Mammalian. Oocytes and Preimplantation. Embryos as Methodological. Components. GEORGE. E. SEIDEL,. JR. Animal. Reproduction. Laboratory. Colorado.
BIOLOGY
OF
REPRODUCTION
28,
Mammalian
36-49
(1983)
Oocytes and Preimplantation as Methodological Components GEORGE
Animal
E.
SEIDEL,
Reproduction
Colorado Fort Collins,
and
in
mammalian
in
some
the
Laboratory
oocyte
along
the
oocyte’s itself into remains
genetic
with
those
epigenetic a fetus.
joining
together
undifferentiated
instructions
until
of
to be leading
Recent have
of the
the
totipotency,
haploid
genomes,
during implantation,
the
excellent
in this
embryos
is
available.
Numbers
of
potential
embryos,
reproductive
both
lated time
are
frequently
increase the by up to a factor
of ovulation,
oocytes,
Use
to seemingly
For
enormously of
exogenous endless
in only
these with
debate
improves
and of
hormones as to
also whether
methodological
be
on
meant
be
abcontrols
four of the that con-
value:
mammals.
to
SIZE
mature
size
and
developproperties. The
examples
representative,
AND
eutherian
2, which
of of
Embryos
not
fetus
this
still
cell types,
The
and
cell
the
mass.
Hesseldahl,
and Nucleic
feasible
of al.,
inner
portions
of to
blastocysts
two
distinct
cell mass. The of the placenthe and
the blastocyst species (Davies
Translation
ova
and,
which
into
the
may nucleus
vary and
and
leads
zygote).
Amphibian
such
this
for
of
are
large
male
enough
substances be
into
more
(known
oocytes,
36
as
have
Acids
to inject
way
known
1971).
Mammalian ovuthe
of ovum
species, develop from timing of implantation
the
The
Transcription Injected
in volumes
equal cells, then a cavity known as the 100-cell stage.
and
morphogenesis from species
greatly
diaover
in
is just
fertilized
they
various
on
a sphere
range
to date
are
trophoblast
depending
inner
stage
is
The
a range
10.
spherical;
proper
ta,
SHAPE
oocyte
gives
about
at
are
and
hormones,
et
to
around embryos
divides into two approximately 4, 8, 16, etc. In most species, the blastocoele forms by
reasons,
(Sugie
increased
compared
0.1 mm in diameter. among species studied
a factor a factor
most once
logistics
in
most
hormonal
processing properties; and developmental
will are
The
and range
number of oocytes of 10 and to regulate
which
their
of
result
is organized oocytes and
sperm potential;
about meter
numbers
normally
treated
to
experimentation 1980).
mature
cycle.
to
not offspring
OVUM
of the fantastic
limited
one to about 15 per female and these few are available
females
paper of
experimental
the
therefore
per
1981a).
This properties
under
application
exhaustive.
and embryos as methods, experimental means to an end, not necessarily related to understanding the biology of the oocyte or embryo. An important constraint on the kinds of experiments that can be done with mammalian
from species,
(Seidel,
chosen
this system. However, in view of the theme of this symposium, Methods in Reproductive Biology, it seemed useful to consider oocytes
and
in
Emphasis
and
endowments of developing embryos. Most studies of the mammalian oocyte developing embryo probe the basic biology
oocytes
does
normalities
shape; mental
interval make the
the ideas field and the
between
Fortunately, this
treatment
advances in preimplantation been remarkable. There seems
synergism researchers
normal.
tribute
female The the
implantation.
size,
two
embryo
material. embryology
from
lumen
of large
the relative accessibility between ovulation and developing
and
modulate
sperm,
are
circumstances,
cells
ovulation,
of the
in the tract
attributes
largest,
tendencies to transform The developing embryo
unattached
unique
of the
maturation,
the
reproductive
is one
most
After
fertilization,
ova
oocyte
respects
body.
JR.
State University Colorado 80523
INTRODUCTION
The
Embryos
oocytes of
important,
directly vesicle
pronucleus have years
in
in the
been and
it is
that
cytoplasm
as germinal
or female
a number
so
the
have
used some
in
MAMMALIAN
advantages
over
their larger (Gurdon
size
those and
et
al.,
for
this
Nevertheless,
the
excellent
of as a very appropriate machinery.
of
sophisticated RNA and RNA can
foreign
1981a)
with
considerably
current point
is that
portant
DNA
sequences
or
also
methodology. the
functional of
embryonic
the with
than
An
im-
activity
of
transcriptionthe stage
development
arrests
at
first
meiotic
division
the first meiotic
polar body, and division, which
metaphase
another, has characterization
et al.,
fidelity
the
in extrusion of of the second
maturation
of
to the postulation of several factors,
promotion
causes
breakdown
chromosome
factor of
the
factor
oocyte
and Markert, remains to
which
metaphase 1971; done
be
(MPF),
which
membrane,
factor
(CSF),
in
and partial including
nuclear
condensation
cytostatic the
(e.g.,
led
which
1). Analysis of events by pipetting from one cell to
(Fig.
intraovum control of these small quantities of cytoplasm
thought
(Brinster
greater
components system varies
oogenesis
is be
37
of
results initiation
test tube, containing protein processing be transcribed from
cell-free
the various translation
oocyte It might
AS METHODS
pletion
primarily
in large numbers et al., 1980).
mammalian purpose.
certain
with
mammals,
availability 1971; Lane
OVA
(CCF), causes
of
meiosis
Masui et al., in characterizing
and
arrest
of
II (Masui 1980). Much these very
Brinster et al., 1981a); therefore, one should choose an appropriate stage for each particular experiment. At certain stages, which vary
important
among
from a common precursor. A very important point is that MPF and CCF also appear to regulate mitotic cell cyles (Wasserman and
species,
functional
there
ribosomes
amplifiction), a very Because
be
numbers
(which
arise
from
make
the
oocyte
productive this system
exploited,
it is unclear
in
introns
function
of
large
which
embryo machine. to
are very
excising
how and
well
similar
RNA
processing; the system valuable for post-translational sing, such as adding carbohydrate In Xenopus oocytes, some proteins foreign that
(Lane
et
oocyte important
RNAS they are
al., and
1980).
understanding
the
able process eukaryotic functional Factors
may
be exprocesto proteins. translated
the
in such the cell
mammalian
destined to components
complexities
it will
of
the
become
will because
the
Cell
remark-
be
value
Cycle
made to the the following ova
of
cell
so
also
oocyte, which is mediated
size.
MPF
and
same
Cytostatic
CCF,
Models
for
The embryos
inducing ovulation from the anterior triggers
final
in amphibians, by progesterone
in the
is a surge pituitary.
maturation but
follicles of
of the
not mammals, produced by
and
their
possible
that
MPF
molecule
or originate
factor
may,
in oocytes much more mitosis than
which
are
were
in
ironically
cryobiology.
because cells
procedures
were
oocytes for
and many
For
some
years,
large, form
cells are
when
about
dynamic
there are principles,
intracellular
with to
a number perhaps
ice
intracellular
freezing
This dehydration in salt solutions
-7#{176}C. In principles,
of the
solutions solution,
first
salt
tonic, crystals.
unfrozen solution This draws water continues, more and
crystals
do
occurs
takes place in the following far is induced
accordance the
in such hypotonic
cooling becomes
worked
inappropriately
way. To prevent super-cooling freezing point, ice formation at
mamfailures,
is that most of the water from cells during cooling, so
damaging 1981). cooled
of were that
applied
fundamental be removed
not
to
elucidated
models
experiments with cryopreservation malian oocytes and embryos
that
universal
first
Cryobiology
huge size of mammalian make them excellent
smaller
however,
(Masui et al., basic research with rneiosis,
probably
both mitosis and rneiosis, in large meiotic cells.
(Leibo,
In vertebrates,
cells. Major events of final oocyte maturation include breakdown of the nuclear membrane (germinal vesicle), condensation of DNA into chromosomes, initiation and cornfollicle
1978).
factors,
is even
be the
be unique to meiosis 1980). Thus, although has been done with
most should
emphasis examples beautifully
oocyte matures within One of the components
complex pathway of gonadotropin surge
the
amphibian
primary ovary.
may
It
these large cells. While important cryobiological
Regulating
illustrate
CCF
interesting
action.
experiments for
proteins.
concerning
This
Clearly,
and
and
of
Smith,
whereby information contained in DNA molecules is used to produce
An exception on mammals,
the the
or
aspects
are processed exported from
embryo are methodological
DNA
RNA translation is just beginning
tremely
from a way
of
modes
with ice
crystals
as
below the (seeding) thermoto form
are composed of relatively leaving a relatively hyper-
the more
between out of the
the cells.
ice As
unfrozen hypertonic
solution as ice
SEIDEL,
38
extracting
grow,
crystals
more
from the cells. When low enough, intracellular if enough
water
has
at that point, will form. A number
the been
no
and
removed
large,
is
ones,
and
of
from
factors
govern
low in large cells especially to the As
to
volume terms,
factors
are
cooled
much
allow
sufficient
it
rate
a system. to volume
compared biconcave-disk
turns
out,
large
more
slowly
time water
cells
must
small
the
cross
large
the
the
principles
extreme
end
last into
a number makes not be
much
work
size
done
as
This well
easily
and
growth
Cryobiology can be
of studied,
teristics
of
the
4) The two cell trophoblast and differing optimal which
makes
its large
size,
new
shrinking
and 2)
1977).
e.g., ice
monitoring crystal
a!.,
for-
1980).
3)
intracellular compartments e.g., cryobiological characcell
nucleus
(Farrant,
1977).
types of the early blastocyst, inner cell mass, probably have cryobiological parameters,
them
a good
model
an
fetal
rat
than
one
embryos also genetic material With
some
species,
developed
for
freezing
but appropriate embryos. Clearly
for
the body mammalian
with cell.
RNA,
or
protein.
unless
it activates
fertilization,
procedures are the mammalian
important research.
methodological
similar
PROCESSING
the
oocyte,
embryo
the smallest It does not
decondensation
of mediated
reagents,
processing
sperm
replacement
rounding Minhas,
of
the DNA with 1982), repair occurred
to
cating
its
DNA
preparing the
the
for
maternal
mixed
together
ability
of
for includes
nucleus,
almost
bond-breaking
the
protamines
sur-
spermatozoal
DNA
the
first
the
the and
first
to
in the be exploited
genetic duplicell
mitosis,
paternal
(Longo,
ovum
and may
DNA
1973). unlock
cycle,
at which become
The the
unique genetic
otherwise inert sperm in a variety of meth-
contexts.
Fertility One meters
fertilized
potential
histones (Wagner of damage that
during it
of by
1980), providing this with a pronuclear membrane,
material
Testing of to
the most measure
fertilizing
ability
sperm. In have been
the past developed
information sperm lations.
Such
disulfide
useless
process
1-cell its
by
any DNA,
processing
a
the
is
the
releases
function.
certainly
in
point
this
of
it
oocyte further
zygote,
sperm is differencells in
nucleus synthesize
Biologically, an
at or
continued
and
the mammalian ovum. It is highly one of the smallest
whereupon
odological
across shape of
(Leibo,
et
cost. yet
SPERM
information nucleus can
measurement
simple;
(RaIl
low not
In many respects the reverse of the tiated, motile, and
time
of fluxes the spherical
intracellular
reliably, for
(Generoso,
the methodological in cryobiology include
feasible,
sperm available
have
cryopreservation
makes
as
sizes. ovum model
that several this system.
monitored
is
measuring
mation
ova
and held
possible that cells. In fact, so
with
of
diameter. are
a com-
of cell
including
embryos evolved with
Cryomicroscopy and
spectrum
1) Measurement is facilitated by
precise
swelling
on
of the
Specific examples value of mammalian
large
from
certain experiments done with smaller
of mammalian concepts have
and
the one just the procedures. to these workers
few years, the mammalian its own as a cryobiological
is being
the following: membranes
et
considerations other cell types
of reasons,
which could
were
as
derived
of theoretical observations
In the has come for
such to develop gratifying
of small
(Whittingham
al., 1972), principles described were used What was especially
to
amount
relatively
successfully
at the
at are
the
of more
mammalian of storing
ovum is playing role in cryobiological
be
ones
frozen
thawed
small form
1981). In if other
embryos
bination empirical
Cryopreserved as a method
procedures
of
is composed et al., 1976).
models of the small
membrane. mammalian
that
ci-yopreservation
mammalian
than
for
to
One of ratio,
surface area of cell When the first
was
which (Mazur
serve
and
to
ratio (Leibo, this means that
equivalent,
intracellular
and
e.g.,
pancreas cell type
cell
crystals
the
and embryos behave as ideal cells when one accounts for
surface functional
the
ice
systems,
indefinitely
of
erythrocytes.
oocytes other
water
becomes freeze, and
damaging
degree of dehydration in such the most obvious is the surface which
more
temperature water will
JR.
about or
to test Frequently,
difficult
in
of
biological
a practical
a sperm few years, that use the
various a
way or
para-
is
the
a sample
of
in vitro systems oocytes to obtain
intrinsic experimental standard in
fertility
of
manipuvitro fer-
tilization system is used, but variations such as using oocytes from human cadavers or removing the zona pellucida to compensate for deficiencies in in vitro methodology have been
MAMMALIAN
exploited. heterospecific
The
from
removed purposes,
hamster
which
oocyte
makes
is not
it appropriate
testing.
This
species
for
procedure
evaluating
has
human
specific,
many
kinds
been
sperm
somes intense
which
X-
and
et
1976) and may eventually become a quality control procedure at artificial insemination centers for a variety of species (Brackett,
of
of chromosome ratio of V to
V-chromosome and
provides
clinically
(Yanagimachi
1982).
in a population of interest in methodology
Seidel
of fertility
used
39
A special case evaluation of the
has been practical zona-free
the
AS METHODS
(Yanagimachi,
involves hamster
zona pellucida (Yanagimachi, 1982). For sperm penetration of the
oocyte
for al.,
most popular scheme fertilization using the
OVA
quick
two great
and
of
direct
especially
to
in species
like
is
(e.g.,
this
system
of
efficacy
evidence
tested
sperm, thus of waiting
expense
sperm and
is
There separating
of
1982),
being
types
sperm.
bearing
Amann,
procedures
analysis X sex chromo-
separate
these
circumventing until parturition,
the
cattle.
1981). A surprising, theme
but
is the
for
use
couples
spermic. number results clinical
important
variation
of in vitro
in
which
on
fertilization
the
this
Androgenesis
systems
ovum,
Many men produce onty a small of primarily abnormal sperm, which in functional sterility. One of the major
spermy sperm
operate to prevent into the ooplasm.
tilization
is
by placing oocyte of percentage fertilize
in
the the of
in
overcoming
vitro
such
cases the which
fer-
infertility
few sperm available wife in vitro. In such ovum,
the
human
next to an a surprising
sperm leads
are to
able to normal
pregnancy.
sperm and
Chromosomes
After fertilization, the chromosomes
Under
suitable
zation,
one
the
first
will
or
subsequent
provide
mitoses
information
of
about
the
of
embryo
the
chromo-
can
there
entry of a second Occasionally two simultaneously, some cases
conditions
of
manipulate
sperm
of 20%
1983).
a mamto poly-
the
one of the sperm, results, a condition in embryonic or fetal
is a relatively
order
Dodds,
karyological examination of the metaphase plate
fertilizes blocks
start fertilizing an oocyte dispermy results. In
on the of Sperm
one
ovum will expel in others triploidy almost always ends
that
Evaluation
of
a sperm or more
after
malian
of
is
Normally,
oligo-
applications
husband
high (Fraser
It
in
is
vitro
and
fertiliso
numbers
rate
possible
but that death.
of
dispermy,
Maudlin, to
1978;
correct
this
lethal pronuclear redundancy microsurgically by removing either one of the male pronuclei or the female pronucleus, so that the embryo becomes
diploid
once
The
chromo-
sensitive
the XX or XV embryos were to the reproductive tract, the would have two genetic fathers,
transferred back resulting young but no genetic
mother.
from
to
a number
sperm chromosomes However, valid regarding
of
from
are
as
sperm 1982),
with or
(Thadani, from
tinguished
of has
an
oocyte.
drawn
if adequate sperm of a
been
done
In
such
cases,
the
sperm
can
clearly
from
those
given from
that men
can be different
for
oocytes (Martin sperm with rat
fertilization
normal
be
sperm,
individual oocyte
already
hamster mouse
lnterspecies hints
of the
can
of
using
1980).
somes
those
used.
Chromosomes studied by species,
muta-
approach is not of an individual distinguish the
conclusions
a population
controls
environmental
1983). This examination one cannot
the
human et a!., oocytes chromo-
usually
be
of
the have
models
a large percentage are chromosomally
female pronucleus be 1XX:2XY:1YY,
If
is selectively the latter
both
sperm
between two males male with a female. be accomplished vitro fertilization resulting
which
the
removed would being lethal. If
are
selfing
genetic
oocyte.
sperm
already
1980).
Thus,
part cally
instead of Crossing
the
same
might
the conventional two males could
by mixing semen and identifying
from of
process jecting
of sperm abnormal
from
male, selfing occurs; the same male is in effect both parents, a condition leading to considerable inbreeding. If the two sperm were from different males, the offspring would be a cross
basis dis-
of embryos
sex
somal
gens (Gledhill, appropriate for sperm, because
composition
more.
somes from the sperm. This procedure could provide an excellent monitor for environmental mutagens for people, as spermatogenesis is very
or
from
markers. be into
the
crossing
oocyte
on
the
Alternatively,
controlled the oocyte
of the methodology motherless mice.
prior to in the progeny
would of
the
by directly in(e.g., Thadani, be producing
an
integral geneti-
40
SEIDEL,
DEVELOPMENTAL
When
the
POTENTIAL
oocyte
is activated,
usually
process of parthenogenetic
fertilization, agents,
but in some it starts the
embryonic
development.
Development
proximately appear
to
bryonic bid,
be
somal
blastocyst
under
genomes diploid,
control
well
of
the
and
Opas,
of
embryos
test
of
Stored
information
in
needed,
of the embryo chromosomes primarily as factors Within
as are (Johnson, likely are placeholders
needed species
variability
for there
before
among
ap-
information embryo
em-
(Fig.
are
differences
depend
on
form
of
RNA
1979). required, which
Intranuclear but probably provide the to proceed. phenotypic
the
stage, and phenotypic
probably
blastocyst majority of
epigenetic, properties of
genetic information differentiated cells.
In
to secrete
it seems
very
of
DNA
(Sakano of cell of
i.e., they oocyte.
DNA and
that
is
offspring result, material from be determined.
totithe The
amphibian
has
been
the
early
oocyte
experiments
since
experiments
have
expansion, the
a great deal of embryonic pecially of the germ layers derm or ectoderm) from
In summary, embryonic extent course genetic
once
of
escape
the
oocytes
are activated,
development
proceeds
with intrinsic of embryonic
momentum. development,
Over the
the epi-
a certain
amount
of
tendencies
genetic
timing
etc.
need
direction.
goes awry Nevertheless, described,
Thus, with the
and
development
the permit
terventions
in this
to
development
a great
eventually
haploids, embryonic
tetrapboids, etc. momentum
epigenetic
characteristics
numerous
of
interesting
in-
shown
of adult although
tissue developed remains to be dedifferentiation In
totipotent zygote,
and that
of Totipotency
of Genetic
Totipotency might be defined of a cell or group of cells to completely normal adult that reproducing. information totipotent. destroyed marked ferred
Normal
embryos,
Instructions as the ability develop into a is capable of with
the
genetic
from the sperm and oocyte, are Three decades ago, Seidel (1952) one of the two cells in genetically 2-cell rabbit embryos and then transthem
to
pseudopregnant
recipients
for
not
large,
such
at least
nuclear
from
diploid
are
also
totipotent,
embryos
all efforts tours
by
to date
These
Markert
trophoblast exploration.
this
are
not
area
requires
parthenoeven
have
and
force,
reviewed
(1981)
and
of lilmensee from cells of
genetically much
to
which
are
Seidel
are
failed
experiments,
de
the have
themselves
Seidel (1982). The experiments and Hoppe (1981) hint that nuclei although
esmesoparent
transplan-
nuclei
this).
fully
cells
by transplanting them into Hoppe and Illmensee (1982)
remarkable
more
have
from
development, (endoderm, which the
on
parthenogenetic (or
nuclei
not genetically totipotent, are capable of programming
studies
demonstrate are
and
They
just begun. Illmensee and Hoppe already shown that nuclei of the inner cell mass are genetically
embryos
though
are geneti-
1981).
(DiBerardino, 1980). Much done to prove that complete is impossible, however.
mammals,
tation have (1981) have mammalian
genetic Tests
by
such These nuclei
that cells
(McKinnell, that,
for
1950s.
embryonic
tissues are such nuclei
shown
process.
of
used
shown
from
also
types
clearly
cyst
and
an
testing whether normal potency of the nuclear differentiated cell can
totipotent
mode
of
and
cally
pellucida,
nucleus cell,
kinds of differences that do occur among species include differences in cell size, amount of stored lipid, pattern of changes in cell cycle length, timing of compaction, degree of blastozona
cell
changes
a differentiated
many
The
in the whether
such
the
the that
for
whether
replacing
of
However, unclear
responsible
if so,
By
with
within cells
differentiation
et al., 1980). types it is still
irreversible.
ovum
antibodies,
irreversible
modification are
questions in of diploid
from
intrinsic
the
that
occurs majority
outside
a
genetic
biology sets of
the central differentiation
differentiation,
from
is really the
of
of
likely
is
nutrients
as
a 2-cell embryo
differentiate
1978).
This
1).
One concerns
intrinsic, ovum
totipotent. totipotency,
offspring some cases
in one blastomere of is identical to that in a 1-cell
eukaryotic nucleus
the cell cycle is only limited
the
species
the
were
epigenetic
not
1981).
probably
to term. Normal, fertile showing that at least in
half
with hapchromo-
is largely an the activated
gestation resulted,
by of
as with numerous various aneupboidies
(Niemierko
development property
(Sherman,
to does
it can occur or tetraploid
as including
trisomies
Thus, early epigenetic
cases course
stage
strict
since triploid
complements defects
genetic and
the
in the
JR.
totipotent, more
thorough
MAMMALIAN
Primary
oocyte
OVA
Germinal
Completion
meiosis
End of S-phase
membrane
formation
of meiosis
of
I
down
Pronuclear
Completion
41
vesicle
break-
Fertilization,
AS METHODS
first
cell
of
cycle
II
Pronuclear break FIG.
maternal and
that
membrane -
First
mitosis
Nuclear
formation
down
1. Chromosome
and
paternal
ploidy
varies
complements
genomes from
membrane
haploid
in the
are separate, to tetraploid.
mammalian
that
oocyte
complements
and
early
sometimes
embryo.
Note
are enclosed
that
at certain
in nuclear
times
membranes,
42
SEIDEL,
Horn
ozygous
Diploid
One of manipulations
the of
mammalian homozygous was
put
by
by
mammals
fertilized surgically.
by
one
embryo The
and
of
this the
(zygote) diploid
in
methodology.
thus,
inbred be
the diploidized pronucleus was chromosome bearing sperm, the genotype with no X chromosome
produced from
five female
derived at least
homozygous
pronuclei
of
totipotent
as
plement periments
with
and
methods
of
ova
considered
two
nucleus
from
a
These elegant exthe power of meth-
pronuclei
removing
microsurgically, diploid
information A third
comes method
second
polar
1982).
A fourth
where
with
cytochalasin
method,
one
of a embryo
the
genetic
was
proposed
the ovum with a sperm. To date, oocyte
fusion
B (Seidel, of
by
(1978). It consists with inactivated Sendai agents, which in
by
without already
from the female pronucleus. involves suppression of the
body
conceptually,
of
both original 2) the homo-
and
procedure
female with another, of the dispermy
Identical
research
periments variation
can does
be not
another none has
the
the
simplest late
Pierre
of
fusing two virus or other effect involves ovum instead of the embryos gone
to
term
proper
the female experiments be females
would
of mammalian
gyno-
gynogenesis,
rescuing
methodology,
triplets,
for
some
will
Multiplets
twins,
valuable
A
adds
that this method selfing females or
All offspring
earlier.
to
development.
crossing
one
is
similar
effecting
it seems likely be useful for
etc.,
subjects designed interfere
are
very
because so with
ex-
that genetic interpreting
treatment effects. In a few mammalian species the problem of genetic variation in experiments has been circumvented by using inbred strains and their F1 inbred lines Recently, has become
crosses, but is impractical
however, feasible.
manufacture who
producing for
producing The first
identical
(1970),
transferred a variety
parthenogenetic
and
than
twins
separated
them
Success
rates
were
2-cell
Hahn
to
Mullen
cultured stages
obtained
much
to The
the
agar
have
of agar oviducts
blocks half
the agar, gestation. ful in horses,
at the 2-cell pellucidae,
blocks ligated
developing
in
gestation.
half
were
which
were for
then
embryos
producing sheep and
stage, placed and embedded
of sheep
and by
before
to new has been
by of
them in them
transferred 4 to 5 days.
recovered, were
and retransferred This technique
been
half and
significant advance was developed (1979), who separated blastomeres
sheep embryos surrogate zonae in small
these in vitro
Moustafa better success
simply cutting mouse morulae transferring them to the uterus. A most Willadsen
et al. embryos
for
low.
very
completely species.
mouse
recipients
were
(1978)
most
identical twins group actually to
into individual blastomeres, embryos to post-compaction
1) transplantation
a diploid
zygote
zygous
com-
sperm
embryonic
of Identical
is genetically
provides
of these:
for
Manufacture
pronuclei
of producing mammals participation of a male. We have
genetic
(other
needed
there
embryos.
the
if
YVY results.
haploid
is that
In any case, will eventually
a
proves that a dipboidized
genome
diploidized,
parthenogenetic
a
Parthenogenesis
Manipulation
produced
sperm a
from
component
develop
likely,
activation,
with allophenic later.
originating
This
critical
proper
parthenotes be considered
be confirmed in Illmensee (1977)
two
dipboid possibility
described
embryos.
Gynogenesis
fertilizing of with
and
the is
from lethal
mice
from the oocyte. nicely illustrate
odology
Soupart oocytes fusogenic
to and
from X-bearing sperm. in some circumstances,
complement
into
remains Hoppe
generation. method;
one this
with
second
Most
with any of the methods genesis. A fifth method of
the first mitosis embryo transferto term. Animals
in by
that
with
many
although
stage.
problem
counterpart
is removed microstate is restored by
animal produced
Although the work other laboratories,
a 1-cell
homozyproduces
transfer,
blastocyst
activation) In
of
this way are completely this methodology
completely Males cannot
in a
However, reported
pronuclei
preventing cytokinesis during with cytochalasin B, and the red to a recipient for gestation produced gous;
outlined
Petters (1977). (1977) first
the
some
production of This concept
Markert
embryo
to
experimental concerning
was the mammals.
forth
procedure,
after
important last decade
Markert and and lllmensee
producing this
most the
embryos dipboid
first
paper Hoppe
Mammals
JR.
and
dissected
the from
recipients for very success-
identical multiplets from cattle. Identical quadruplets
produced
4-cell stage, although are lower for quarter
with
blastomeres
probabilities than for
at
of pregnancy half embryos.
the
OVA
MAMMALIAN
technique
Willadsen’s
suboptimal with
in
vivo
effect
in
in vitro
culture
culture.
replaces
techniques
Because
produce
identical
morulae Very
(Willadsen recently
greatly
et al., 1981). this methodology for
simplified
al.,
1982;
collected
et
blastocyst
cracked,
microsurgical
stage,
from
placed
in
from
an
ready
for
the
a cracked,
are the
One
by
conceptual
most identical Willadsen
basis
work
by
Kelly
there
is
slight
divisions
that
develops
to
for et
al.
15
dividual cells were two that resulted higher chance than the two
of
of
from
the
The
actual
the
minutes,
which
portive embryos
with in
an
ly
from
they
inner
by
who in
back
found the
a
to that
two
2-cell
the had
a
into inner cell mass the slight head start.
the
have
from
cells
8-cell
embryos which
of
the
results
8-cell
embryo,
developmental 4-cell embryos information has for producing
slightly
lower
those
from
considerably embryos, clining
near pregnancy
lower; zero. rates
than
those
are
probably
due
of
developed embryo
and
played
sup-
The original and although
were usually
chimeric, the were derived
original
8-cell
have This
using
embryo.
already
embryos
kinds
been
is clearly
an
which
will
of experiments.
from
to
an
has
tually there this technology modify the
it the
is any
will
appears
be
passed
the
one of
of the
the
genome,
to
all
the
may shown
1981; T. al., 1981). DNA
be
especially DNA
passed
into
of have
on
of
Church 1981;
Wagner et In these copies been
to
al., exof
injected
the pronuclei, and in many sequences were incorporated and
the
by a number
thousands
naked
of Luckily,
of foreign
been
1-cell at the
cells
(Brinster et al., 1981b; 1982; Costantini and Lacy,
some of
into some
Even-
embryo.
zygote
and Ruddle, Wagner et
periments,
on that
incorporation as has
groups recently and Tamaoki, E.
modifying
with gametes or genetic modifications from
that to
genome,
Gordon 1981;
and markedly.
be countless applications of to mammals. If one wanted to mammalian genome, the obvious
developing
amenable
sequencing improved
will
start since
sequence
eighth de-
for
acids
DNA
Genome
Methodology nucleic
stage
embryos,
and those from These progressively
identi-
cells. marked,
of Heterologous Mammalian
1-cell
way.
quarter
the
animal
details of this method are presented pertinent points will be considered. rates from transferring half are
into
head
Before the a few other Pregnancy
embryos;
Incorporation
since
(also see been used identical
remarkable
other
to
in
whole
absolutely
many
Fehilly
produce blastomeres
embryo
procedure. of
(Markert and
8-cell
sheep
method
place embryo,
exclusive-
multiplets
embryos
an
embryos
is derived
a considerable
start over cells from Spindle, 1982). This to improve methodology
sheep frequently
the
this
of them, a support
in-
that
division
4-cell
from
facilitate
these
found
exclusively
very
placenta
the
resulting embryos and resulting lambs
elegant
cell
embryo
When
fetuses
is that
then using Willadsen’s technique (Willadsen, cases, the inner cell
roles as trophoblast were genetically
produced
goes
marked, they from the first
mass
the
quintuplet
of developing cells without
cell
from
Identical
method
when
of 4-cell
cells
a given
given and
embryos of
the
them one at a time with a blastomere
three-eighth
blastomere
the
embryo.
blastomeres those
such
initial a
most have
to the
embryo and host oviduct 2). In most
the
This effect was even clearer at the 4- to 8-cell division. Willadsen and Fehilly (unpublished) have taken this a step further and found that mixing
by
from a 4-cell intermediate 1969) (Fig.
of
as
differenti-
Willadsen
principles separating
these
lambs
mass
point
the
1978).
exciting developments in multiplets was recently and Fehilly (1983). The (1978),
occur
as forming
used
all
after
actually
8-cell embryos and placing in surrogate zonae pellucidae
practical.
asynchrony a 4-cell
half
A further
the fetus proper; inner cell mass,
such
of
make
occur
blastocyst
a is
are
calves,
to
cal
and
50%
the
Petters,
(1983)
to
blastocysts,
is
pellucida
embryos
pairs.
to
very
the of
reported
twin
procedure
or
About into
10
morulae
pellucida
in
function and
are
zona The
develop takes
of
of zona
ovum.
et
These
the
divisions, not when are present (Tarkowski
1967).
cells
ate to form even in the
in half with the embryo
cut
cell cells
Wroblewska,
been
pellucida
immediately.
identical
production
is
surrogate
transfer
microsurgery
late zona
original
embryos
which
the
the
unfertilized
half
makes
at
of of
few
(Ozil
1982).
the embryo blade. Half
and
removed
embryos
al.,
nonsurgically
early
the
bovine
Williams
has
of appears
number number
bovine
of cells
parts
differentiation
blastomeres
from
number
functional
they may also form a viable this method to
multiplets
43
insufficient
the
available
the
are encased in a zona pellucida, interact more effectively to embryo. Willadsen has also used
AS METHODS
cases,
succeeding
into
a
SEIDEL,
44
Manufacture One 8-cell
of
JR.
3/8
Embryos
Two 4-cell
+
Eight
=
embryos
3i
FIG. 2. Manufacture of three-eighth sheep embiyos. One cell from an 8-cell embryo and one 4-cell embryo are placed together in a surrogate zona pellucida. In most cases, inner cell masses arise from the blastomere originating from the 8-cell embryo. From Willadsen and Fehily (1983).
generations.
In a few
evidence of genetic DNA, i.e., RNA and transcribed mous
and of
of
has
even
Although
work
First,
remains,
an
this
enor-
is a most
try to inject the DNA into the because this may be the most in the life cycle for modifying
there
is a massive
chromatin,
as
the
replaced other cells.
with Second,
the
are
reprogramming
protamines
of
histones the ovum
the
1981). Third, there genetic reprogramming understood. In any pronucleus
(and
modified
more
imagined
a few
mammalian
easily
ago,
embryos
technology
the than
years
in the
sperm
female) most
which
will
application
would will
be
a
can insure
critical
embryos
be have that
DNA
(Tarkowski,
Two
with decades
Chirneras ago,
demonstrated
that
cleavage-stage
mouse
two
or Allophenes it
was
genotypically
inject cells into the blastocoele stage. A variety of cell types
can
used
area
successfully
inner cell impossible
in a few
and
embryos
would
different form
1981;Russell,
are
juxtaposed
the
methodology
value the
that
it must
context methods
has
already
been
considered,
of three-eighth embryos multiplets (Willadsen and
1983). cations
There are of allophenic especially of the first
literally hundreds animals; only
illustrative applications
the basic biology muscle is composed
to make Fehilly, of applia few that
will be presented. was to elucidate
of myogenesis. of multinucleate
Skeletal cells, but
whether this is a consequence of nuclear division without
Mintz and Baker (1967) made mice from strains with differing forms
Another
that
of
isocitrate
muscle cells. of subunits muscle area
be
of using (Papaio-
1978).
application
showing one
However, of such
especially in as experimental
manufacture identical
seem One
if they
allophenic
mass (Gardner, 1978). It is to do justice to this important
pages.
is so powerful,
making
1965,
is to blastocyst
be
to
Mintz,
mammals at the
then studied composed
dramatically
1961;
resulting of both
approach
mic Methodology
the cells
in if
Another
division. phenic
PROPERTIES
EMBRYOS
each other pellucida;
a suitable recipient, be composed of
it was unclear cell fusion or
DEVELOPMENTAL
to zona
a
1971).
One
to mammals.
OF
transferred to mouse would
annou,
com-
of recombinant
embryo if juxtaposed after removal of the
considered, embryos
enzymes, to repair Brandriff, Pedersen,
may be other types of events that are not yet case, the DNA in the male
possibly
mosaic vitro
to the clearly
characteristic of appears to have a
system of very active DNA repair which in fact go over the male genome accumulated damage (Pedersen and 1980; Generoso, 1980; Brandriff and
ponent
been
of the injected protein have been
beginning.
Some workers male pronucleus vulnerable stage DNA.
there
translated.
amount
encouraging
cases, expression resultant
cell from exclusively
forms
in which
of cell alloisozy-
dehydrogenase, They from
found both
by fusion allophenic
and enzymes strains, of cells. techniques
MAMMALIAN
have
been
genetic
especially
potential
of
thenogenetic mice, for into
various
to
ductive 1974).
tracts There
genetic
defect,
term
cell
types.
when
sometimes
tissues
of
(Stevens Moustafa, suggests basically retarded
develop
the et
genotype
a!., 1978).
Since
or
cells
of
primordial
the
animal,
and
cells
although
they
development more
carcinoma characteristics
host,
embryonic
to
that
cell
virtually
primordial can
Martin,
kinds
cells
become 1980;
chimeras.
chimeras meres
were such
which
that
would
the
studies concern
the of
by
outside
the
Interspecies
ancient embryo
to too,
placenta,
were
recipient,
to
Greek transfer
Some chimeric
are
two in-
primarily
gen. males
are to
the
the
reproductive
defect
in cells
of
the
Therefore, embryos
with
those The
allele.
Such
the same testicular although require the
were mice
germ
study animal
of these animals sense. and
et
cells
strain
rates
One
between
with
somatic
of
both
normal
the
fertile strain This
between allele
(Lyon et a!., the resulting
with
produced
aberrant
sperm
with
of embryo proved
with that,
such as Sertoli cells to function normally,
themselves
1978).
the
testosterone.
made feminization
cells
do not.
technology
normal chimeras
and
composed
cells receptor
growth
al.,
testosterone
receptor some of
germ
to andro-
is required for interest to know
cells
can
factors contributing production, such
chimeras with get
the
genotype as the feminization. somatic androgen
androgen
of such sterile female external
chimeras were the testicular
and
model.
of the use the testicular X-linked allele
required
with normal testes of
cells
transfer
respond
testosterone was of germ
of skin
produce such genotypes,
intracellular
testis
males
somatic
system. tolerant reciprocal
of this
because
it
both
sexual
embryo
cannot
develop
This
of
usefulness
the
masculin-
study
With
so that
gonads
a bull.
possible to of specified
the
twins
cases,
females
Even though the testes secrete testosterone,
differing
the
of Each from males
when
all
immunologically for example,
increase
Allophenic
embryo,
sheep
versa
for
accepted.
a
chimeric In
blasto-
which
exchange
co-twin
it is now embryos
will
1975).
1978;
mythological between
of are also so that,
technology twins with
and
cancer
mixing
of
model
twins other
grafts
part-goat
the
a
vice
born
tissue
from successful
of the
are
normal
1981).
the
embryos would be transferred. have gone to term, producing in
cells
genitalia
they
the
including
part-sheep,
if
genitalia
have already contributed of some aspects of caris undoubtedly much left
manufactured
internal
whether
thus,
Mintz,
In nearly
normal
(lllmensee, and
These
form
species
tissues
parents
and
and
sex.
Since
next
cases,
vasculature
causes
cells
has no effect. spermatogenesis,
They,
gametes;
Stewart
these ways, embryos to our understanding cinogenesis, and there for them to contribute. Recent unpublished European laboratories terspecies
placed
of
and
teratoall the lethality
in
in a blastocoele.
all
germ
part
when
mass
of
that bovine
most
placental
which
female
receptor of
completely
take
development
inner
kinds
usually have cells including
revert
cells
embryonic
some
twins,
opposite
causes
1978),
parents,
incapable
In
in the
One of the nicest demonstrations of allophenic mice concerns feminization syndrome. This
form
become are
which of cancer
of
which
allophenic
(Stevens,
can
bizzare,
cells,
the
cells
embryos are damaged or cannot muster
resulting
themselves
have
are
Such each
1977; rescue
to term.
Even
form
a!.,
1981).
occur
differentiation
parthenote
form a complete other embryos.
gametes
embryos
some
sometimes
in the
thus,
parthenogenetic
for
the
parthenote
germ
to
provides
have
and
the first models of chimerism in mammals was fraternal
bovine
that of
to from
the par-
(Warwick
circulating cells during fetal development. resulting calf has some cells originating the co-twin, so that it is not unusual for
ized
1977; Surani et Such albophenic
resources support
a
Hoppe
successful
(Benirschke,
of
repro-
that When
twins
aggregated with to recipients,
that parthenogenetic normal, but perhaps in such a way that they
the strength fetus without
anastomoses
earlier,
are transferred
One of studied
but
not
ordinarily
1949).
was
do
45
is not
Berry,
(Graham, to suspect
have shown totipotent.
goats
par-
to
mothers reason
as described
embryos and
of
AS METHODS
strains of to develop
transferred
lllmensee (1982) are genetically
young
study
Diploid
blastocysts,
recipient is no known and
the
developmental inbred appear
of
thenogenetic normal embryos
of
the
normal
develop
and nuclei
is
and
embryos from example, frequently
completely
the
valuable
totipotency
OVA
also to
as
among
animals
experiment
embryos
be
from
used
to
efficiency the causes
of of
(Falconer
is strains
to
make of mice
and rapid growth rates. One might that have muscle cells of the normal
growth
rates
and
liver
cells
SEIDEL,
46 of the fast grew rapidly,
growing strain. If such one could conclude that
of muscle growth in should
were not the strain
study
rapid
the
responsible of interest,
liver
Embryo
allophenes properties
for but
animal
increased that one
to understand
of
the
production this
is
biggest
problems
embryonic
problem,
mortality.
chimeras
the uterus mortality embryonic
in
might
To
be made
material,
the organ responsible interest. Of course,
some cases, clear answers will not because several organs are involved some sort However,
of interaction in other cases,
be obtained It
with
these
be
unfair
would
that
these
methods
biggest problem date is that the fairly
random
genotypes
cases and,
have
in
obtained, or there
to
give
the
foolproof.
impression the
with allophenic technology resulting mosaic animals
to are
of
the
used.
two
However,
only tissues of one genotype in many other cases, all but
or
more
in
many
accuracy Emhryo
of
will
provide desired
can be found a few percent
and
repeatability.
the ability composition
to
flexibility embryos
from
results the
One
able tract
sheep.
Anderson
plet
et al. (1981)
pregnancies
in
inheritance It might
apsects
using
some
gestation
from
a breed
with
short
some length.
embryos Mixed
from breed
a breed with pregnancies
lengths
midway
and
the
not
merely
other,
between by
triggered
but may inhibitor. perimental
be
examples
are
those
demonstrating
controlled There are uses of given
by
of
that an
inducing
as well hundreds embryo Seidel
multiembryos length
long had
in
and
gestation gestation one
breed
parturition
is
(1979).
in of
a
1981b).
transfer
or more circumgenetic
alleles
(Seidel,
These life
numerous
the other
examples
examples
clearest aspects
of
are
probably
also
very
interesting
to
cytoplasm
without
being cytoiminject mito-
or embryos of one strain While this can also be fusion of more differen-
methodology more controlled
enormous of
with
ova in methodidea is making
with embryos proapproach to some
kinds
of studies
value
in
will
probably
understanding
many
processes.
ACKNOWLEDGMENTS I am grateful to numerous friends and colleagues for discussing ideas with me. Related work in our laboratory has been supported by the Rockefeller Foundation, Colorado State University Experiment Station through Regional Project W-1 12, and Genetic Engineering, Inc., Denver, CO. REFERENCES G.
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