THESIS

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This is to certify that the

thesis entitled

Blastomere separation of preimplantation embryos in
the golden hamster and squirrel monkey as a method
for production of monozygotic twins.

presented by
Richard G. Rawlins

has been accepted towards fulfillment
of the requirements for

Ph.D. . Zoology

degree m

wfl/ABLM

Major professor

Dr. W. R. Dukelow

 

 

[hue May 2, 1984

 

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BLASTOMERE SEPARATION OF PREIMPLANTATION EMBRYOS IN THE
GOLDEN HAMSTER AND SQUIRREL MONKEY AS A METHOD FOR
PRODUCTION OF MONOZYGOTIC THINS

By

Richard Graham Rawlins

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology
1984

 

ABSTRACT

BLASTOMERE SEPARATION OF PREIMPLANTATION EMBRYOS IN THE
GOLDEN HAMSTER AND SQUIRREL MONKEY AS A METHOD FOR
PRODUCTION OF MONOZYGOTIC THINS

By

Richard Graham Rawlins

DevelOpment of techniques for blastomere separation,
production of viable multiplet embryos, and the in vitro
culture of isogenic and control embryos to development
stages suitable for embryo transfer were investigated using
hamster (Mescocricetus auratus) and squirrel monkey

(Saimiri sciureus) embryos. Experimental protocols were

 

first applied to hamster embryos of 2-, 4- and 8-cells, fer-
tilized in vivo; they were then applied to monkey embryos
fertilized and cultured in vitro.

Viable multiplet hamster embryos (assayed by morphology
and the exclusion of the vital dye trypan blue) were pro-
duced by mechanical separation and by chemical and mechani-
cal separation of blastomeres. Pretreatment of parent
embryos with a 0.25% solution of pronase lysed the zona
pellucida and a solution of calcium and magnesium-free

phosphate buffered saline with 0.02% EDTA reduced

intercellular adhesion prior to microsurgery and increased
the post-surgical survival of multiplet embryos. Mechanical
separation was used to produce a viable half-embryo from an
in vitro fertilized 4-cell squirrel monkey embryo. Chemical
pretreatment had no detrimental effect on control embryos.
None of the multiplet embryos produced underwent further
development after transfer to in vitro culture at 37°C in 5%
COZ in air. Control embryos of the same developmental sta-
ges were successfully cultured to morula and blastocyst
stage in TALP. A total of l93 hamster embryos were sub-
jected to blastomere separation and l285 were cultured in
vitro to test the efficacy of four different culture media.
These included TC-199 with pyruvate, TC-199 with 20-30%
pregnant hamster serum, Tyrodes solution with 20-30%

pregnant hamster serum, and TALP.

Hith love and gratitude for their patience and support:

To my parents,

Herbert Lee and Eleanor Graham

Rawlins

ii

ACKNOWLEDGEMENTS

I would like to thank the following individuals for
their personal and professional support over the last
several years. Without their help and interest, the task
would not have been completed. Drs. William T. London,
Peter Marler, Robert Hinde, Norman Maldonado, and
Leo Hhitehair provided invaluable encouragement as well as
friendship. My graduate committee, Drs. L. Clemens, J.
King, J. Johnson and G. Dawson supplied both an interest in
the research and in the candidate. Their help is greatly
appreciated. I am indebted to all the ERU's, especially to
Ms. L. M. Cleeves for her friendship, invaluable assistance
in preparing munuscripts and memorable ERU lake parties.
There is no way to adequately express my gratitude to my
advisor, Dr. N. R. Dukelow. His efforts on my behalf were
legion. He has educated by example and left an indelible
mark. Most important, he has been a great friend as well as
professor. Finally, I would like to thank two very special
peOple - Matt Kessler and Cindy Evans - they know the

reasons why.

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . .

LIST OF FIGURES . . . . . . .
INTRODUCTION. . . .

LITERATURE REVIEH . . . . . . . . . . .

An historical perspective. .
Use of mammalian embryos for
blastomere separation and
new microsurgical methods . . . .
Live births from embryos produced
from isolated blastomeres . . .
Laser destruction of blastomeres. .
The role of the zona pellucida
and chemical dissociation
of the blastomeres. . . . . . .
Chemical removal of the zona pellucida
and chemical dissociation of the
blastomeres . . . . . .
Production of monozygotic multiplets
in domestic species by blastomere
separation. . . . . . . . . . . .
Production of monozygotic multiplets
in domestic species by blastomere
separation. . . . . . . . . . . .
Applications of blastomere separation
and monozygotic multiplet embryos
to research on nonhuman primates
and humans. . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . .

Production and collection of embryos .
Embryo microsurgery. . . . . . . . .
Embryo culture . . . . . . . . . . . .
Statistical analyses . . . . . . .

RESULTS . . . . . . . . . . . . . . . . .

Production and viability of hamster
half-EMbT‘YOS. o o o o o o o o o 0

iv

vi

vii

10

T4
16

20

The effect of chemical pretreatment
on half-embryo viability. . . . . . . . . . . 5l
The effect of culture in vivo on
half-embryo development . . . . . . . . . . . 55
Differential develOpment of control
embryos in vitro. . . . . . . . . . . . . . 55
Efficacy of the culture media for
control embryo development. . . . . . . . . . 57
The effects of chemical pretreatment
on the development of intact
embryos . . . . . . . . . . . . . . . . . . . 59
The effect of temperature change
on embryo development. . . . . . . . . . . . . 64
Micromanipulation of a squirrel
monkey 4-cell embryo . . . . . . . . . . . . . 67

DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . 70
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . 76
LITERATURE CITED. . . . . . . . . . . . . . . . . . . . 79

APPENDICES
A. Publications by the author . . . . . . . . . . 89

8. Curriculum vitae . . . . . . . . . . . . . . . 94

 

Table

LIST OF TABLES

Page

Blastomere separation of hamster
embryos (both methods pooled). . . . . . . . 50

A comparison of multiplet embryo

production in the hamster by

mechanical and chemical separation

of blastomeres . . . . . . . . . . . . . . . 53

A comparison of culture in vitro
of hamster embryos by developmental
stage (pooled for all media) . . . . . . . . 56

A comparison of hamster embryo
culture by developmental stage and
culture medium . . . . . . . . . . . . . . . 58

A comparison of the effects of

treatment with pronase and with

calcium and magnesium-free PBS

plus EDTA on the development in

vitro of 8-cell hamster embryos in

TALP . . . . . . . . . . . . . . . . . . . . 60

vi

LIST OF FIGURES

Figure Page
1 The micromanipulators used for
blastomere separation . . . . . . . . . . . 39
2 Procedure for mechanical
separation of blastomeres . . . . . . . . . 4l
3 A comparison of the morphology

of hamster half-embryos derived

from blastomere separation of

8-cell embryos by mechanical (A)

and chemical/mechanical (8)

methods. . . . . . . . . . . . . . . . . . 54

4 An example of a blastocyst, within
the zone pellucida, cultured in
vitro from an 8-cell hamster
embryo in TALP. . . . . . . . . . . . . . . 61

5 An example of spontaneously
dissociated blastomeres from an 8-
cell hamster embryo denuded with
pronase and washed in calcium and
magnesium-free PBS (with 0.02%
EDTA). . . . . . . . . . . . . . . . . . . . 65

6 Microsurgical removal of a single
blastomere from a 4-cell squirrel
monkey embryo fertilized in vitro. . . . . . 69

vii

 

INTRODUCTION

Microsurgical production of viable monozygotic
multiplet embryos by blastomere separation of single parent
embryos has been routinely performed in rodents and has
resulted in the live birth of normal, identical offspring.
In the last five years, artifically produced sets of normal
twins, triplets and quadruplets have been derived from
domestic livestock. The technique has not been applied to
nonhuman primate embryos, but the recent success of embryo
transfer in two species of nonhuman primates now offers the
possibility that monzygotic embryos generated by micro-
surgery can now be transferred and maintained in utero to
term. The present study was directed at the development of
techniques for blastomere separation, production of
multiplet embryos, and in vitro culture of the isogenic
embryos to cell stages suitable for embryo transfer in the
hamster (Mesocricetus auratus) and squirrel monkey (égimigi
sciureus). Experimental protocols for blastomere separation
and embryo culture were first attempted using hamster
embryos fertilized in vivo prior to use on the less readily

available in vitro fertilized monkey embryos.

The goals of the study were to develop a successful
micro-surgical procedure for blastomere separation in the
hamster and squirrel monkey, to determine the viability of
the surgically produced multiplet embryos, and to evaluate
in vitro culture methods that would support continued growth
and differentiation of the monozygotic embryos.

The first objective of using blastomere separation to
produce multiplet primate embryos is maximization of output
of in vitro fertilization systems utilizing primates by
increasing the numbers that can be generated. Blastomere
separation and subsequent embryo culture in vitro also per-
mits sex determination and assay of chromosomal and bioche-
mical normality of preimplantation embryos while identical
multiplets are maintained undisturbed and continue to deve-
lop without risk in culture. Monozygotic multiplet embryos
provide a valuable resource as isogenic animals for experi-
mental use in physiology and behavior. Finally, the suc-
cessful use of blastomere separation to produce multiplets
in primates has direct application to the growing field of
human in vitro fertilization and embryo transfer, since the
methods developed to assay the normality of half embryos in
nonhuman primates can also be used to determine viability of
human embryos as well. Thus removal of blastomeres and
subsequent culture in vitro can provide a valuable tool for
the early detection of genetic or metabolic abnormalities in
in vitro fertilized human embryos prior to the costly and

often unsuccessful transfer procedure.

LITERATURE REVIEW

BLASTOMERE SEPARATION

 

An Historical Perspective

Modern techniques for the micromanipulation of mam-
malian preimplantation embryos are derived from experimental
work on embryonic development and differentiation which
began 100 years ago. The experimental analysis of develop-
ment was inaugurated largely by Wilhelm Roux, whose work on
the physiology of amphibian embryological development in the
l800's provided the first proof that early embryonic growth
is self-differentiated (Roux, l888). Roux demonstrated that
once division had begun, the component blastomeres were
totipotent and capable of further development. By intro-
ducing a hot needle into one of two blastomeres of a frog
(Rana esculenta) 2-cell embryo, half of the embryo was
killed and the capacity of the other blastomere to develop
was tested (Roux, 1888). The intact blastomere survived and
developed while the destroyed blastomere decayed. The
experiment produced the first known viable half-embryo and
was subsequently replicated many times (Morgan, l895).

Roux's work was criticized because the killed blasto-
mere had not been removed from contact with the viable half

embryo, and it was argued that total isolation of the living

blastomere had not been obtained. Embryologists were not
concerned about the dead blastomere interacting metaboli-
cally, but they worried that there may have been mechanical
constraints placed on the development of the intact blasto-
mere by the presence of the decaying tissue (Spemann, l938).

Driesch (l89l, l892) was the first to completely
separate and maintain two viable blastomeres. Sea urchin
embryos (Parechinus 3p.) at the two cell stage were placed
in sea water and violently shaken for several minutes until
the blastomeres dissociated. The isolated blastomeres con-
tinued to develop at first as if still attached, producing a
half blastula with an opened blastocoele, but the open edges
then curved together and produced two normally proportioned
embryos of half the normal size. Similar results were later
shown for amphibians (Morgan, l895; Spemann, l938).

Hertwig (l893) made the first attempt at production of
twin embryos in amphibians by bisection of the newt (Triton
'taeniatus) two-cell embryo. Although he did not succeed at
isolating the blastomeres, he introduced a new method for
embryo microsurgery which improved upon the crude disso-
ciation of cells by agitation. He constricted the two cell
embryo along the first cleavage plane with a hair loop to
cut the blastomeres apart. The same technique was later
used by Endres (1895) to produce the first set of viable
Triton twins by blastomere separation, and by Herlitzka

(l897) and Spemann (l938) who confirmed Endres' work.

Again, the twins produced were morphologically normal, but

 

of half the normal size.

It is important to note that these early experiments
were directed at understanding the regulation of early
embryonic development rather than the production of monozy-
gotic half embryos. At that time, little was known about
the capacity of the early embryo for self-regulation during
differentiation and it was the work of both Roux and
Driesch which gave rise to the concept of totipotency for
early embryonic development. Driesch (l89l) thought the
isolated blastomeres were of equal developmental potential
and could regulate development, once disturbed, towards a
whole embryo, providing its initial structure was not
complex. It was Roux (1888) who provided the evidence that
self differentiation in early embryonic development sub-
sequently gave way to what he called dependent differen-
tiation which resulted in the loss of totipotency of the
blastomeres and subjected them to the limiting influences of
the other cells such that independent development was lost
and mosaic development occurred (Spemann, 1938; Nicholas and
Hall, 1942).

Studies of half embryo development in echinoderms and
amphibians continued into the l900's, but the emphasis
shifted from examination of the developmental potential of
the half embryo toward consideration of the interactions
between blastomeres as assessed by isolation of individual
cells. The goal was resolution of the conflict between

Driesch's concept of totipotency and Roux‘s theory of mosaic

develOpment. In time, it was shown that neither concept was
correct. Species-specific differences in the capacity of
embryonic tissue to reconstitute itself following manipula-
tion were identified, and attention turned away from earlier
work, which had shown only the potency of an isolated part
of the embryo to develop, toward more detailed analyses of
the mechanics of total organization of the embryo during
ontogeny (Spemann, l938; Nicholas and Hall, l942).

In the first three decades of this century, blastomere
isolation was confined experimentally to echinoderms and
amphibians. Huber (19l5) suggested the use of mammalian
embryos in isolation studies, but there are no reports of
any such work available. The prevailing view was that
higher life forms could not be used for blastomere separa-
tion studies since, from the beginning of embryogenesis,
potentiality was limited as a result of the increased spe-
cialization of mammalian species relative to lower life
forms (Nicholas and Hall, l942).

Use of Mammalian Embryos for Blastomere Separation and New
Microsurgical Methods

 

The first known attempt to use mammalian embryos for
blastomere separation studies was undertaken by J. S.
Nicholas and B. V. Hall at the Osborn Zoological Laboratory
of Yale University in l933. They removed 2-cell stage rat
embryos from the oviducts, rinsed them in acidified calcium-

free Ringer's solution to remove the zona pellucida, and

separated the blasomeres mechanically with a fine hair or
glass needle. Once bisected, the cells were transferred to
the left uterine horn of a host female nat for culture.

Most of the blastomeres were lost, but a few did implant and
produce embryos which were dissected from the uterus at l0
days (Nicholas and Hall, l933).

Nicholas and Hall (1933, 1942) introduced important new
techniques for embryo manipulation. A glass pipette was
developed for holding and stabilizing the embryo during
surgery. The pipette was drawn to a distal aperture of
about 25% the diameter of the embryo and placed in a univer-
sal joint clamp so that the point would lie in the field of
view of the microscope. The zone pellucida of the embryo
was then aspirated into the pipette and the suction tube
closed with a clamp to anchor the embryo in place. Then a
needle pipette was introduced into one blastomere through
the zona pellucida and the blastomere removed to produce a
half embryo (Nicholas and Hall, 1942). The incidence of
damage to the surviving blastomeres due to the effects of
mechanical separation and aspiration was apparently high
since, of half embryos produced, none ever developed
following transfer (Pincus, 1936).

, Hall's (1935) finding that the zona pellucida dissolved
in the presence of an acid solution at a rate proportional to
the degree of acidity led to development of an acid bath
(0.0002 M HCl in Calcium-free Ringer's solution) which would

remove the zona without damage to the blastomeres over a
period of about three hours. Once denuded, the blastomeres
were washed in Calcium free Ringer's solution which had been
buffered to pH 7, and blastomere separation induced by
cutting with a glass needle or eyelash, or by forcing them
apart with a jet of fluid from a capillary pipette (Nicholas
and Hall, 1942). Acid removal of the zona pellucida of the
embryo was employed because the resiliency of the zona pre-
vented application of the microsurgical instruments (either
a fine knife, glass needle, or eyelash) and reduced the
chance of successful blastomere isolation. The chemical
method was preferred because it produced two intact half
embryos from the same parent embryo and eliminated the risk
of mechanical damage to one or both blastomeres. The
methods developed by Nicholas and Hall for embryo micro-
surgery are virtually identical to those employed today.

Despite improved microsurgical techniques and the fact
that removal of the zona pellucida did not inhibit further
embryonic development, the separated rat blastomeres did not
develop beyond the egg cylinder stage after transplantation,
and viable offspring were never produced.

During this same period, Pincus (1936) claimed to have
tranSplanted single blastomeres of 2-cell stage rabbit
embryos into a host oviduct and produced normally differen-
tiated, but small blastomdermic vesicles from the pseudo

pregnant uteri of the recipient does. However, there are no

published data to support the work.

Live Births from Embryos Produced from Isolated Blastomeres

From 1942 to 1952, no additional studies of half embryo
production and development were published, but the results
of Nicholas and Hall were noticed by Frederick Seidel at the
Max Planck Institute, who began working on the isolation and
transfer of blastomeres from rabbit 2-cell and 4-cell
embryos. Seidel obtained two live rabbits from 2-cell half
embryos produced by destruction of one blastomere with a
needle (Seidel, 1952). Later work focused on the develop-
ment of isolated blastomeres from 4-cell stage rabbit

(Oryctolaggs cuniculus) embryos, but only midterm fetuses

 

were obtained (Seidel, 1956, 1960). Identical twins were
not produced since Seidel was destroying blastomeres rather
than separating them. The work was significant because it
was the first report of live births resulting from
transplantation of half embryos in mammals and because it
represented the first known attempt to culture these embryos
in vitro to study embryogenesis after blastomere destruction

(Seidel, 1960).

Laser Destruction of Blastomeres

Blastomere isolation by selective destruction and in
vitro culture of the resulting embryos was carried a step
further by Daniel and Takahashi (1965). who used a ruby laser

as a microsurgical tool to destroy individual blastomeres of

 

10

2-, 4-, and 8-cell rabbit embryos. The remaining blastomeres
did survive and were cultured in vitro, but did not progress
beyond midmorula stage (32-64 cells). Typically, the 1/2
blastomeres cleaved four times to produce 16 cells, the 1/4
blastomeres cleaved 3 times and reached 8 cells, and the 1/8
blastomeres divided to 4 cells (Daniel and Takahashi, 1965).
The laser produced embryos were not transferred to recipient
does to test their viability in an in vivo system, but the
results of in vitro culture of these isolated blastomeres
were not particularly good in comparison to Seidel's success
with in vitro culture of isolated rabbit blastomeres.

The Role of the Zona Pellucida in the Development of

Manipulated Embryos

Moore et a1. (1968) extended Seidel's work by examining
the developmental potential of single blastomeres from rab-
bit embryos at the 2-, 4-, and 8-cell stage to determine if
blastomeres isolated from the later developmental stages
remained totipotent. Blastomeres were by mechanical removal
of the zona pellucida and mechanical separation of the cells
with a glass or silvered steel needle attached to a micro-
manipulator. Blastomeres were also left within the zona, and
these were produced by needle destruction of the surrounding
blastomeres after puncture of the zona pellucida. Rabbit
serum was injected into the embryo after destruction of the
blastomeres to disperse the cytoplasm and the damaged

blastomeres were removed. The dissections were carried out

11

in homologous serum with 300-500 I.U. of pencillin per ml.
of fluid in a welled glass slide. The isolated blastomeres
of 2-, 4-, and 8-cell embryos, zona-free, with zona, or
injected into a surrogate zona pellucida, were then trans-
ferred to the oviducts of synchronized recipient does. None
of the zona-free single blastomeres survived, but 30%, 19%,
and 11% of the 2-, 4-, and 8-ce11 blastomeres left in the
zona developed into normal live offspring. About 34% of the
4-cell blastomeres placed in surrogate zonas underwent one
or two additional cleavages, but none survived to blastocyst
stage. No 8-cell blastomeres were placed in host zonae in
these experiments. The results of mechanical separation of
rabbit blastomeres showed a progressive decrease in the sur-
vival rate of single blastomeres with increasing cell stage
of the parent embryo. It is also worth noting that mechani-
cal separation of the 4- and 8-cell stage embryos resulted
in greater damage to the blastomeres because of increased
intercellular adhesion and the diminished size of the
blastomeres, so that the results may not have actually
related to a loss in potential for independent development
with advancing cell stage, but rather to an artifact of the
experimental procedure. Failure of the zona-free blasto-
meres to develop after transfer was attributed to leucocytic
invasion and digestion of the cells (Moore et al., 1968).
These studies did little to extend Seidel's findings,

since the only live births were also produced from

12

blastomeres isolated by destruction of surrounding blasto-
meres. However, they are significant because they represent
the first attempt to produce identical offspring by mechani-
cal separation of individual blastomeres from rabbit
embryos. They also represent the first use of the technique
of reintroduction of isolated blastomeres into surrogate
zonae pellucidae to protect the cytoplasm from attack by the
maternal immune system.

Seven years after Seidel (1952) published the first
results of blastomere isolation experiments with rabbits,
Andrzej Tarkowski of the University of Warsaw Zoological
Institute developed isolated blastomeres of mouse embryos
(Tarkowski, 1959a, 1959b). The work was intended to
investigate the regulatory capacity of half embryos and to
determine the fate of cells arising from blastomeres iso-
lated at simple and complex cell stages.

Experimental manipulation of the embryos was unchanged
from Seidel's earlier report. Blastomeres were destroyed
under a dissecting microscope by piercing them with a glass
needle held in a micromanipulator. The 2-cell and 4-cell
embryos were anchored by suction through a patent glass
pipette. The microsurgery was done in mouse serum diluted
1:1 with normal saline under a dissecting microscope, and
the procedure lasted from 16 to 28 minutes (Tarkowski,

1959b).

13

After the blastomeres had been destroyed, the embryos
were transfered to the oviducts of host females mated the
previous night by vasectomized males. The embryos were
introduced by making a slit in the ovarian capsule and
injecting the material directly into the lumen of the ovi-
duct through the infundibulum. The females received embryos
from donor females of a different coat color for genetic
confirmation of the transfer should young be produced
(Tarkowski, 1959a).

Of 175 half embryos produced from 2-cell embryos, 30.8%
implanted and 17.1% developed normally but were not allowed
to go to term. 0f 21 3/4 embryos transferred, 47.6%
implanted and 33.3% developed normally. Three females gave
birth to young derived from 1/2 embryos and one female deli-
vered a litter from 3/4 embryos. All of the offspring were
normal and reproduced (Tarkowski, 1959b).

In addition to the transfer of blastomeres isolated by
destruction of adjacent cells and left within the zona, a
series of transfers of denuded half embryos derived from 2-cell
parent embryos was attempted. The zona pellucida was removed
mechanically by aspirating the embryo into a micro-pipette
(70u diameter) and expelling it to rupture the zona. The
blastomeres were then separated by a glass needle or a jet of
medium from the pipette. The denuded blastomeres did not
develop in vivo (Tarkowski, 1959b); Tarkowski and Hroblewska,
1967), but would culture to the blastocyst stage in vitro
(Tarkowski, 1971).

14

Both Seidel (1959, 1960) and Tarkowski (1959) found
that isolated blastomeres of the 2-cell and 4-cell embryo
would develop to normal blastocysts that were reduced in
size, but culture of later cell stage blastomeres resulted
in a high incidence of formation of trophoblastic vesicles
(blastocysts without an inner cell mass).

Chemical Removal of the Zona Pellucida and Chemical
Dissociation of the Blastomeres

Tarkowski and Hroblewska (1967) hypothesized that the
cause of trophoblast formation was an epigenetic effect and
that the fate of an embryo cultured from an isolated blasto-
mere was linked to its spatial position within the embryo at
the morulae stage. Therefore, both the number and arrange-
ment of the blastomeres were significant variables.

Because blastomere isolation induced by destruction of
adjacent cells did not permit a test of the epigenetic
hypothesis, Tarkowski and Hroblewska (1967) returned to the
chemical isolation of blastomeres, as devised by Nicholas
and Hall (1942), to collect data on developmental capacities
of all blastomeres from a single parent embryo. The study
was particularly important methodOIOgically, since 1) chemical
removal of the zona pellucida by lysing with a 0.5% solution
of pronase (Mintz, 1962) was employed, 2) the blastomeres of
the denuded embryos were dissociated by soaking the embryos
in a 0.02% concentration of Versene (EDTA) in calcium- and

magnesium-free Dulbecco's solution, and 3) the denuded and

15

isolated blastomeres were cultured in vitro using Brinster's
medium (Brinster, 1963). Hithin 48 hours of incubation,
blastocysts formed from the denuded blastomeres, but only
15% of the 8-cell derived embryos were true blastocysts.
Transfer of the denuded embryos was not tried due to the
previous failure of zona-free embryos to develop in the
mouse. Despite the occurrence of trophoblastic vesicle for-
mation instead of normal development to blastocyst stage in
the 1/8 embryos, the experiment showed that at least up to
the 8-cell stage, the blastomeres remained totipotent. This
finding was later substantiated by Kelly (1975, 1977).
Tarkowski did not attempt to replace the zona pellucida of
denuded embryos derived from in vitro culture of single
blastomeres, but he did suggest, probably as a result of

the successful in vivo development of single blastomeres
which were left within the zona, that such transfers might
implant and undergo further develOpment in utero (Tarkowski,
1971).

Tarkowski was apparently unaware that Moore, et al.
(1969) had already attempted embryo transfer of pig embryos
produced by mechanical separation of 4- and 6-cell parent
embryos with subsequent replacement of the zonae pellucidae.
They suggested that the failure of the microsurgically pro-
duced embryos to develOp might relate to a critical interac-
tion of the blastomeres with the parent zona pellucida such

that introduction of a blastomere into a foreign zona would

16

result in cell death, that damage to the zona itself could
be responsible, or that leucocytic invasion and digestion of
the blastomeres via the rent made in the zona killed the
embroys.

The first two ideas were discounted because isolated
and denuded blastomeres developed in vitro without any
inEeraction with the parent zona (later substantiated by
Menino and Hright, 1979a, 1979b) and because damage to the
zona, in the absence of manipulation of the blastomeres, did
not significantly decrease embryo survival in vivo following
transfer. They concluded that a significant change must
occur in either the embryo or the uterine environment once
the blastocyst hatches from the zona pellucida in order for

the embryo to survive (Moore et al., 1969).

Production of Monozygotic Embryos by Blastomere Separation

Shortly after publication of Moore's (1969) paper,
Mullen et al. (1970) produced the first set of identical
twins in the mouse produced by microsurgically splitting a
2-cell embryo and culturing each half embryo in separate
containers until compaction occurred. The embryos were then
transferred to the reproductive tract of a surrogate mother
and live offspring were born (Mullen et al., 1970). Similar
results were obtained by Fiser and Macpherson (1975) who
produced 54 pairs of identical twin blastocyst embryos by
chemical dissociation (Calcium- and Magnesium-free

Brinster's medium plus 0.02% EDTA) of 2-cell mouse embryos.

17

They argued that the reproductive potential of selected
females of a mammalian species could be multiplied by a pro-
cess of superovulation, embryological cloning, and embryo
transfer (Fiser and Macpherson, 1975).

Trounson and Moore (1974) continued attempts to produce
identical offspring in domestic animals by embryo splitting.
The earlier work with rabbits and pigs clearly showed that
survival and development of single blastomeres in recipient
females required the enclosure of the half embryo in a rela-
tively intact zona pellucida to protect the denuded blasto-
meres from desruction in utero.

Two approaches were considered. The blastomeres could
be protected from direct eXposure to the uterine environ-
ment, or denuded blastomeres could be cultured in vitro to
blastocyst stage, when they would normally shed the zona,
and then be transferred to a host female. The second method
produced live offspring. Fertilized ova from superovulated
Merino ewes were reocovered on days 4-7 by lavage with
Dulbecco's phOSphate buffered saline (PBS) and the morula
were treated with a 5% solution of pronase (Sigma) for 1.5
to 8 minutes to remove the zona pellucida or reduce it to a
thin shapeless membrane. The embryos were then washed in
PBS with 20% serum to remove residual pronase and trans-
ferred to a glass container in the same medium for micro-

manipulation at 35°C. Sterile glass needles were used to

remove any remnants of the zona and to cut the inner cell

 

18

mass of each morula in two. In later trials, the enzymatic
removal of the zona was discontinued because it was found
mechanical removal did not damage the blastomeres (Trounson
and Moore, 1974).

Once divided, the half embryos were rinsed in PBS with
20% serum, then incubated for 48 hours at 37.5°C in
polystyrene tubes. Over 80% of the embryos continued devel-
Opment to expanded blastocysts. The embryos were then
transferred to recipient ewes. Fourteen half embryos, pre-
pared from seven parent embryos, reached blastocyst stage
and were transferred individually to surrogate mothers.

Only two of these females lambed (14.3%), but the experiment
produced the first live births of domestic livestock from
half embryos. They were not identical twins (Trounson and
Moore, 1974).

Tarkowski and Rossant (1976) extended microsurgery on
mouse embryos to include production of haploid mouse blasto-
cysts by mechanical separation of the pronucleate embryo
into two havles, each containing one of the pronuclei.
Haploid embryos remained viable and developed to blastocysts
with a distinct inner cell mass. The haploid cell lines
provided a new source of material for the study of gene
action. Tarkowski (1977) also reported that preimplantation
development in these embryos required the presence of the X
chromosome since none of the Y bearing, androgenetic embryos

developed beyond the 4-cell stage.

19

Epstein et al. (1978) published an additional and prev-
iously unreported use for blastomere separation in genetic
analysis the following year. Because the most reliable method
for embryonic sex determination is chromosome analysis, and
because preparation of the cells for karyotyping is destruc-
tive, embryos could not assayed if other cytological or
metabolic work was to be done. To circumvent this problem,
half embryos were prepared by dissociation of 2-cell mouse
embryos and culturing them in vitro, reaching twin blasto-
cyst stage after 2.5 days. One half embryo was frozen for
later use in enzyme assays and the other prepared for chro-
mosome analysis. The twin blastomeres developed to blasto-
cysts 71% of the time and readable karyotypes were obtained
from 47% of the half-blastocysts. Frozen embryos were
pooled in groups of five each of the same sex and tested for
HGPRT and APRT activity (hypoxanthine-guanine-phosphoribosyl
transferase and adenine phosphoribosyl transferase) to assay
x chromosome function in vitro. No embryo transfers were
done (Epstein et al., 1978).

Moustafa and Hahn (1978) improved on Mullen's (1970)
success with mouse blastomere separation and embryo develop-
ment by splitting morulae. Sixteen cell embryos were
collected from superovulated females, and divided with
micromanipulators following mechanical removal of the zona
pellucida or chemical digestion with pronase (Moustafa and

Brinster, 1972). Half embryos were cultured in Brinster's

20

medium at 37.5°C in 5% C02 and air. Over 80% of the embryos
developed to the blastocyst stage after 72 hours of culture.
Twenty pairs of half embryos, incubated for 30 hours
following surgery, were transferred to host females and 8
sets of identical twins were delivered (40%). Fourteen of
these reached puberty and showed normal reproductive perfor-
mance. The results showed the production of monozygotic
twins in mice could be routinely accomplished in at least
one mammalian species, but only under conditions where the
bisected embryo could be cultured in vitro prior to
transplantation and a relatively low success rate tolerated.
Later work confirmed these results (Dyban and Sekirina,
1981; Tsunoda and McLaren, 1983).

Tsunoda and McLaren (1983) improved the survival rate
of mouse half embryos produced by bisection of 2-cell parent
embryo by avoiding in vitro culture following surgery and
using the ligated oviduct of a host female to culture the
half embryos for two to four days before transfer to surro-
gate mothers (8-12% in vitro vs. 65% in vivo culture). The
methods used were developed by Hilladsen (1979, 1982).

Production of Monozygotic Multiplets in Domestic Species
by Blastomere Separation

Hilladsen (1979) introduced new techniques for blasto-
mere separation which permitted production of monozygotic

twins (sheep) by intermediate culture of the half embryos in a

host oviduct, rather than in vitro, prior to embryo transfer.

 

21

The methods were devised to provide protection for the
embryo in vivo following surgery (Trounson and Moore 1969).

Preliminary experiments showed that 2-cell sheep
embryos with damaged zonae pellucidae could be embedded in
an agar chip, transferred to a ligated rabbit oviduct for in
vivo culture, and would continue normal development as long
as the agar plug remained intact (Hilladsen, 1979). Two
cell embryos were then collected and mechanically separated
in PBS at room temperature. The half embryos were produced
by slicing the zona pellucida along the interblastomeric
groove with a glass needle mounted on a micromanipulator.
aspirating the blastomeres out of the zona with a micropi-
pette, separating the blastomeres by suction rather than
cutting, and injecting the isolated cells back into surro-
gate zona prepared from previously evacuated embryos. The
half embryos were then embedded in a 1.2% solution of agar
(DIFCO) in 0.9% NaCl in distilled water cooled to 36-38°C.
Pairs of monozygotic embryos were embedded and expelled into
PBS as a solid cylinder. Several sets of twin embryos were
then embedded into a larger cylinder of agar and transferred
to the ligated oviducts of recipient ewes on the first or
second day of the estrous cycle. The embryos were removed
by lavage after 3.5 to 4.5 days and were dissected out of
the plug. Embryos which reached late morula or early

blastocyst stage were transferred to host females in days

5-7 of the estrous cycle. Sixteen sets of monozygotic twins

 

22

were transplanted, one pair to a single ewe. Eleven of 16
host females became pregnant (68%) and 10 went to term, pro-
ducing 5 sets of identical twins and 5 single lambs
(Hilladsen, 1979). The success paved the way for further
experiments in species that could not be cultured in vitro
to develOpmental stages adequate for embryo transfer.

Hilladsen next investigated the use of a surrogate zona
pellucida, the agar embedding technique and cryopreservation
for supporting development of multiplet embryos derived from
single blastomeres obtained from 2-cell, and pairs of
blastomeres from 4-cell, and 8-cell sheep (9115,32.)
embryos. In this series, the ligated oviduct of a non-
synchronized host ewe was used to incubate the embedded half
embryos until they reached an age of 5.5 to 6.5 days. The
embryos were recovered and if they had developed to late
morulae or early blastocysts, one of each monozygotic pair
was transferred immediately to a ewe in day 6 of the estrous
cycle (all but a few of the host females entered estrous
naturally, but some were induced with prostaglandin). The
other half was frozen in 1.5 M DMSO in PBS to test the abi-
lity of the manipulated embryos to withstand
cryopreservation.

Embryos were frozen by cooling at 0.3 C/min to -36°C,
then at O.1° C/min to -60°C before placement at 196°C in

liquid nitrogen. Frozen embryos were thawed by rapid

warming from -196° to -50°C, then a rate of 4°C/min to

 

23

-10°C prior to rapid thawing to room temperature (Nilladsen,
1977). Frozen embryos were transplanted when the recipient

of one of twin halves did not return to estrous (Hilladsen,

1980).

Use of the nonsynchronized ewe as the primary recipient
of the half embryos for the brief period of culture in the
ligated oviduct had no detrimental effect on development.
All sets of monozygotic embryos recovered developed at a
normal rate, but were reduced in size relative to ordinary
day 7 sheep embryos and contained only half the number of
cells. The size reduction did not affect embryo survival.
The pregnancy rate (78.6%) and survival rate (80.0%) were
comparable to those observed for transfer of intact sheep
embryos. There was no difference in survival rates for half
embryos derived from 2-, 4- or 8-cell embryos, suggesting
that at least up to the 8-cell stage the blastomeres remain
totipotent. Out of 30 half embryos transferred, 25 lambs
were born (83.3%). Of these, three sets of monozygotic
twins (2 sets from 8-cell parent embryos) were produced.
Nine of the 12 frozen half embryos were transferred and
three lambs, each a monozygotic twin of a lamb produced in
the first transfer series, were born (Nilladsen, 1980). The
success of this experiment proved that multiplet monozygotic
embryos could be produced from domestic species from
preimplantation embryos at a late stage of development and

marked the first attempt to freeze and thaw half embryos for

24

subsequent embryo transfer. Although the survival rate of
the cryopreserved twins was below that of both intact and
half embryos transferred immediately to host females, the
delivery of 25% of the frozen embryos as normal term neo-
nates demonstrated that isogenic animals of different ages
could be produced by delay of embryo transfer through
freezing.

Having shown the utility of the embedding and in vivo
culture techniques for production of monozygotic twins in
sheep, Nilladsen and Polge (1981) used the same methods to
attempt preparation of monozygotic quadruplets in cattle

(Bos taurus) since cattle twins had been shown to be very

 

valuable as experimental animals for physiological research.
Quarter cow embryos were derived by separating 8-cell
embryos into four groups of two, injecting the blastomeres
into surrogate zonae pellucidae, and embedding them in agar
plugs for culture in the ligated oviducts of sheep for 90
hours. Of 44 quarter embryos transferred to the sheep, 91%
continued to develop and 77% became blastocysts. Twenty-six
blastocysts, 13 pairs of monozygotic embryos, were trans-
ferred to the tip of the uterine horns of each surrogate
mother (cows) on day 7 of the estrous cycle. Nine of the 13
females became pregnant (69%) with six carrying twins (46%).
Eight went to full term and delivered one set of monozygotic
triplets, two sets of monozygotic twins and one singleton

(31%). Normal development was obtained even though the

25

multiplet embryos had been cultured to blastocyst stage in a
foreign species prior to transfer. Also, the first artifi-
cially produced quadruplet embryos developed to term.

Hilladsen et al. (1981) next integrated the microsurgi-
cal production of multiplet embryos in cattle to routine
embryo transfer in livestock following artificial insemina-
tion. This required evaluation of half and quarter embryos
generated by Splitting intact parent embryos at the 32 to 60
cell stage. Previous blastomere separation had been done on
2- to 8-cell embryos recovered by laparotomy, but the objec-
tive here was to flush day 5 and day 6 embryos at morula
stage for manipulation without resort to standing surgery
for embryo recovery. The embryos would be obtained just as
they are for commercial embryo transfer.

Hereford x Friesian heifers were superovulated with
2000 i.u. of PMSG on day 9 to 14 of the estrous cycle and
injected 48 hours later with 750 g Cloprostenol. Ten hours
after the onset of estros the females were inseminated with
fresh or frozen Hereford semen. Embroys were flushed from
the oviducts with PBS on day 5 or day 6 and manipulated as
before. Noticeable differences in the microsurgery were
noted. Early cell stages could be separated more easily
than the morulae and considerable cell damage was induced by
the microsurgery. However, the morulae tolerated the
splitting and the survival rate of half and quarter BMPFYOS

did not differ significantly from that of intact transferred

26

cow embryos (75%). Fourteen pairs of monozygotic twins were
transferred and 10 twin pregnancies were reported (71%).
Four groups of quadruplet embryos gave rise to three twin
pregnancies, but no more than two offspring were derived
from the original set of four isogenic embryos.

The results argued against the utility of producing
quadruplets, but did demonstrate that late stage embryos
would retain the capacity to develop into whole embryos after
separation into half and quarter embryos by microsurgery.
Live births of monozygotic twin horses were also obtained
from quarter embryos produced by Splitting 4-cell parent
embryos into single blastomeres (Hilladsen, 1982). It was
subsequently shown that multiplet embryos from cows would
survive cryopreservation at a rate (72%) equivalent to that
obtained with intact cow embryos (Lehn-Jensen and Nilladsen,
1983). For all practical purposes, artificial sets of twins
from domestic species could now be consistently produced
from nonsurgically recovered embryos and stored indefinitely
by freezing.

Hilladsen's methods transformed blastomere separation
of mammalian embryos from a laboratory exercise directed at
understanding the regulation of development in the
preimplantation embryo to a manipulative technique of great
utility and commercial value in animal husbandry.

Monozygotic twinning was formerly treated as a biologi-

cal curiosity of the metatherian mammal, the nine-banded

 

27

armadillo (Patterson, 1913; Enders, 1966), an event of
exceptionally low natural incidence in eutherian mammals
(Hamilton and Mossman, 1972; Markert and Seidel, 1981), or
the pathological result of induction by introduction of exo-
genous teratogens (Tuchmann-Duplessis and Mercier-Parot,
1961; Hay, 1964; Ferm and Hanover, 1969; Ferm 1978; Kaufman
and O'Shea, 1978; Hsu and Gonda, 1980; Naruse et al., 1983).
Nilladsen demonstrated that monozygotic multiplet embryos
could be artificially generated and transplanted at rates
comparable to those obtained from transfer of intact
livestock embryos and that normal and reproductively viable
offspring would result from the procedure. The discovery
was of major significance since the only repeatable methods
for induction of twinning in cattle had previously involved
the transfer of multiple embryos and polyzygotic embryos did
not result in isogenic offspring (Rowson et al., 1971;
Heyman and Renard, 1978).

Markert and Seidel (1981) conjectured that production
of identical twins would benefit livestock producers by
generating twice the number of offspring per embryo. The
prospect of delayed transfer of a twin embryo offered the
additional opportunity to evaluate phenotypic information
from the transfer and maturation of one of the half embryos
so that an estimate could be made concerning the value of
transfer and maintenance of the frozen twin against the

clone which had matured. They also suggested that the cost

28

of research could be substantially reduced by using artifi-
cial multiplet embryos since by eliminating genetic
variation in experimental trials, the number of animals
needed in a particular research series could be reduced
without loss of experimental power.

Further simplification of bovine embryo microsurgery
occurred with the discovery (Williams et al., 1982) that
morulae could be bisected, introduced into host zonae pellu-
cidae, and cultured in vitro for 24 hours prior to transfer
to recipient females without the intermediate steps of
embedding in agar and culture in a host oviduct. Even in
vitro culture was abandoned when Ozil et al. (1982) showed
that bisected morulae and early blastocysts from cattle
could be directly transferred to the surrogate and still
result in viable monozygotic twins. Although the survival
mate of the half embryos, when produced by direct transfer,
was lower (51%) than that obtained by Hilladsen, the simpli-
fication of the microsurgical procedure and embryo culture
phase offset this difference in terms of the labor required
to generate multiplet embryos (Ozil, 1983; Williams, et al.,
1983). Elimination of half embryo embedding and in vitro
culture prior to transfer has not met with success in other
mammalian species to date.

Today, bovine blastomere separation is routinely used
in commercial embryo transfer facilities in the United

States, France and England for increasing the numbers of

29

purebred livestock embryos available for transfer and for
the generation of multiple embryos of preselected parentage
(Hilliams et al., 1983; Baker et al., 1984).

Along with commercial applications, experimental work
with blastomere separation and production of half embryos
continues. Half embryo, conspecific chimeras have been pro-
duced by splitting 4- and 8-cell sheep embryos and joining
the blastomeres of the two developmentally different embryos.
The result is the differentiation of cells derived from the
8-cell blastomeres into the inner cell mass of the chimera
and the generation of the trophectoderm from the younger
4-cell blastomeres (Nilladsen and Fehilly, 1983). The use
of blastomere separation and fusion to produce interspecific
hybrids in domestic animals has also been reported, but the
results are not yet published (Seidel, 1983a). Sheep-goat
chimeras have been generated by joining the isolated blasto-
meres of 8-cell embryos from one species with blastomeres
from 4-cell embryos of the other, so that the inner cell
mass derives from the 8-cell line of blastomeres and develops
into the fetus and the trophectoderm from the 4-cell line
differentiates into the chorionic membranes, allowing
the foreign species to be hosted in the surrogate mother
since the placenta which supports the fetus matches the host
(Seidel, 1983a; Nilladsen and Fehilly, 1983). Finally,
serial production of monozygotic embryos from the same

parent embryo has been attempted in mice by culture of

3O

daughter embryos cloned from the parent, but the method
shows only limited potential. Of 174 2-cell embryos resplit
after initial derivation from parent 2-cell embryos, only
19% continued development to the blastocyst stage (O'Brien
et al., 1983). Continued reduction in cell mass to levels
below a metabolic threshold may account for the failure of
serially cloned embryos to develop further.

Applications of Blastgmere Separation and_!gnozygotic
Multiplet Embryos to Research on Nonhuman Primates and
Humans.

To date, there have been no reports of the use of
blastomere separation to produce monozygotic multiplet
embryos in nonhuman primates. Although the South American
marmosets (Callithricidae) routinely twin (Epple, 1967), the
natural incidence of twinning in most primate species is
very low (Wilson, 1978; Rawlins and Kessler, 1983) and
usually produces fraternal rather than identical offspring.
Artificial production of isogenic primates would allow gene-
tic control in physiological and behavior research, and
increase the reproductive potential of genetically valuable
females. Microsurgical production of monozygotic primates
could also be used to increase the numbers of embryos
availabe for transfer to recipient females as a means of
multiplying the numbers of endangered primate species.
However, the utility of producing artificial twins through
bastomere separation in primates is currently limited by the

very low survival rates of the embryo transfers which have

31

been attempted in nonhuman species. Only five nonhuman pri-
mates have been produced by embryo transfer of in vivo and
in vitro fertilized embryos to surrogate mothers. Before
monozygotic primate embryos can be used in husbandry and
conservation, a considerable amount of work must be done to
enhance the success of intact nonhuman primate embryo
transfers.

Blastomere separation and production of monozygotic
nonhuman primate embryos has immediate use in the study of
development of the preimplantation embryo. Long term
studies of the biochemical and genetic normality of in vitro
fertilized squirrel monkey (Saimiri sciureus) embryos have
established the utility of nonhuman primates as an animal
model for investigation of fertilization and differentiation
of the human embryo (Dukelow et al., 1983). In vitro fer-
tilized embryos from the squirrel monkey have been subjected
to biochemical (Ghosh et al., 1982; Hutz et al., 1983) and
chromosomal assays (Mizoguchi and Dukelow. 1981; Asakawa
et al., 1982) to determine the normality of embryonic deve-
lopment. Given the rapid growth of human in vitro fer-
tilization and embryo transfer programs world wide, these
studies are of critical importance to the identification of
abnormal embryonic development since current ethics and
legislation prevent experimental investigation of human

embryogenesis.

32

Despite high levels of fertilization, only a small per-
centage of the embryos fertilized in vitro develop beyond
the 8-cell stage (Chan, 1983). This limits embryo produc-
tion and research opportunities. Blastomere separation and
the generation of multiplet embryos could double the number
of embryos that can be obtained from the in vitro fertili-
zation of a limited number of oocytes.

Blastomere separation may also be useful in assessing
the genetic normality of primate embryos or chromosomal
activation. A half embryo can be held in culture white its
twin is subjected to gradual fixation and chromosomal analy-
sis or placed in culture for investigation of biochemical
and metabolic normality. Minimally, removal of blastomeres
and their subsequent in vitro culture to morulae or blasto-
cysts would provide sufficient material to determine the sex

of the embryo by destructive karyotyping while retaining a

viable identical twin for transfer and gestation.

 

MATERIALS AND METHODS

Production and Collection of Embryos

Mature female hamsters (8-10 weeks old) were used as
embryo donors. They were maintained on a 14L:100 (lights on
from 0600-2000 h) cycle. Females were examined for the pres-
ence of a postovulatory vaginal discharge and if a plug was
present, they were placed on a superovulatory regimen. Each
animal was injected with 30 I.U. of pregnant mare's serum
(PMS) (Serotropin, Teizo, Tokyo, Japan) i.p. at 1000 h on
the morning of Day 1 (day of ovulatory plug) and injected 76
hours later at 1400 h on Day 4 with 30 I.U. of human
chorionic gonadotropin (HCG) (A.P.L., Ayerst Laboratories,
Inc. New York, NY) i.p. (Mizoguchi and Dukelow. 1980). The
females were mated with males on proven fertility on the
evening of Day 4 following the injection of HCG.

At varying intervals after mating, the females were
sacrificed by cervical dislocation to obtain embryos at dif-
ferent stages of development. Two-cell embryos were recov-
ered from the oviducts 40 hours post coitum and 4-cell
embryos at 60 hours. Eight-cell embryos were obtained from
the uterine horns 72 hours after mating and morulae at 78

hours (Ghosh et al., 1982).

33

34

Embryos were collected by oviductal and uterine lavage.
The oviducts were dissected away from the uterine horns.
Once isolated, 0.5 ml of culture medium was injected into
the fimbria of the oviduct with a blunted 25 gauge needle on
a 1.0 ml sterile plastic tuberculin syringe (Becton-Dickinson)
to flush the embryos into a welled glass slide. A retrograde
flush with 0.5 ml of culture medium was also used to wash
embryos from the uterine horns. Embryo recovery was
carried out at room temperature, but the medium injected
into the oviduct or uterine horn was maintained at 37°C
by warming in a temperature controlled water bath.

Embryos were sorted according to developmental stage and
counted, under stereoscopic magnification (105x), by
aspirating them into the a 5 ul micropipettor (SMI,
Emeryville, CA). They were then transferred to an eight
chamger LAB-TEK culture slide (Miles Laboratories, Inc.,
Naperville, IL) filled with 0.5 ml of medium per chamber at
37°C and incubated at 37°C in 5% C02 in humidified air, or
subjected to experimental manipulation.

Squirrel monkey embryos were obtained by fertilization
in vitro. Adult (500 to 600 grams body weight) Bolivian and
Guyanan squirrel monkeys (Charles River Research Primates,
Port Washington, New York) were housed indoors from November
through May on a 12L:12D light cycle (lights on 0600 h to
1800 h) at 211 2°C, and outdoors in gange cages from June to

October (Jarosz and Dukelow. 1976). The animals were fed

35

commercial high protein New World monkey diet
(Ralston-Purina, St. Louis, MO) daily along with fresh
fruit. Water was avilable ad libitum.

Females were induced to ovulate by exogenous hormone
administration. Because the squirrel monkey does not
menstruate, stages of the estrous cycle cannot be determined
for indivudal animals without the use of vaginal smears or
steroid assays. Therefore, induction of follicular growth
and oocyte maturation was arbitrarily started five to six
days before the scheduled time of laparoscopic oocyte reco-
very (Dukelow, 1970).

Four daily injections of 1.0 mg of follicle stimulating
horomone (FSH-P, Burns-Biotec Labs., Inc., Omaha, NE) were
given i.m., followed by a single injection of 250 I.U. of HCG
i.m. on the final day of FSH treatment, 16 hours prior to
laparoscopy for follicular aspiration (Dukelow, 1979).
During the nonbreeding season (July through September) five
days of FSH injections were required to induce follicular
development (Kuehl and Dukelow, 1975).

Oocytes were recovered by laparoscopy (Dukelow et al.,
1971). The monkeys were anesthetized with sodium pentobar-
bital (15 mg/600 gm animal) 16 hours after the HCG injec-
tion. A small midline incision was made in the abdominal
wall with a scalpel and the trocar-cannula of the
laparoscope (Karl Storz, Tuttlingen, W. Germany) was pushed

into the peritoneal cavity through the incision. The

r:

36

trochar was then removed and the 4 mm 0.0. laparoscope
inserted. The peritoneal cavity was then insufflated with
C02 through the cannula.

Once the ovaries were visualized, a 25 gauge needle on
a 1 ml sterile plastic tuberculin syringe was pushed through
the abdominal wall and used to move the fimbria of the ovi-
duct aside to expose the ovary. The ovarian follicles were
then penetrated by the needle and the oocytes aspirated into
the syringe, which contained 0.05 ml of tissue culture
medium (TC-199, GIBCO, Grand Island, NY) modified to contain
1 mm pyruvate, 100 ug/ml gentamicin (Schering Corp.,
Kenilworth, NJ), 1 U/ml heparin and 20% heat inactivated (30
min at 56°C) fetal calf serum (FCS, GIBCO) (Asakawa et al.,
1982; Chan et al., 1982). The medium was sterilized prior
to use by filtration through a 0.45 um Millex@ filter
(Millipore Corp., Bedford, MA) and stored in 10 ml
Vacutainer (Becton-Dickinson Co.) tubes at 4°C, then warmed
to room temperature before laparoscopy.

Aspirated oocytes were transferred directly into 0.25
ml of TC-199 per chamber in sterile 8-chamber culture slides
and incubated at 37°C in 5% C02 in humidified air. Oocytes
were matured for 21 hours in culture prior to insemination.

Semen was collected from adult male squirrel monkeys by
electroejaculation (Kuehl and Dukelow, 1974). The animals
were placed under physical restraint, and a rectal probe was

inserted to deliver short pulses of current (120 pulses/sec

37

at 5 msec duration). Voltage was varied at 3 sec intervals
by a cyclic increase and decrease. The ejaculated plug of
coagulum was diluted in modified TC-199 (as above) and held
at 37°C for 5-10 minutes to permit release of sperm. Sperm
counts were made with a hemocytometer and the oocytes in
each chamber of the culture slide were inseminated with 0.05
ml of a sperm cell suspension of 105 to 106 sperm/ml, 21
hours after collection of the ova (Chan, 1983).

Twenty four hours after insemination and incubation,
the oocytes were reexamined on an inverted compound
microscope at 450x to determine if fertilization had
occurred. The following were used as criteria for fer-

tilization (Chan, 1983).

1.) Presence of two or more polar bodies in the perivi-
telline space.

2.) Presence of two or more polar bodies and two pronuclei
or two or more equal sized blastomeres by 24 hours post
insemination.

3.) Observation of the sperm tail or midpiece within the

cytoplasm.

Pyknotic and morphologicaly abnormal (shrunken or asym-
metrical) oocytes were classed as degenerated. One cell
monkey embryos were obtained 24 hours after insemination,
2-cell at 48 hours, and 4-cell embryos at 60 hours (Hutz,
1983).

38

Embryo Microsurgery

 

Mechanical removal of the zona pellucida and blastomere
separation was based on a modification of techniques deve-
loped by Nicholas and Hall (1942) and Willadsen (1979). The
zona pellucida was opened and the blastomeres removed by
aspiration for division and subsequent replacement within
the zona pellucida or culture as denuded embryos.

First, a 5 ul glass micropipette was drawn to a patent
needle on an electric microelectrode puller (Stoelting,
Chicago, IL.). The tip diameter was approximately 90 u.
Second, another 5 ul glass micropipette was pulled to a non-
patent needle of about 30 u diameter. Two right angles,
opposed and in the same plane, were bent into the pipettes
over the open flame of a gas microburner constructed from a
10 gauge stainless steel syringe needle. The bends were
required to correct for the difference in height between the
micromanipulators and the stage of the microscope (Figure
1). The needles were placed in the instrument holders
(patent) of two Emerson Model B micromanipulators (J.H.
Emerson Co., Cambridge, MA) which were mounted on either
side of the stage of a 7x zoom stereomicroscope. Three
dimensional movement of each instrument could be indepen-
dently controlled, in the vertical Z axis by helical gear
adjustment and vertically and horizontally in the X and Y
axes of the microscope stage by movement of a single

joystick control.

39

 

Figure 1: The micromanipulators used for blastomere
separation.

40

For microsurgery, the needles were lowered to the sur-
face of a 150 mm glass petri plate containing a 1 ml drop of
culture medium in the center. Intact embryos were then
transferred from a welled glass slide with a micropipette to
the medium in the petri plate. No more than five embryos
were removed from culture at any time. The remaining
embryos were maintained at 37°C on a slide warmer while
microsurgery was carried out at room temperature on the
others.

After transfer, the embryos were located under magnifi-
cation in an erect image field and each needle was then
brought to either side of a single embryo to begin surgery.
The patent (left side) pipette was connected to a ruber hose
with a spring clamp and suction applied by mouth. When the
tip of the 90 u patent needle was brought near the embryo,
the suction aspirated the zona pellucida a slight distance
into the pipette and the suction line was clamped, anchoring
the embryo to the end of the moveable probe. Next the 30 u
nonpatent needle was pushed through the zona pellucida on
the right side and moved laterally to tear a linear cut
across half the circumference of the zona (Figure 2). Care
was taken not to damage any of the blastomeres.

Once the zona pellucida had been opened, the nonpatent
probe was replaced with a second patent needle 45 u in
diameter which was also attached to a suction line. The tip

of this probe was blunted by snapping off the end with a

 

F?

Figure 2.

41

 

 

 

 

 

 

Procedure for mechanical separation of
blastomeres. A) Needle is used to open
the zona pellucida. B) Patent pipette is
used to separate blastomeres. C) Isolated
blastomeres are replaced into a host zona
pellucida prior to culture or embryo

transfer.

42

pair of microsurgical forceps. The 45 u probe was pushed
through the rent in the zona pellucida and brought up
against the blastomeres. Under gentle suction, the blasto-
meres were aspirated from the zona pellucida. While the
objective was separation and removal of only half of the
blastomeric contents of the embryo, the entire embryo was
usually withdrawn due to strong intercellular adhesion.

When this occurred, the denuded intact embryo was released
from the micropipette and allowed to settle to the surface
of the petri plate. The 90 u pipette was then used to pre-
vent movement of the embryo by placing the tip lengthwise
behind the blastomeres, and the 45 u probe was replaced with
the 30 u needle which was used to divide the blastomeres by
pressing down with the tip of the probe along the first
cleavage plane of the embryo against the surface of the
glass. The divided blastomeres were then picked up with the
45 u patent probe and replaced into a zona pellucida or left
denuded.

A micropipette was used to collect the half embryos and
transfer them directly to 0.5 ml of culture medium (21-22°C)
per chamber in an eight chamber tissue culture slide for
incubation, or to Dulbecco's phOSphate buffered saline (PBS)
(GIBCO) for viability assay by vital dye staining with trypan
blue (Kodak, Rochester, NY), followed by culture. A 0.2%
solution of trypan blue was used to assess mechanical
damage to individual blastomeres incurred during surgery.

Exclusion of the dye indicated the plasma membrane of the

43

embryo was intact and the blastomeres normal. Failure to
incorporate the dye also provided an indirect measure of
metabolic normality since exclusion of trypan blue correlates
strongly with normal metabolic activity as measured by
uptake of flourescein diacetate (Hutz, 1983; Ridha, 1983).
After treatment with trypan blue, the embryos were rinsed
twice in PBS for 30 seconds and placed in culture. All
vital embryos were incubated for 24 hours at 37°C in 5% C02
and humidified air, then checked for further development.
Intact embryos of the same developmental stage were cultured
in parallel with the half embryos as controls.

Due to the high incidence of structural damage to indi-
vidual blastomeres from mechanical removal of the zona
pellucida and blastomere separation, chemical removal of the
zona pellucida from the embryo and chemical reduction of
intercellular adhesion of the blastomeres prior to mechani-
cal separation (Tarkowski and Wroblewska, 1967; Fiser and
Macpherson, 1975) was also used.

Initially, treatment consisted only of removal of the
zona pellucida from the intact embryo by lysing in a
filtered (45 um Millex@ filter) 0.25% solution of pronase
(Grade B - Lot 71879, Calbiochem, Los Angeles) in PBS for
30 seconds at room temperature. The zonae pellucidae disap-
peared within 15 to 20 seconds. Denuded embryos were very
sticky and adhered to the surface of the glass petri plate

(even when the glass had been treated with organosilane

44

(Prosil-28, PCR, Inc., Gainesville, FL) as well as to the
glass micropipette. This made it very difficult to separate
and transfer the embryos, so intracellular adhesion was che-
mically reduced in later experiments. Denuded embryos were
rinsed twice in PBS and transferred to a solution of calcium
and magnesium - free PBS (GIBCO) containing 0.02% EDTA as a
calcium chelating agent. After 5 - 15 minutes, the embryos
were placed in culture medium for microsurgery.

Blastomere separation was accomplished without
anchoring the embryo by suction to avoid damage to the
cells. The shaft of the 90 u pipette was used to minimize
embryo movement by placing it beside the blastomeres length-
wise. The 30 u needle was used to make a vertical cut down
and across the cleavage plane of the embryo and the blasto-
meres usually dissociated without visible structural damage
using this method. If an embryo rolled away from the
needle, suction was applied to several blastomeres with the
90 u blunt pipette to stabilize the embryo. The half
embryos were then assayed for structural damage with trypan
blue and if viable, transfered to the incubator for 24
hours. Chemical pretreatment of the embryos prior to micro-
surgery reduced blastomere injury during manipulation and
increased the speed with which the embryo could be bisected.
This reduced the total amount of time the half embryo spent
out of culture and the method improved experimental results.

None of the half embryos was embedded in agar for culture

45

since it had been previously demonstrated (Seidel, 1960;
Moustafa and Hahn, 1978; Menino and Wright, 1983) that zona—
free mammalian embryos, especially those of rodents, could

be successfully cultured in vitro.

Embryo Culture

 

Hamster embryos were cultured at 37°C in 5% C02 and
humidified air for a minimum of 24 hours of incubation.
Embryo development was assessed for the 2-, 4-, and 8-cell
stage and for microsurgically produced multiplet embryos,
with and without zonae pellucidae. Four culture media were
tested. These were TC-199 (GIBCO) with 20% fetal calf serum
plus pyruvate, TC-199 with 20% and 30% pregnant hamster
serum (PHS), Tyrodes solution (GIBCO) with 20% and 30% PHS,
and TALP (Tyrode's solution, albumin, lactate and pyruvate)
(Bavister et al., 1983).

Modified TC-199 was used because it had previously been
shown to support development of in vitro fertilized squirrel
monkey embryos (Kuehl and Dukelow, 1979; Dukelow et al.,
1983). It was prepared by adding 25 ml of heat inactivated
(30 min at 56°C) fetal calf serum (GIBCO) to 100 ml of stock
TC-199. After mixing, 1.05 mM of pyruvate (14.4 mg pyruvic
acid) were added along with 1.25 ml of gentamicin (Schering
Corp., Kenilworth, NJ). The medium was sterilized by
filtration through a 0.45 um MillexO filter and stored in
10 ml Vacutainer tubes at 4°C for no longer than 30 days.

Before use the medium was rewarmed to 37°C in a water bath.

46

The pH ranged between 7.2 and 7.5.

TC-199 with 20 and 30% PHS was used under the assump-
tion that the addition of homologous serum might enhance
development of hamster embryos. It was prepared from a
stock solution of TC-199 to which pregnant hamster serum was
added just before use. The stock solution comprised 100 ml
of TC-199, 14.4 mg of pyruvate (1.05 mM), 0.87 ml of sodium
lactate syrup (50 mM) and 1.25 ml of gentamicin. The stock
solution was filtered (see above) and stored in 10 ml
Vacutainer tubes at 4°C for no more than 30 days. Pregnant
hamster serum was obtained from embryo donors killed by cer-
vical dislocation. About 2 ml of blood were drawn from each
animal by cardiac puncture with a 20 gauge needle mounted on
a 3 ml sterile plastic syringe. The blood was transferred
to a 3 ml Vacutainer and held in an ice bath for 15 minutes
to allow clot formation. Serum was separated by centrifuga-
tion for 30 minutes at 5000 rpm in a refrigerated (2°C)
Sorvall RC2-B centrifuge with head type HS-4 and pipetted
into 3 ml Vacutainers for indefinite storage at -20°C. For
each experiment, 2 or 3 ml of frozen PHS were thawed and
heat inactivated. Then 8 or 7 ml of TC-199 stock solution
were added to the 2 or 3 ml of PHS and mixed for 30 seconds
on a tube shaker. The medium was then sterilized by filtra-
tion and maintained at 37°C. The pH after addition of PHS
was 7.2. Unused TC-199 with PHS was discarded after each

trial.

47

The test of modified Tyrode's solution (GIBCO, Grand
Island Biological Co., Grand Island, NJ) was based on the
successful use of this medium by Whittingham and Bavister
(1974) for culturing fertilized hamster ova to the 2-cell
stage. It contained 9.5 g of Tyrode's powder in 1 l of
distilled water, with 4.0 g of bovine serum albumin (BSA,
Sigma Chemical Co., St. Louis, M0), 1.0 g of sodium bicar-
bonate, 0.10 mM of sodium pyruvate, 800 I.U. of gentamicin
(12.5 mg in 1.25 ml) and 20% or 39% PHS, heat inactivated
and added immediately before use (Fan, personal
communication). The pH of the medium was 7.6 after addition
of PHS and equilibration with 5% C02 in air at 37°C.

TALP (Tyrode's solution, albumin, lactate and pyruvate)
was the last culture medium tested. Bavister et al. (1983a,
1983b) had shown it to effectively support development of

both hamster and rhesus monkey (Macaca mulatta) embryos in

 

vitro and given the requirement of in vitro culture of
rodent and primate embryo in the present study, its use was
warranted. TALP was prepared according to Bavister et al.
(1983a). It contained 114.0 mM NaCl, 3.2 mM KCl, 2.0 mM

C3612: 0-5 m" "9912, 25.0 mM NaHC03’ 0,4 mu Nauzpo4, 5.0 mM

glucose, 10.0 mM sodium penicillin-G, 1 mg per 100 ml of
phenol red and 3 mg per ml of BSA. In addition, four basic

amino acids required for the maturation of hamster oocytes

in vitro (Gwatkin and Haidri, 1973) were added from a 100%

concentrate stock solution which combined the four. Final

 

48

concentrations were glutamine (146 mg/l), phenylalanine

(16 mg/l), methionine (8 mg/l), and isoleucine (26 mg/l)
(Bavister et al., 1983A). The sodium pyruvate and BSA were
added immediately before use and the solution was sterilized
by filtration (45 um MillexO). The pH ranged between 7.6
and 7.8.

In all experiments, the same culture medium was used
for embryo recovery, microsurgery, and embryo culture.
Statistical Analyses

All data were analysed using non-parametric statistics.
Media efficacy and experimental protocols were evaluated by
Chi-square (contingency table or differences among propor-
tions) tests calculated according to Siegel (1956) and
Freund (1971).

RESULTS

Production and Viability of Hamster Half-Embryos

A total of 193 hamster embryos were subjected to
blastomere separation (Table 1). Two, 4-, and 8-cell
embryos were used to produce multiplet embryos by splitting
each embryo in half with the micromanipulators with or
without chemical pretreatment. Of a possible 386 demi-
embryos, 194 survived surgery, as assayed by either morpho-
logical integrity of the blastomeres (Tarkowski, 1971) or by
exclusion of the vital dye, trypan blue (Hutz, 1983; Ridha,
1983). The survival rate before culture in vitro was 50.3%.
Of all the multiplets produced, 27% were successfully
replaced within homologous zonae pellucidae. There was a
significant difference in the surgical survival rate of the
embryos which varied according to the developmental stage of
the parent embryo. The 2- and 8-cell parent embryo survival

rates of 63.2% and 55.9% respectively did not differ signi-
ficant1y (x2 = 1.18, p > than .05), but both survival

rates were significantly different from the 33.6% rate of
4-cell embryos (X2 = 20.94, p < .001). There was no
significant difference by developmental stage in the propor-
tion of viable multiplets returned to zonae pellucidae (X2 =
1.18, p > .05).

49

50

TABLE 1

BLASTOMERE SEPARATION OF HAMSTER EMBRYOS
(Both Methods Pooled)

Stage Embryos Multiplets ‘1 Multiplets 1 Development

 

 

 

 

 

 

 

Surviving Placed in in Culture
z_qn_e_P.
2-Cell 38 48/76 63.2 4 8.3 0.0
4-Cell 61 41/122 33.6 20 48.7 0.0
8-Cell 94 105/188 55.9 29 27.6 0.0
Total 193 194/386 50.3 53 27.3 0.0

Multiplet Survival by Developmental Stage

x2 = 20.94
df= 2
p < .001

Note: Viability assayed by morphological appearance and
exclusion of the vital dye, Trypan Blue (Kodak,
Rochester, NY).

51

The differential survival of 2-cell and 8-cell half-
embryos was thought to be an experimental artifact since
the procedure for separation of blastomeres was changed
during the study. Intercellular adhesion increases from the
2- to 8-cell stage and the expected result would be to see
fewer numbers of the more developed embryos giving rise to
viable multiplets than was observed, since separation of
blastomeres should be progressively more difficult as deve-
10pment of the parent embryo proceeds. The survival rates
for multiplet embryos produced by mechanical and chemical
procedures were compared to determine if the change in pro-

tocol influenced the pooled results on survivorship.

The Effect of Chemical Pretreatment on Half-Embryo Viability

Table 2 shows the numbers of multiplet embryos derived
from 4- and 8-cell parent embryos by mechanical and chemical
separation. Survivorship of multiplets from chemically
pretreated 4-cell embryos was 87.5% versus 25.4% for those
divided mechanically. The difference was significant (x2 =
23.97, p < .001). Similar results were found for the 8-cell
embryos, of which 46.3% of the mechanically separated
embryos survived versus 82.0% for the pretreated half-
embryos. This difference was also significant (X2 = 18.89,
p < .001). Six times the number of 4-cell parent embryos
were divided mechanically relative to chemically pretreated
embryos, but only 3 times more 8-cell parents were

separated. The pooled results for 4-cell embryonic

52

mortality following surgery reflect these proportions, but
the factor responsible for the difference in survival for
mechanically separated 4- and 8-ce11 embryos is not
apparent. The survivorship of mechanically bisected 8-cell
embryos was significiantly greater than that of the 4-cell
derivatives (X2 = 11.2, p < .001). There was no significant
difference in multiplet survival between 4- and 8-cell
embryos when chemically pretreated (X2 = 0.26, p > .05).
The data in Table 2 clearly show the efficacy of chemical
removal of the zona pellucidae and reduction of inter-
cellular adhesion in facilitating blastomere separation.
Multiplet embryos produced by chemical pretreatment con-
sistently had less injury following surgery and showed less
structural deformation thatn did embryos produced by mecha-
nical separation. Figure 3 compares the morphology of half
embryos produced by both methods.

Despite development of a successful procedure for
separating blastomeres from parent embryos without apparent
cell damage, none of the multiplet embryos underwent further
development in vitro. Retention of the zona pellucida had
no effect. Morphologically normal half-embryos, assayed by
trypan blue, placed immediately into four different types of
culture media, and with or without the protection of a zona
pellucida did not reorganize to whole embryos and died in

vitro.

53

TABLE 2

A COMPARISON OF MULTIPLET EMBRYO PRODUCTION IN THE HAMSTER

BY

MECHANICAL AND CHEMICAL SEPARATION OF BLASTOMERES

Developmental Stage of Embryo

 

 

 

 

9.3;st essay.
Mechanical
Embryos 53 69
Survivors 27/106 64/138
Percentage 25.4 46.3
Chemical
Embryos 8 25
Survivors 14/16 41/50
Percentage 87.5 82.0
x2 = 23.97 x2 = 18.89
df = 1 df = 1
p < .001 p < .001

 

 

 

Note: Survivorship assayed by morphological normality and
by exclusion of the vital dye Trypan Blue (Kodak,
Rochester,

NY).

 

 

54

 

Figure 3. A comparison of the morphology of hamster
half-embryos derived from blastomere separa-
tion of 8-ce11 embryos by mechanical (A) and
chemical/mechanical (8) methods.

55

The Effect of Culture In Vitro on Half-Embryo Development
Five viable half embryos, derived from 8-cell parents,

were replaced in zonae pellucidae and transferred to the tip
of the left uterine horn (ligated) of a synchronized female
(Rihda, 1983) which had been mated with a vasectomized male.
Five days later the female was sacrificed and the uterus was
flushed with culture medium. No embryos were recovered.
The uterine horn was then opened and examined for implana-
tion sites, but none were found. Half-embryos within the

zonae pellucidae did not develop in vivo.

Differential Development of Control Embryos In Vitro

Given the differential survivorship of multiplet
embryos by developmental stage, a comparison of in vitro
culture of intact control embryos of the same cell numbers
was made. Table 3 shows the proportion of 2-, 4-, 8-cell
and morula stage embryos which underwent further development
after 24 hours of in vitro culture (results from all culture
media were pooled). A total of 1285 embryos were tested.
The results show a significant increase (X2 = 160.4, p <
.001) in development with advancing embryonic age. Note
that none of the 353 2-ce11 embryos placed in culture deve-
loped, regardless of the culture medium used. When the 2-
cell embryos were excluded from analysis, the increased
development of morulae relative to 8-cell embryos in culture
was still significant (X2 = 19.89, p < .001). The data

suggested the 8-cell embryo would be the most suitable

———-"

56

TABLE

3

A COMPARISON OF CULTURE IN VITRO OF HAMSTER EMBRYOS BY
DEVELOPMENTAL STAGE (POOLED FOR ALL MEDIA)

 

 

Stage Embryos Development 24h + Percentage
2-Cell 353 0/353 0.0
4-Cell 297 72/297 24.2
8-Ce11 593 379/593 63.9
Morula 42 41/42 97.6

All Stages B-Cell vs. Morula
x2 = 160.4 x2 = 19.89

df = 2 df = 1

p < .001 p < .001

57

parent for blastomere separation given the rate of develop-
ment of 8-cell controls. Morulae were not used in this
study, despite their routine bisection for monozygotic twin
production in domestic cattle, because this developmental
stage exceeds the maximum deve10pment obtained with in vitro

fertilized squirrel monkey embryos.

Efficacy of the Culture Media for Control Embryo Development

Next, the ability of the four culture media to support
continued development of intact preimplantation hamster
embryos was examined as an indirect test of ability to sup-
port half—embryo growth. Table 4 compares the development
of 2-, 4-, 8- cell and morula stage control embryos in
TC-l99 with pyruvate, TC-l99 with PHS, Tyrodes solution with
PHS, and TALP.

TC-l99 with pyruvate supported no further embryonic
development regardless of developmental stage and was aban-
doned as a culture medium. It is excluded from statistical
analysis here. The addition of 20-30% PHS to TC-l99
markedly improved in vitro culture of B-cell embryos, but
had no effect on the 4-cell stage. Only 23.4% of the 8-cell
controls underwent additional cleavage in TC-l99 with PHS.
Tyrodes solution with 20-30% PHS more than doubled devleop-
ment relative to TC-199. In this medium 35.3% of 4-ce11 and
58-1% of 8-cell embryos developed to the 8-cell and morula
stages respectively. None became blastocysts. In TALP,

2-cell embryos would not continue growth, but 30.1% of the

58

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59

4-ce11 and 81.2% of the 8-cell embryos developed to 8-cell
and early morulae stages. In addition, 39.6% of the 8-cell
embryos became blastocysts (Table 5). This stage was not
reached by 8-ce11 embryos in the other media. Finally,
97.6% of the morulae cultured in TALP developed to blasto-
cyst stage. The differences in the percentage of embryos
developing in the three culture media (excluding TC-l99 with
pyruvate) were significant for both 4-cell (X2 = 11.95, p <
.001) and 8-cell embryos (X2 + 145.39, p < .001). There was
no significant difference in 4-cell development between
Tyrodes solution with PHS and TALP (X2 = 0.198, p > .05),
but TALP was superior for the culture of B-cell embryos (X2
= 9.45, p < .001). Based on this finding, TALP was used as
the culture medium for control and manipulated 8-ce11
embryos exclusively. However, none of the half-embryos
developed in TALP, despite the fact that the control embryos
routinely cultured into blastocysts (see Figure 4).

The Effects of Chemical Pretreatment on the Development of

Intact Embryos
Detrimental effects on embryonic development from chem-

ical pretreatment were evaluated next. Table 5 compares

the development of 8-cell embryos left intact within the
zona pellucida, denuded by removal of the zona pellucida
with 0.25% pronase, and of 8-cell embryos denuded and washed
from 5 to 15 minutes in a solution of calcium and magnesium-
free PBS with .021 EDTA at room temperature (22° C) prior to
incubation in TALP at 37° C in 5.0% CO in air. Of the

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61

 

Figure 4: An example of a blastocyst, within the zona
pellucida, cultured in vitro from an 8-cell
hamster embryo in TALP.

62

controls, 81.2% underwent further growth and 39.6% developed
into blastocysts. The embryos without zonae pellucidae were
inclubated in TALP immediately following a 30 second treat-
ment with pronase followed by two rinses in PBS at room tem-
perature. Of these, 79.2% continued development and 49.6%
became blastocysts. There was not significant difference in
the general development of these two groups (X2 = 0.41, df =
1, p > .05), but a significantly larger number of zona-free
embryos developed into blastocysts (49.6%, X2 = 6.54, df =
l, p < .02).

Embryos denuded and treated in calcium and magnesium-
free PBS with EDTA did not achieve the percent development
seen in the other two groups. Only 55.9% of the 8-cell
embryos continued cleavage, and showed a significant reduc-
tion relative to control (X2 = 30.48, p < .001). However,
the proportion of treated embryos which developed into
blastocysts did not differ significantly (x2 = 2.42, p >
.05) from controls. Both of these groups differed signifi-
cantly from the higher percentage of blastocysts developed
from the zona-free embryos (X2 = 12.25, p < .01).

The improved deve10pment of the zona-free 8-cell
embryos in vitro is unexplained, but similar results have
been observed in mice (Tsunoda and McLaren, 1983). This may
be due to improved uptake of required nutrients in the
absence of a barrier from the zona-pellucida. Decreased
deve10pment of the denuded 8-cells with reduced inter-

cellular adhesion could result from disintegration of the

63

cell-cell gap junctions which support compaction and
spreading of 8-cell blastomeres into early morulae (Hillman,
et al., 1972; Deuchar, 1975; Balakier and Pedersen, 1982;
Trosko et al., 1982). However, the equivalent percentage of
development in this group to blastocyst stage, relative to
controls, argues against this as a detrimental effect.

Temperature effects were rules out since all embryos
were maintained at 22°C prior to culture in TALP. Duration
of the experimental treatments was not considered a factor
since enzymatic removal of the zona pellucida requires less
than 30 seconds, and the 8-cell embryos, denuded and chemi-
cally dissociated over a period of 15 minutes, developed
just as well as the control embryos which were immediately
transferred to the incubator.

Given the substantial improvement in multiplet survival
following chemical pretreatment and the minimal difference
in development of intact and denuded 8-cell parent embryos,
chemical pretreatment was retained for microsurgical produc-
tion of half-embryos. Interestingly, in two experiments
where 8-cell embryos were chemically denuded and subjected
to reduction of intercellular adhesion, three of the embryos
spontaneously dissociated while in culture into isolated
blastomeres or groups of two and four blastomeres (Figure
5). None of these developed further. This suggests that
the physical contact of the microsurgical instruments on the
chemically pretreated embryos was not adversely affecting

the development of the multiplets, but that the loss of

64

than 30 seconds, and the 8-cell embryos, denuded and chemi-
cally dissociated over a period of 15 minutes, developed
just as well as the control embryos which were immediately
transferred to the incubator.

Given the substantial improvement in multiplet survival
following chemical pretreatment and the minimal difference
in development of intact and denuded 8-ce11 parent embryos,
chemical pretreatment was retained for microsurgical produc-
tion of half-embryos. Interestingly, in two experiments
where 8-cell embryos were chemically denuded and subjected
to reduction of intercellular adhesion, three of the embryos
spontaneously dissociated while in culture into isolated
blastomeres or groups of two and four blastomeres (Figure
5). None of these developed further. This suggests that
the physical contact of the microsurgical instruments on the
chemically pretreated embryos was not adversely affecting
the development of the multiplets, but that the loss of

intercellular contact might be responsible.

The Effect of Temperature Change on Embryo Development
Another factor which could cause developmental failure
of the multiplets was the delay of entry into culture caused
by the microsurgical procedure itself. While the 8-cell
embryos were routinely split within 5 to 10 minutes after
chemical pretreatment, the half-embryos were placed into 0.5

m1 of TALP in a culture slide and held on a slide warmer at

37° C until all half-embryos in a series had been completed.

 

65

 

 

Figure 5. An example of spontan-
eously dissociated blastomeres
from an 8-cell hamster embryo
denuded with pronase and washed
in calcium- and magnesium-free
PBS (with 0.02% EDTA).

66
intercellular contact might be responsible.

The Effect of Temperature Change on Embryo Development

Another factor which could cause developmental failure
of the multiplets was the delay of entry into culture caused
by the microsurgical procedure itself. While the 8-cell
embryos were routinely split within 5 to 10 minutes after
chemical pretreatment, the half-embryos were placed into 0.5
ml of TALP in a culture slide and held on a slide warmer at
37° C until all half-embryos in a series had been completed.
In groups of 5 embryos at a time, this took from 30 to 60
minutes to complete. A preliminary test of the delay of
entry into culture with intact 8-cell embryos was made using
both TC-199 with PHS and TALP. A total of 33 embryos were
held out of culture at room temperature for two hours in 0.5
ml of medium in the covered wells of glass depression slide.
They were then incubated as previously described. None of
the embryos developed further. The results indicated that,
although the embryos of other species have been held at room
temperature for lengthy periods during micromanipulation or
prior to embryo transfer of intact embryos in both rodents
and domestic cattle (Moustafa and Hahn, 1978; Williams et
al., 1983), the exposure to temperatures below 37°C was
detrimental to hamster embryos.

This conflicted with earlier experiments in the study
which had shown that dhilling hamster embryos to 4°C for 2

hours prior to microsurgery did not kill them. A total of

67

28 2-cell hamster embryos were tested for viability with
fluorescein diacetate (Hutz, 1983) after being cooled to 4°C
and maintained for four hours. All remained metabolically
normal. The damage incurred by delay of incubation was pro-
bably linked to prolonged exposure of the culture medium to
the atmosphere instead of temperature changes. Phenol red,
used as an indicator of pH change in TALP and TC-199, shows
a definite color shift from pink to purple as the medium is
exposed to air and becomes more basic. Similar changes were
observed in the medium during the delayed culture
experiments and during microsurgery. A gradual change of
the culture medium to a more basic pH may be responsible for
the lack of embryonic development, but microsurgery under
oil-covered microdrops of medium was not tried in this
study, nor was in vitro culture under oil used since intact

embryos were deve10ping well in culture without it.

Micromanipulation of a Squirrel Monkey 4-Ce11 Embryo
Because of the lack of success with in vitro and in

vivo development of the multiplet hamster embryos, the more
difficult to obtain squirrel monkey embryos were diverted to
other research. However, the methodology for blastomere
separation developed on the hamster embryo was applied to
the monkey in one experiment. Blastomere separation
(mechanical\only) was performed on an in vitro fertilized
and in vitro cultured 4-cell squirrel monekey (Saimiri

sciureus. The parent embryo was obtained by laparoscopic

68

collection of the oocyte following the ovulatory induction
regimen given. The oocyte was incubated 21 hours prior to
fertilization. Fresh sperm were colelcted by electroejacu-
lation and suspended in TC-l99 with pyruvate, and added to
the oocyte in culture. At 60 hours after fertilization, a
4-cell embryo was obtained. The embryo was transferred to a
1.0 ml drop of TC-l99 with pyruvate in the petri dish of the
micromanipulator unit and one of the four blastomeres
(degenerate - see Figure 6) was removed by aspiration
(Figure 6). The manipulated embryo excluded the vital dye
trypan blue and was cultured in TC-l99 with pyruvate for 24
hours at 37°C in 5% CO2 in air. The embryo did not develop
further. However, the experiment resulted in the production
of the first viable microsurgically produced nonhuman pri-
mate embryo (Rawlins et al., 1983). Subsequent experiments
completed on oocytes recovered from squirrel monkeys indi-
cate that a 0.25% solution of pronase will remove the zona
pellucida from the ova within 30 seconds at room tem-

perature.

69

 

Figure 6. Microsurgical removal of a single blastomere
from a 4-ce11 squirrel embryo fertilized in
vitro. A) all blastomeres inact. B) degen-
erate blastomere removed.

DISCUSSION

The objective of this study was to develop a method for
the production of monozygotic multiplet embryos in the
hamster and squirrel monkey by blastomere separation. It
has been shown that viable half embryos can be produced from
4- and 8-cell hamster embryos by either mechanical, or
mechanical and chemical, separation of blastomeres; but the
combination of the two methods results in the greatest per-
centage of post-surgical survival of the half-embryos. The
methods developed on the hanster embryos for blastomere
separation are also effective on in vitro fertilized
squirrel monkey embryos. The use of the vital dye trypan
blue proved effective as an assay of embryonic viability for
both intact and manipulated embryos of both species. The
results are consistent with other studies on the isolation
of individual blastomeres in preimplantation embryos of
eutherian mammals (Nicholas and Hall, 1942; Seidel, 1960;
Tarkowski and Wroblewska, 1967; Moustafa and Hahn, 1978;
Willadsen, 1979; Ozil, 1982; Williams, 1982).

Failure of the hamster multiplet embryos to develop in
vitro is not surprising given the known sensitivity of
hamster embryos to any form of manipulation in culture and

especially to a basic pH (Whittingham and Bavister, 1974),

70

 

71

but there is no explanation for why this is the case. It is
difficult to attack experimentally since the multiplet
hamster embryos produced in this study were viable prior to
entry into culture and control embryos, as well as embryos
subjected to all phases of the separation protocol except
physical dissociation, developed to blastocysts in the in
vitro culture system. The problem rests with development of
a suitable system for culture of the half embryos, not with
the methods used to separate the blastomeres.

The lack of development in multiplet embryos returned
to homologous zonae pellucidae and transferred to synchro-
nized hamster females is also interesting. It has been
known for many years that murine embryos would develop in
vitro without a zona pellucida, but not in vivo (Tarkowski,
1959, 1971). Similar results were reported with sheep
(Trounson and Moore, 1974). However, half-embryos have
developed to full term and resulted in live births when
transferred immediately following microsurgery (Ozil, 1982;
Williams, 1982, 1983). In these experiments, the manipu-
lated embryos were developed to morulae before being sec-
tioned, and because they were advanced developmentally, they
may have been more capable of survival. From a maturational
standpoint, they were near the point in embryonic develop-
ment when protection of the zona pellucida is no longer
required. Transfer of half-embryos derived from hamster
morulae and replaced in zonae pellucidae would likely have a

better chance for further development in vivo than those

72

derived from 8-cell embryos since transfer of 8-cell embryos
with intact zonae pellucidae has been successful in the
golden hamster (Bavister el al., 1983a; Hoppe and Bavister,
1983; Ridha, 1983), but lowered survival and development
occurs when the zonae pellucidae are removed (Hoppe and
Bavister, 1983). From these studies, the transfer and
implantation of hamster multiplet embryos should be
possible.

The results of the tests on culture media for sup-
porting further development of hamster embryos are clear.
The more advanced embryonic stages show better rates of
development in vitro than do early 2- and 4-ce11 embryos,
regardless of the culture medium used. TALP is signifi-
cantly better for maintaining growth in both 8-cell and
morula embryos. Althoguh there are no published reports of
successful culture of 2- and 4- cell embryos (Bavister el
al., 1983), 30.1% of the 4-cell embryos cultured in TALP in
this study underwent additional development. The signifi-
cantly greater development of zona-free 8-cell embryos in
TALP compared to controls in this study differs from the
results of Hoppe and Bavister (1983) which, using the
hamster and TALP, showed no difference between the groups.
The percentage of embryos devleoping into blastocysts in
their study ws 46% for controls and 44% for the denuded
8-cells (pronase - 0.25%), compared to 39.6% and 49.6% for
the same groups in this study. TALP is a desireable culture

medium for experimental work because, unlike the media which

73

contain serum, it is chemically defined and can be modified
according to experimental design. Further, it is useful
because the medium has been shown to support both hamster

and monkey (Macaca mulatta) embryo growth in vitro, thereby

 

providing a single nutrient system for both rodent and pri-
mate species.

Given the low numbers of 4- and 8-cell embryos obtained
from in vitro fertilization of squirrel monkey oocytes
(Chan, 1983), chemical pretreatment of these embryos prior
to microsurgery should be used to minimize the risk of
damage to the blastomeres and to maximize post-surgical sur-
vival of the multiplets. Although only one primate embryo
was manipulated, the results indicate that production of
monzygotic embryos from nonhuman primates is possible with
existing technology. The recent success of embryo transfer

in two primate species (Papio sp. and Macaca mulatta) makes

 

transfer of half-embryos in nonhuman primates feasible.
Based on the consistent sucdess of twin production by
blastomere separation of morula and blastocyst embryos in
domestic cattle, and if in vitro fertilized primate embryos
can be developed in culture to morulae or early blastocysts,
blastomere separation should be attempted on these later
developmental stages instead of 4-cell and 8-cell embryos,
as the probability of successful embryo transfer is greater.

While generation of artifical sets of monzygotic twins
in exotic primate species has great utility for breeding

programs, and the use of isogenic primates of a single

74

species for experimental research holds great promise for
both physiological and behavioral work, and it is in gene-
tics that blastomere separation provides the most novel
application.

As mentioned previously, separation of blastomeres into
twin embryos allows for both delayed implantation of one of
the embryo and or the use of genetic assays on half of the
daughter embryos of the parent (Epstein et. al., 1978;
Epstein, 1981). Sex determination prior to embryo transfer
by karyotyping one of the multiplets is a simple applica-
tion. Chromosome activation can also be examined (Hoppe and
Whitten, 1972; Epstein, 1978). Another interesting applica-
tion is the assessment of in utero differences on subsequent
embryonic development and animal behavior. This has been
examined in mice by examining resulting differences in the
body weight and stature of monozygotic multiplets carried to
term in one or several different surrogate mothers. Clear
differences in morphology were identified despite isogene-
city of the embryos which correlated with in utero differen-
ces among the surrogate mothers. Behavioral differences
were not considered (Gartner and Baunack, 1981). Similar
studies of the influence of the genome relative to nurture,
in utero and post-natal, could easily be carried out on
nonhuman primates in conjunction with production of monozy-
gotic multiplets by blastomere separation and embryo

transfer.

75

Another important use of blastomere separation in
primates is the detection of chromosomal abnormalities in in
vitro fertilized human embryos. This problem is now
receiving considerable attention (Schlesselman, 1979; Angell
et al., 1983). Identification of monosomy, trisomy,
mosaicism and polyploidy in preimplantation human embryos
has not been accomplished despite the possibility that their
occurrence after fertilization in vivo and in vitro may be
very frequent. Production of half-embryos would permit
chromosomal examination of one multiplet while the other
remained in culture and useable for transfer.

As Seidel (1983a) has pointed out, one of the most
important constraints on experimentation with mammalian
embryos is the limited numbers which can be produced.
Blastomere separation and the production of monozygotic
multiplet embryos from a single parent embryo provides an
elegant method to alleviate the shortage. It also allows

for twinning by choice instead of chance.

SUMMARY AND CONCLUSIONS

This study was directed at the development of tech-
niques for blastomere separation, production of viable
multiplet embryos, and the in vitro culture of isogenic and
control embyros to developmental stages suitable for embryo
transfer in the hamster (Mesocricetus auratus) and squirrel
monkey (Saimiri sciureus). Experimental protocols were
first tried on in vivo fertilized 2-, 4-, and B-cell hamster
embryos prior to use on the less easily obtained, in vitro
fertilized and in vitro cultured monkey embryo. The
following conclusions can be made from the data:

1.) Blastomere separation can be used to produce
viable monozygotic half-embryos in both the
hamster and squirrel monkey.

2.) Mechanical removal of the zona pellucida and
mechanical separation of the blastomeres of
hamster embryos results in significantly greater
post-surgical losses of multiplet embryos than
that seen when embryos are chemically treated
prior to surgery.

3.) The zona pellucida of both hamster and squirrel
monkey embryos can be removed by lysing in a 0.25%

solution of pronase in phosphate buffered saline

76

4.)

5.)

6.)

7.)

80)

9.)

10.)

77

without damage to the embryo.

A solution of calcium and magnesium-free phosphate
buffered saline with 0.02% EDTA as a calcium
chelating agent can be used to reduce intercellu-
lar adhesion between blastomeres prior to micro-
surgery without damage to hamster embryos.
Mechanical separation of the blastomeres of a 4-
cell squirrel monkey embryo resulted in production
of a live 3/4 embryo.

The vital dye, Trypan Blue, provides a rapid and
effective means of assaying control and half-
embryo viability prior to and after incubation.
Despite the successful micromanipulation of
hamster embryos into viable multiplets, none deve-
loped after 24 hours of incubation at 37°C in 5%
C02 in air.

Temperature changes that occurred during micro-
surgery were not responsible for the failure of
multiplet hamsters to develop in culture.

Transfer of hamster multiplet embryos to surrogate
mothers did not result in implantation or develop-
ment of the embryos.

The success of culture in vitro of control hamster
embryos was a function of their developmental
stage at the time of entry into culture. Early
2-cell embryos did not develop and 4-cell embryos

showed little growth, while 8-cell embryos and

11.)

12.)

13.)

78

morulae regularly developed further, regardless of
the medium used.

In vitro culture of control hamster embryos was
best in TALP, compared to TC-l99 with pyruvate,
TC-l99 with pregnant hamster serum, and Tyrodes
solution with pregnant hamster serum. TALP regu-
larly supported development of 8-cell embryos to
morula and blastocyst stages.

Chemical pretreatment of intact 8-cell hamster
embryos had no effect on development to blasto-
cyst stage compared to controls, but denuded
8-cell embryos showed greater overall development
than did controls.

Additional work is needed to isolate the factors
responsible for failure of multiplet embryos to
develop in culture. There is no obvious reason
why blastomere separation and culture should not
work in this rodent. The extreme sensitivity of
hamster embryos to any form of manipulation may be

responsible.

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85

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Ozil, J.P., Heyman, Y. and Renard, J.R. (1982). Production
of monozygotic twins by micromanipulation and cervical
transfer in the cow. Vet. Rec. 110: 126-127.

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Tatusia novemcincta. J. Morph. 24: 559-650.

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New York, NY.

Rawlins, R.G. and Kessler, M.J. (1983). Congenital and
hereditary anomalies in the rhesus monkeys (Macaca
mulatta) of Cayo Santiago. Teratology 28: 169-174.

Rawlins, R.G., Fan, B., Chan, P.J., and Dukelow, W.R.
(1983). Blastromere separation of hamster
(Mesocricetus auratus) and in vitro fertilized squirrel
monkey (Saimiri sciureus) embryos. Amer. J. Primatol.
4: 353.

 

Ridha, M.T. (1983). Embryo production, freezing and
transfer in the golden hamster (Mesocricetus auratus).
Ph.d dissertation. Michigan State University, E.
Lansing, Michigan.

 

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Translated by Hans Laufer. In: Foundations of
Experimental Biology (B.H. Willier and J.M.
Oppenheimer, eds.). Hafner Press, NY.

Rowson, L.E.A., Lawson, R.A.S. and Moor, R.M. (1971).
Production of twins in cattle by egg transfer. J.
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Schlesselman, J.J. (1979). How does one assess the risk of
abnormalities from human in vitro fertilization? Am.
J. Obstet. Gynecol. 135: 135-148.

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isolierten blastomere des zweizellenstadiums im
saugetierei. Naturwissenten 39: 355-356.

86

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306-307.

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Furchungszellen aus dem Ei des Kaninchens (Oryctolagus
cuniculus). Roux Arch. Entw. Mech. 152: 3-130.

 

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embryos as methodological components. Biol. Reprod.
28: 36-49.

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354.

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In: Methods in Mammalian Embryology (J.C. Daniel,
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mouse embryos produced by bisection of one-cell
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blastomeres of mouse eggs isolated at the 4- and 8-
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procedures on the viability of mouse embryos containing

half the normal number of blastomeres. J. Reprod.
Fert. 69: 315-322.

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of hamster eggs fertilized in vitro or in vivo. J.
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sheep embryos during deep freezing and thawing. In:
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stages containing half the normal number of blastomeres
in the sheep. J. Reprod. Fert. 59: 357-362.

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the large domestic species. In: Mammalian Egg
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Boca Raton, FL.

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Berlin.

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monozygotic quadruplets in cattle by blastomere
separation. Vet. Rec. 108: 211-213.

88

Willadsen, S.M., Lehn-Jensen, H., Fehilly, C.B., and
Newcomb, R. (1981). The production of monozygotic
twins of preselected parentage by micromanipulation of

nonsurgically collected cow embryos. Theriogenology
15: 23-29.

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Identical twin bovine pregnancies derived from bisected
embryos. Theriogenology 17: 114.

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Bisecting bovine embryos: Methods, applications, and
success rates. In: Proc. Ann. Conf. Artific. Insem.
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eds.). pp. 1818-1946. Springer Verlag, Munchen.

APPENDIX A
PUBLICATIONS BY THE AUTHOR

Full Papers:

 

Age changes in the pubic symphysis of Macaca mulatta. R.G.
Rawlins. Amer. J. Phys. Anthr0p. 42: 477-487, 1975.

Forty years of rhesus research. R.G. Rawlins. New
Scientist 82: 108-110, 1979.

Parturient and post-partum behavior of a free-ranging rhesus
monkey (Macaca mulatta). R.G. Rawlins. J. Mammalogy
60: 432-433, 1979.

 

A five year study of tetanus in the Cayo Santiago rhesus
monkey colony: behavioral description and
epidemiology. R.G. Rawlins and M.J. Kessler. Amer. J.
Primatol. 3(1): 23-39, 1982.

Age and pregnancy related changes in serum total cholesterol
and triglyceride levels in the Cayo Santiago rhesus
monkeys. M.J. Kessler, R.G. Rawlins. Exp.
Gerontology 18: 1-4, 1983.

The Hemogram, serum biochemistry and electrolyte profile of
the free-ranging Cayo Santiago rhesus macaque (Macaca
mulatta). M.J. Kessler and R.G. Rawlins. Amer. .
Primatol. 4: 107-116, 1983.

Congenital and hereditary anomalies in the rhesus monkeys
(Macaca mulatta) of Cayo Santiago. R.G. Rawlins and
M.J. Kessler. Teratology 28: 169-174, 1983.

 

Aging in the musculo-skeletal system of rhesus monkeys: 1.
Passive joint excursion. C.J. DeRousseau, R.G. Rawlin
and J. Denlinger. Amer. J. Phys. Anthr0p. 61: 483-494,
1983.

The hemogram, serum biochemistry, and electrolyte profile of
aged rhesus monkeys (Macaca mulatta). M.J. Kessler,
R.G. Rawlins and W.T. London. J. Med. Primatol. 12:
184-191, 1983.

89

Blood values of free-ranging patas monkeys (Erythrocebus
patas). M.J. Kessler, E.C. Phoebus, R.G. Rawlins, J.E.
Turnquist and W.T. London. J. Med. Primatol. 12: 209-
217, 1983.

 

Preimplantation development of the primate embryo after in
vitro fertilization. W.R. Dukelow, P.J. Chan, R.J
Hutz, F.J. DeMayo, V.D. Dooley, R.G. Rawlins and M.T.
Ridha. J. Exp. 2001. 228: 215-221, 1983.

Early development of the in vitro fertilized preimplantation
primate embryo. W.R. Dukelow, P.J. Chan, R.J. Hutz,
F.J. DeMayo, V.D. Dooley, R.G. Rawlins and M.T. Ridha.
In: Proc. Kroc. Foundation Conf. Santa Ynez, CA.
March 7-11, 1983. W.H. Stone and C. Markert eds. In
Press 1983.

Tetanus in monkeys: symptomatology. epizootiologY. treat-
ment and prophylaxis. M.J. Kessler, R.G. Rawlins, W.T.
London and M.C. Hardegree. Proc. Amer. Assoc. Zoo Vet.
New Orleans, 1982. M.E. Fowler (ed.) 1983 p. 37-39.
Plenum Press, N.Y.

Animal Model of Human Disease: Tetanus, M.J. Kessler and
R.G. Rawlins. Comparative Pathlogy Bulletin 15(4): 3,
1983.

Submitted Papers and Books:

Intestinal parasites of the free-ranging rhesus monkeys
(Macaca mulatta) of Cayo Santiago. M.J. Kessler,
B. Yarbrough, R.G. Rawlins and J. Berard. J. Med.
Primatol. In press, 1984.

 

Absence of naturally acquired tetanus antitoxin in Cayo
Santiago macaques. M.J. Kessler, R.G. Rawlins, M.C.
Hardegree. R.E. Fornwald, and W.T. London. Submitted,
1984.

The Cayo Santiago Macaques. (eds.) R.G. Rawlins and M.J.
Kessler. SUNY-Albany Press, Primatology Series.
Submitted, 1984.

Abstracts and Presented Papers:

Metamorphosis of the pubo-ischiadic symphysis in Macaca

mulatta. R.G. Rawlins. Amer. J. Phys. Anthr0p. 41:
500, 1974.

90

Locomotor ontogeny in Macaca mulatta I: behavioral strate-
gies and tactics. R.G. Rawlins. Amer. J. Phys.
Anthr0p. 44: 201, 1976.

Locomotor ontogeny in Macaca mulatta II: locomotor dimen-
sions of the shoulder girdle and forelimb. R.G.
Rawlins. Amer. J. Phys. Anthr0p. 47: 156, 1977.

 

Locomotive and manipulative use of the hand in Macaca
mulatta. R.G. Rawlins. Amer. J. Phys. Antrhop 48:
427-428, 1978.

Sampling positional behavior of Cayo Santiago macaques
(M. Mulatta). R.G. Rawlins and D. Decarie. Amer. J.
Phys. Antrhop. 50: 473, 1979.

Population dynamics of the Cayo Santiago colony. R.G.
Rawlins. Biannual Meeting of the Regional Primate
Centers Directors. 1979, San Juan, Puerto Rico.

Locomotor ontogeny in Macaca mulatta: biomechanical impli-
cations of scaling effects and the onset of degener-
ative joint disease. R.G. Rawlins. Research Week -
University of Puerto Rico School of Medicine. 1980.
San Juan, Puerto Rico.

Population dynamics of the Cayo Santiago colony (1977-1980):
implications for the management and conservation of
primate populations. R.G. Rawlins. Symposium on
progress in the conservation and supply of nonhuman
primates. American Soc. Primatologists. 1980.

Tetanus in monkeys: clinical description, treatment and
prophylaxis. M.J. Kessler, R.G. Rawlins, W.T. London
and C. Hardegree. Proc. American Assoc. for Laboratory
Animal Science. 1980, Indianapolis, Indiana.

Tetanus in monkeys: clinical description, treatment and
prophylaxis. M.J. Kessler and R.G. Rawlins. Research
Week - University of Puerto Rico School of Medicine.
1981, San Juan, Puerto Rico.

Behavioral description and epidemiology of tetanus in the
rhesus monkeys of Cayo Santiago. R.G. Rawlins and M.J.
Kessler. Intern'l. J. Primat. 3(3): 326, 1982.

Hair mineral content in rhesus monkeys. B.M. Marriott, J.C.

Smith, Jr., R.M. Jacobs, A.O. Lee Jones, R.G. Rawlins,
and M.J. Kessler Fed. Proc. 41: 770, 1982.

91

Lactate and pyruvate interactions in the development of
hamster and squirrel monkey oocytes fertilized lg
vitro. P.J. Chan and R.G. Rawlins. Proc. Society for
the Study of Reproduction. 1982.

Tetanus in monkeys: symptomatology. epizootiology, treat-
ment and phrophylaxis. M.J. Kessler, R.G. Rawlins,
W.T. London, M.C. Hardegree. Proc. Am. Assoc. Zoo
Veterinarians. 1982.

Blastomere separation and culture techniques in hamster and
squirrel monkey embryos. R.G. Rawlins. Proc. Michigan
Academy of Science. 1983.

Golden rhesus macaques on Cayo Santiago. M.J. Kessler and
R.G. Rawlins. Amer. J. Primatol. 4: 330, 1983.

Demography of the free-ranging Cayo Santiago Macaques
(fl. mulatta): 1976-1982. R.G. Rawlins and M.J.
Kessler. Amer. J. Primatol. 4: 331, 1983.

Findings of the blood group genetics studies on the Cayo
Santiago Colony, 1972 to present. C. Duggleby,
P. Haseley, D. Sade, R.G. Rawlins and M.J. Kessler.
Amer. J. Primatol. 4: 330, 1983.

Blastomere separation of hamster (Mesocricetus auratus) and
in vitro fertilized squirrel monkey (Saimiri sciureus)
embryos. R.G. Rawlins, B. Fan, P.J. Chan and W.R.
Dukelow. Amer. J. Primatol 4: 353, 1983.

 

Xenogenous and in vitro fertilization of frozen-thawed
primate oocytes and blastomere separation of primate
embryos. W.R. Dukelow, F.J. DeMayo and R.G. Rawlins.
Fertility and Sterility. 41: 325, 1984.

Dominance rank and serum total cholesterol levels in free-
ranging adult male rhesus macaques of Cayo Santiago.
R.G. Rawlins and M.J. Kessler. Amer. Soc. Primatol.
1984.

Kin-structured migration and genetic differentiation. C.R.
Duggleby, D.S. Sade, R.G. Rawlins, and M.J. Kessler.
Amer. J. Phys. Anthr0p. 63: 153, 1984.

Management of a harem breeding colony of rhesus monkeys
(Macaca mulatta) requiring periodic removal and return
of adult females. M. Kessler, W. London, R. Rawlins,
J. Gonzalez and H. Martinez. Amer. J. Primatol.

(In press).

 

92

Use of pronase and Ca1+, MgTT-free PBS to enhance survival
of microsurgically produced half-embryos. R.G. Rawlins
and W.R. Dukelow. Amer. J. Primatol. (In press).

Book Reviews:

The Socioecology and Psychology of Primates. R.H. Tuttle,
Ed. Aldine 1976. Human Biology 49: 651-654, 1977.

 

93

APPENDIX B
CURRICULUM VITAE

Name: Richard Graham Rawlins

Born: January 15, 1950

Birthplace: Joliet, Illinois

Formal Education:

Degrees Received:

Northwestern University, 1968-1973
Michigan State University, 1982 to
present.

Bachelor of Arts with Distinction and
Departmental Honors, Northwestern
University, 1972

Master of Arts, Northwestern University,
1973

Honors and Scholarships:

John Merrill Scholarship - University of
Illinois, 1968

Illinois State Scholarship 1968 - 1972

National Merit Finalist 1968

Northwestern University Academic Scholar-
ships 1968 - 1972

PhilBeta Kappa - Northwestern University
972

National Science Foundation Fellow

National Science Foundation -
Dissertation Improvement Grant

Sigma Xi Meritorious Research Award
Michigan State University-1984

Professional Societies:

American Association for the Advancement
of Science

American Society of Primatologists

International Primatological Society

Animal Behavior Society '

94

03 75 8372