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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.

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