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
