis.

INTRODUCTION

The preimplantation development is crucial mammalian fertilized

species. egg goes

divisions

and

studies

In

mouse,

cell

fertilization

that

some

some

contain

1970). Dagg

Gates (1962)

stage

there

Timing subject

cells

et have

immediately

a certain

the

than

mice (1977)

inbred

fewer

time

than

addition,

mice

have

either

BALB/c

McLaren

shown

that

and

strains suggest early and

and that haplotype, the

or

H-2

preimplantation

both led

are

embryo

mice.

In have

mouse Laboratory,

strains Bar

METHODS

inbred and congenic from the Jackson

of Embryos mice 2-6 months old were superovulated with 5 IU pregnant mare’s serum (PMS) followed 48 h later by 10 IU human

chorionic

gonadotropin

immediately

(hCG)

Determination

are of the H-2 that gene(s) in

(ICN,

Nutritional

All injections were either at 1500 h or at 1600 h daylight saving time. placed individually with a single male after

the

hCG

the presence of a vaginal Embryos were collected (1968) medium at specified

the CBA strains,

timing

AND

Female by injection (Organon)

Biochemicals). standard time Females were

These studies the timing of

the

following obtained

Collection

a given

(1973)

that both developing

influence

Accepted December 23, Received July 28, 1981. Correspondence.

at

this

in to that

preimplantation

JU. affect

The fact “slow”

mouse

cells

C57BL

the

of these strains us to hypothesize

complex

and

fewer cells per embryo Q strain, or the inbred

C57BL, Rill, and that genetic factors development. C3H mice

cells

support

Harbor, ME: A, C57BL/lOSn, DBA/2, CBA, C3H/He, C57BR/cdj, SJL, and B10.BR. The C3H.B10 mice were obtained from Dr. D. Shreffier, Washington University, St. Louis, MO. The mice were housed in a day-night cycled room with controlled temperature and food and water ad libitum. The clock was never altered during the year so that the ioom was light either from 0600-2000 h standard time or from 0700-2 100 h daylight saving time.

after

compared has shown

Bowman

throughout

period C3H mice have than mice of the outbred

The were

(Rafferty,

fewer

that

Mice

and Whitten and at the blastocyst

average,

data

MATERIALS

in few

fewer

others

the embryos of BALB/c strain 129 mice. Titenko CBA

time

contain

al. (1961) shown that on

of events of very

is almost at

presents

of

leading

particularly

embryos

more

are,

events

mammals,

division so

mammalian survival

this time the single a series of cleavage

the

1979).

asynchronous,

of the

differentiation

formation. has been

(Soll,

the

During through

cell

to blastocyst development

period for

injection, and checked for plug the next morning.

into Whitten times post-hCG

of Cell Number

and Biggers injection.

Per Embryo

with more than 4-5 cells were subjected Tarkowski (1966) procedure to assess the of cells per embryo. Briefly, the embryos were washed free of culture medium and placed in 0.8% sodium citrate for 5-15 mm at room temperature. Next, the embryos were transferred to a clean slide Embryos

to the number

of

development.

and 0.02 absolute

ml drops of freshly ethanol: I part glacial

Usually,

at least

5 drops

prepared acetic

of

Final scattering of nuclei was the embryos during air drying. stained in lactic-acetic-orcein

1981.

591

fixative

fixative (3 parts acid) were added.

were

required.

achieved by blowing on The preparations were or Giemsa, and the

592

GOLDBARD

TABLE

1. Association

of slow development

with

the H2k

ET AL.

haplotype.

No.

Mean

Mouse strain

H-2 haplotype

embryos scored

embryo (SE) 89 h posthCGa

A C57BL/lOSn

a b

54 48

32.9 33.1

DBA/2 CBA C3H/He

d k k

53 45 40

30.5 (1.4) l8.p(i.O)b 24.3 (04)b

C57BR/cdj DBAJ1 SJL

k q

55 47

18.7 30.2

(i.O)b (1.5)

s

48

31.9

(2.0)

were

aEmbryos

of Tarkowski bsigficantiy

nuclei at 400

collected

(1966). lower

counted using X magnification.

The female previously mice were

than

the

a Zeiss

of Time

Assessment

89 h post-hCG other

injection

inbred

phase-contrast

and

with PMS and hCG, not mated. Groups 8 h post-hCC to 16 of ovulated eggs removed

and the clutch by teasing apart the oviduct. The clutch was then treated with 300 units/mI of hyaluronidase (Sigma), to and

allow

the

counting

of

first

set

of

experiments

was

designed

of cells per embryo in a variety of inbred

89 h mouse

strains

H-2

results

are

shown

different in Table

haplotypes

tested.

Student’s haplotype each different

other, from

blastocyst developing

of

demonstrate the

but the

than

were other

chosen stage

strains,

for but

most

the

haplotype

those

of was

highly strains.

(89

empiri-

H2k

Significance

h

the

other

assessed

of the different

or other factors, ment of strains accomplished

of

morula

stage

experiments genes in the

at only

the

H-2

and

this

composition H2k to H2b) ment

of

and for

the

to

H-2

2. Group composition

complex

(from

gene. For and inbred First,

we

these

strains,

In

not did

may

the

cleavage Esworthy from

was undercomplex, developThis was congenic A shows of a H2b

to

time

results

3 strains that the marked

measure

these

of

of it

at this

mating

1. It

which time

of

later). that

fertilized

of

that there in rate of

strains

(discussed has reported

time.

or time

is possible

in the

are

of

in Fig.

penetration

differences

strains

Ped

ovulation

shown

among

division (1979)

the

linked

the congenic A of Table 2.

expression

or

Warner, gene(s)

ovulate within 2 h of H-2 linked Ped gene

time

differences for

report

H-2

we used in Group

the the

transport

account

mouse

to discern

of the

Therefore,

be

sperm

is

early

experiments

with

show not

(from develop-

a preliminary

action studies listed

all so

genetic

interpretation

influences

measured

is seen that each other,

(Group in

(Verbanac and this H-2 associated embryo development.

earliest these strains

studied

The

fast.

complex

we planned

of the

was

a change

the H-2 complex the pattern of

development.

Next, time

of the embryos this observation,

pair too,

only changed

slow H-2

embryo

outbred slow

the method

by

development To confirm

case,

at

from

that

We

the

determined

congenic

in

fertilization.

is

account for the slow of the H-2’ haplotype. by using 2 sets of

strains, as shown in Table that changing the genetic

reciprocal

B),

does

H2k from

significantly The time

post-hCG)

inbred

by

strains.

The second set taken to test whether

mouse

The

t test. The 3 strains were not significantly

development mouse

haplotypes.

1, and

cally that embryos develop more slowly

changes the fast to slow.

the

to

assess the number post-hCG injection of

no./embryo

of this observation 1981) we named Ped: preimplantation

RESULTS

The

cell

(1.5) (2.1)

level.

H-21) from

microscope

of Ovulation

disperse the cumulus cells the number of ova ovulated.

mean

at P