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This is to certify that the

dissertation entitled

XENOGENOUS FERTILIZATI'ON OF CRYOPRESERVED GOLDEN HAMSTER
AND SQUIRREL MONKEY OVA

presented by
FRANCESCO JOHN DeMAYO

has been accepted towards fulfillment
of the requirements for

Ph.D. Physiology

degree in

 

 

w. R. Dukelow

Major professor

 

Date 7'29‘83

 

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

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/V/—~ alt/(>3

XENOGENOUS FERTILIZATION 0F CRYOPRESERVED GOLDEN HAMSTER
AND SQUIRREL MONKEY OVA

By

Francesco John DeMayo

A DISSERTATION

Submitted to
Michigan State University
in partiai fquiIIment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physioiogy
1983

ABSTRACT

XENOGENOUS FERTILIZATION OF CRYOPRESERVED GOLDEN HAMSTER
AND SQUIRREL MONKEY OVA

by

Francesco John DeMayo

The fertilization of frozen hamster and squirrel monkey ova in
the rabbit oviduct was the objective of this investigation. A total
of 2,842 (87.0%) of the 3,267 hamster ova were judged viable by their
ability to exclude trypan blue after cryopreservation. Analysis of
the factors involved in the freezing of hamster ova showed that
successful hamster ova cryopreservation can be accomplished using PBS
or TC-l99 as the freezing medium, l.5 M or 2.0 M DMSO as the cryo-
protectant, initial slow cooling to temperatures of -l0°C, -20°C, or
—30°C, terminal slow cooling temperatures of -40°C to —80°C, and
recovery of the stored ova by thawing at l-4°C/min or 92°C/min. Cryo-
preserved hamster ova were fertilized at a significantly lower rate
(ll%) than the nonfrozen controls (35.7%).

Cryopreservation of squirrel monkey ova by the same procedure as
hamster ova showed significantly lower viability. The viability of
cryopreserved squirrel monkey ova was increased by adding DMSO at 20°C
instead of at 0°C. However, viability of cryopreserved squirrel
monkey ova was not affected by increasing the concentration of DMSO

from l.5 M to 2.0 M or 3.0 M added at 20°C. Recovery of frozen

 

Francesco John DeMayo
squirrel monkey ova at rates of l-4°C/min, l7.6-27.6°C/min, or 96°C/
min did not alter the viability of these ova. Frozen—thawed squirrel
monkey ova yielded xenogenous and jn_yitrg_fertilization rates similar

to those of the nonfrozen controls.

45;; ._,_...v- '

 

To my grandmother,

Katherine T. Weber

 

 

 

ACKNOWLEDGEMENTS

Although the inscription under the title lists only one author,
the efforts of many people are incorporated in its pages. I wish to
express my gratitude to Dr. w. R. Dukelow, my advisor, for the gui-
dance, and friendship given to me during my 4 years at the ERU.
Gratitude is also due to those who served on my graduate committee,
Drs. M. Ozaki, R. Bernard, S. Walsh, J. King, L. Ross and E. Convey.

I am indebted to all those who worked with me at the ERU and made
this work a real pleasure. ERU's deserving special mention for their
help are: Philip Chan, Yutzke Yoruzu, Reinhold Hutz, and Bonnie
Cleaves.

I wish to thank Ms. Diane Hummel for her assistance in the typing
of this manuscript.

Finally, a special thanks is owed to my fiance, Janet Thomas.

Her help and support were available when I needed it most.

TABLE OF CONTENTS

LIST OF TABLES

 

LIST OF FIGURES

 

INTRODUCTI N

 

LITERATURE REVIEH

 

 

Cryopreservation
A Historical Perspective

 

 

Cooling and Thawing Rates

 

Stages of Embryonic Development

 

Freezing Medium
Cryoprotectants

 

Nucleation Temperature

 

Assessment of Viability

 

Xenogenous Fertilization

 

MATERIALS AND METHODS

 

 

Animal Care

 

Cryopreservation
Medium

 

 

Ovum Collection
Cryoprotectant

 

Nucleation of the Medium

 

Slow Cooling

 

Thawing

 

 

Removal of Cryoprotectant

 

Assessment of Viability

 

Xenogenous Fertilization Procedurw
Ovum Collection

 

Sperm Collection

 

 

Rabbit Surgery

 

Embryo Recovery

 

Statistical Analysis

Page

vi

TABLE OF CONTENTS (continued)

RESULTS

 

DISCUSSI N

 

SUMMARY AND CONCLUSIONS

 

BIBLIOGRAPHY

 

APPENDICES

A. Publications by the Author
B. Vita

Page
38
54
59
6l

 

Table

lO

ll

l2

l3

LIST OF TABLES

Page

The cryopreservation of mouse ova and embryos at -196°C
by slow cooling (<l°C) and slow thawing (<lOO°C/min)--- 5

The cryopreservation of hamster and rabbit ova and
embryos at -196°C b slow cooling (<l°C/min) and slow
thawing (<lOO°C/min{ ----------------------------------- 6

The cryopreservation of rat ova and embryos at -196°C
after slow cooling (<l°C/min) and slow thawing (<lOO°C/
min --------------------------------------------------- 9

The cryopreservation of goat and sheep embryos by slow
cooling (<l°C/min) and slow thawing (<l00°C/min) ------- ID

The cryopreservation of cow embryos at -l96°C after
slow cooling (<l°C/min) and slow thawing (<l00°C/min)-- ll

The two step cryopreservation of mouse and hamster ova

and embryos -------------------------------------------- l3
The two step cryopreservation of sheep and cow morula

and blastocysts ---------------------------------------- l4
The culture of mammalian ova in the rabbit oviduct ----- 25

Xenogenous fertilization of mammalian ova in the
rabbit oviduct ----------------------------------------- 26

The composition of the supplemented TC-199 used for
gamete manipulation ------------------------------------ 29

The analysis of variance of the effects of initial slow
cooling temperature and plunging temperature on the
viability of hamster ova ------------------------------- 44

The xenogenous fertilization of frozen-thawed hamster
and squirrel monkey ova -------------------------------- 5l

Ig_vitro fertilization of cryopreserved squirrel monkey
ova ---------------------------------------------------- 53

vi

 

LIST OF FIGURES

 

Figure Page
l Slow cooling apparatus used for cryopreservation ------- 32
2 The viability of hamster ova after cryopreservation in

l.5 M DMSO-PBS or 1.5 M DMSO-TC-l99 -------------------- 39
3 The viability of hamster ova after cryopreservation in
l.5 M DMSO and recovery by slow or rapid thawing ------- 4l
4 The viability of hamster ova cryopreserved in 1.5 M
DMSO-TC-l99 or 2.0 M DMSO-TC-l99 ----------------------- 42
5 The viability of hamster ova rapidly cooled to -l0°C,
-20°C or -30°C and slow cooled to -40°C to -80°C before
plunging in liquid nitrogen ---------------------------- 43
6 The viability of squirrel monkey ova cryopreserved in
l.5 M DMSO-TC-l99 or 2.0 M DMSO-TC-l99 ----------------- 45
7 The effect of the temperature of the addition of DMSO
on the viability of cryopreserved squirrel monkey ova
immediately upon recovery ------------------------------ 47
8 The effect of the temperature of the addition of DMSO
on the viability of cryopreserved squirrel monkey ova
after being cultured for 3 hr -------------------------- 48
9 The effect of the concentration of DMSO on the viabili-
ty of crypreserved squirrel monkey ova ----------------- 49
10 The effect of the thawing rate en the viability of
cryopreserved squirrel monkey ova ---------------------- 50
vii

 

INTRODUCTION

The production of human offspring by in_vitro fertilization and

 

embryo transfer has heralded the need for a non-human primate model to
study the morphological, physiological, and biochemical events of
early development. Such a primate model could be used to screen for
pharmacological or environmental factors which might be beneficial or
detrimental to the developing embryo. The development of techniques
for the nonsurgical recovery of primate ova and the extracorporeal (i_
yitrg_and xenogenous fertilization) production of primate embryos has

aided in the study of primate development and the establishment of

this screening system.

 

The efficacy of jn_yitrg fertilization and xenogenous fertiliza—
tion systems, to study primate development, would be enhanced by a
readily accessible pool of ova for use in the developmental studies.
The intent of the present investigations was to develop a system for
the cryopreservation of squirrel monkey (Saimiri sciureus) ova. In
order to avoid loss of expensive primate ova, preliminary cryopreser-
vation trials utilized golden hamster ova.

Since the purpose of this investigation was to preserve the via—
bility of ova for eventual fertilization, the viability of the frozen-
thawed ova was tested by the ability of the ova to be fertilized by
homologous sperm in the oviduct of the pseudopregnant rabbit (xeno-

genous fertilization).

LITERATURE REVIEW

Cryopreservation

A Historical Perspective:

 

The first report of the successful storage of a fertilized mamma-
lian embryo at a temperature below the normal body temperature for the
species was by Chang (l947). One cell rabbit embryos, which were held
at l0°C or 5°C for l20 to l44 hours, cleaved after being in culture at
37°C for 24 hours and yielded live offspring after transfer. The
optimum temperature for storage of rabbit embryos held in rabbit serum
was reported to be 10°C. Furthermore, these stored embryo were viable
after being stored for l44 to l68 hours (Chang, l948a,b,c)

Early attempts at freezing rabbit ova and embryos to the tempera—
ture of —76°C by rapid cooling and thawing met with complete destruc—
tion of the ova and embryos (Smith, l949). However, a small percen—
tage of rabbit embryos (6/556) did continue to develop after slow
cooling to -79°C or to —l90°C and rapid thawing when the freezing
medium was supplemented with l5% glycerol (v/v) (Smith, l952; l953).
But the development was minimal since only one ova developed to the 6-
cell stage (Smith, l952).

Furthermore, the addition of glycerol to the storage medium of
unfertilized mouse ova was shown to aid in their cold storage at 5°C

(Lin §t_ l., l957). Unfertilized mouse ova were stored at —l0°C in

 

3
5% glycerol in saline for times of 0.25 to 2 hours. Survival was
limited and decreased with prolonged storage (Sherman and Lin, l959).
These mouse ova, which were able to survive storage at ~lO°C, were
transplanted to mated females and did result in live births (Sherman
and Lin, l958a,b). Sherman and Lin (l958b) observed in some cases
that mouse ova which were frozen to -l0°C showed intracellular freez-
ing. Upon thawing, these ova showed severe damage. In l963, Sherman
showed that this intracellular ice formation could be avoided by
slower cooling of the ova. When mouse ova were cooled to —20°C at a
rate of 0.7°C/min, 90% were structurally unaltered as compared to
those frozen at a more rapid rate. Furthermore, when rabbit embryos
were slow cooled in l0% glycerol to -79°C, a high proportion of
embryos developed to birth after thawing and embryo transfer (Ferdows
et_al., l958).

The need for the addition of glycerol to the storage medium and
the beneficial effects of slow cooling on oocyte survival after
storage at subzero temperatures have also been demonstrated by the
viability of grafted ovarian tissue after low temperature storage.
Rat ovarian tissue was soaked in l5% glycerol and cooled to -79°C or
-l90°C. After thawing, the ovarian tissue was grafted to recipient
animals and the endocrine activity of the tissue resumed. The resump-
tion of activity only occurred when l5% glycerol was present in the
freezing medium and slow cooling was employed (Smith et_al,, l95l;
Parkes gt_al., l953; Parkes, 1958). And although the endocrine acti-
vity of the ovarian tissue was restored, a high proportion of the
oocytes in the ovarian grafts were destroyed (as many as 90%) (Parkes

and Smith, l953; Deanesly, l954, l957; Green et_al., 1956). When the

4

same experiments were done in mice, Parrott (1960) observed similar
results. However, even though a great deal of oocyte destruction
occurred, a small proportion of the graft—recipient mice were fertile.
But the extent of reproductive life in these mice was greatly reduced.

The early attempts at preserving mammalian embryos met with
limited success until 1971 when Whittingham reported that 55% to 65%
of mouse eight cell embryos and early blastocysts suspended in 7.5%
pyrolidine in a modified Dulbecco's phosphate buffered saline (PBS)
survived freezing to -79°C. This method allowed for storage for only
30 minutes and the results have not been duplicated. Then, in 1972,
Whittingham gt_al, evaluated the factors involved in the survival of
cryopreserved mouse ova. Mouse one—cell, two-cell, eight-cell, and
blastocyst embryos survived freezing in 1.5 M DMSO in PBS or 1 M
glycerol in PBS if cooled at a rate of 0.3°C/min to l.9°C/min to
-80°C before being plunged into liquid nitrogen or liquid oxygen.
When the embryos were thawed at relatively slow rates (4° to 25°C/min)
and the glycerol or DMSO removed, 50% to 70% of the embryos survived
cryopreservation. The transfer of the recovered embryos which were
viable resulted in 40% of the embryos developing into newborn mice.
These findings have led to the successful cryopreservation of mouse
embryos (Table l), hamster and rabbit embryos (Table 2), rat embryos
(Table 3), goat and sheep embryos (Table 4), and cow embryos (Table
5).

The success of these attempts at cryopreservation relied on the
fact that the ova and embryos were cooled slowly (<l°C/min) to very

low subzero temperatures before storage in liquid nitrogen (-l96°C)

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12
and that recovery of the frozen embryos was accomplished by slow
thawing (<100°C/min). Mouse embryos did not survive cooling at a rate
greater than 7°C/min (Leibo et_al,, 1974). However, mouse and sheep
embryos did not survive if they underwent slow cooling to above -50°C
before being plunged into liquid nitrogen and were recovered by slow
thawing (Leibo et_al,, 1976; Willadsen, 1977). Furthermore, rapid
thawing led to a decreased survival of mouse embryos if storage was
accomplished by slow cooling to temperatures above -45°C before immer—
sion into liquid nitrogen (Whittingham et 21,, 1972; Wilmut et_§l:,
1972; Whittingham gt_al,, 1979). Consequently, in 1979, Whittingham
et_al: observed that a high proportion of viable mouse embryos could
be recovered from cryogenic storage by rapid thawing if slow cooling
was terminated at a relatively high subzero temperature (—40°C) before
storage at -190°C. This gave rise to the two-step freezing system,
where ova and embryos could be slowly cooled to high subzero tem-
peratures (-30°C to -40°C), then, rapidly cooled from -30°C and —40°C
to —l90°C and stored. Recovery could then be accomplished by rapid
thawing (>100°C/min) (Wood and Fairand, 1980). Since this discovery,
mouse and hamster ova and embryos (Table 6), and sheep and cow ova and
embryos (Table 7), have been frozen by the two-step method.

The factors involved in cryopreservation of mammalian ova and
embryos are the stage of embryonic development, the freezing medium,
the cryoprotectant, the nucleation temperature, the cooling and thaw—
ing process, and the assessment of viability after recovery (Leibo and
Mazur, 1978; Whittingham, 1980). The principles of each of these

factors will be discussed in the following sections. Because of its

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16
ultimate importance, the cooling and thawing process will be discussed

first.

Cooling and Thawing Rates

 

The proper cooling and thawing rates are crucial for embryo sur-
vival during cryopreservation.

The rates at which embryos can be cooled and thawed have pre-
viously been discussed. The rationale of why the embryo is restricted
to slow cooling rates requires an understanding of the physical events
that occur during the embryo's exposure to subzero temperatures. As a
cell is cooled in medium in which ice formation has occurred, more ice
forms as the temperature is lowered. As ice formation increases the
concentration of solute particles in the fluid surrounding the cell
increases. The increase in solute causes an osmotic force upon the
cell and cellular dehydration occurs (Mazur, 1970; Leibo, 1979).
Cellular dehydration causes cellular shrinkage. This phenomena has
been observed by the aid of a cryoscope (Leibo, 1977). Injury to
cells during freezing can be caused by both supraoptimal and subopti-
mal freezing rates (Mazur, 1965; 1977a).

Freezing of cells at supraoptimal rates causes inadequate dehy-
dration of the cells. This allows the formation of intracellular ice
(Mazur, 1965; l977a,b). Intracellular ice formation in mouse ova has
been correlated with ovum death when cells were cooled too fast during
cryopreservation (Leibo et_al,, 1978; Rall et_al,, 1983).

Suboptimal freezing rates have been defined as rates in which the

intracellular water remains in equilibrium with the extracellular

17

water and ice (Mazur, 1977a,b). The damage to the ovum during sub-
optimal freezing could be the result of the removal of water as ice,
the concentrating of solutes of high and low molecular weight, the
shrinkage of the cell, or the precipitation of solutes. The damage
done to cells during slow freezing is termed a solution effect.
Solution effects in mammalian ovum cryopreservation are prevented by
the addition of glycerol, DMSO, or other permeating compounds which
function to prevent the concentrating of solutes (Mazur, 1970).

Mouse embryos in the presence of a cryoprotectant survive rapid
cooling to -196°C only if they are slow cooled to -50°C prior to being
plunged into liquid nitrogen. Embryos, which are frozen by such a
method only survive cryopreservation if they are slowly thawed (<100°C/
min). Damage incurred by rapid thawing of embryos occurs between
-70°C and -20°C. The lethality of rapid thawing in such a system
results from the rapid change in solute concentration as the cryo-
protectant and medium components melt. Slow thawing allows sufficient
equilibration time of solutions as the medium is warmed (Leibo, 1974).

Mammalian ova and embryos can survive rapid thawing if slow
cooling is terminated at high subzero temperatures and the ova and
embryos are rapidly cooled to -l96°C (Tables 6 and 7). Observation of
mouse embryos cooled slowly to -40°C, followed by rapid cooling to
-l96°C, and subsequent slow thawing revealed the lethality of the slow
thawing. No intracellular ice was formed in slow cooling of ova by
such a method. But upon slow thawing, intracellular ice formed in the
ova at -80°C and ice crystallization spread beyond the boundary of the

cell plasma membranes at -55°C. This spreading of ice crystallization

18

was determined to be the lethal factor when slow thawing was employed.
Ice formation was not observed with rapid thawing (Rall et_al,, 1980).

In summary, successful cryopreservation of mammalian embryos can
occur only at optimal combinations of cooling and thawing rates. Slow
cooling to low subzero temperatures must be followed by slow thawing
' and slow cooling to high subzero temperatures must be followed by
rapid thawing after storage at -196°C to yield viable embryos. The
proper cooling and thawing rates allow for the proper exchange of
water and solutes between the intracellular and the extracellular
compartments. This exchange prevents damage due to intracellular ice

formation or solution effects.

Stage of Embryonic Development

 

The stage of embryonic development does not appear important in
the cryopreservation of mice, rabbit, rat, and sheep embryos, as can
be seen in Tables 1 through 4. Unfertilized ova and embryos from the
one—cell stage to the blastocyst stage of development can be success-
fully cryopreserved with no apparent differences among stages of
development. However, the preimplantation embryos of other species
are more labile during cryopreservation. Pig embryos from eight—cell
stage to blastocyst stage of development cannot survive cooling to 0°C
(Polge et_al., 1974). Furthermore, the cryopreservation of cattle
embryos is dependent upon the stage of development. Early cleavage
stages of cow embryos, 8—ce11 to 16-cell stages (Wilmut et_al,, 1975)
and 8-cell to early morulae (Trounson §£.El-’ 1976a) are damaged by
cooling to 0°C. The later stages of development of cattle preimplan—

tation embryos, morulae to blastocysts, survive cooling to 0°C

19

(Trounson et_al,, 1976a,b) and 4°C (Bon Durond et 21,, 1981, 1982;
Linder et_al,, 1982) without a decrease in viability.

The presence of the zona pellucida around the preimplantation
embryo has been speculated as an important factor in cryopreservation
of mouse embryos (Wilmut, 1972). Hatched bovine blastocysts, freed of
their zona pellucida, as well as, blastocysts retaining their zonae
pellucidae, did not survive cryopreservation (Trounson et_al,, 1978).
The viability of the zona pellucida-free bovine blastocysts may be due
to the stage of development of the embryo rather than the lack of any
protective action stemming from the zona pellucida. Hamster ova,
which have had their zona pellucida enzymatically removed, have sur-
vived cryopreservation at a similar rate as the zona pellucida intact

ova (Flemming et_al,, 1979).

Freezing Medium

 

Mammalian embryos were first successfully cryopreserved by Whit-
tingham et__l, (1972) who used Dulbecco's phosphate buffered saline
(PBS) to which a cryoprotectant, DMSO, had been added. Since this
report, PBS has been the medium of choice for cryopreservation (Whit-
tingham, 1980). A modification of PBS (PBS supplemented with 0.33 mM
pyruvate, 5.56 mM glucose, and 3 mg/ml bovine serum albumin) has,
also, been used in the cryopreservation of mammalian embryos (Whit-
tingham and Adams, 1976; Flemming §t_al,, 1979; Wood and Farrot,
1980). The addition of 10% fetal calf serum (Massip et_al,, 1979),
10% sheep serum (Willadsen et_al,, 1978), and polyvinylalcohol

(Creighton and Lindo, 1983) to PBS does not decrease embryo viability

20
after cryopreservation. The substitution of PBS with Hams F-10 medium
did not give any significant difference in the viability of bovine
embryos after freezing and thawing. But the development of the thawed
embryos in culture was impeded by the Hams F-lO. Zona pellucida-free
hamster ova, however, showed a preference for cryopreservation in
HEPES buffered Tyrode's solution. Viability of hamster ova was not
different after cryopreservation but human sperm did not bind as well
to the ova frozen in PBS as to those ova frozen in the modified
Tyrode's solution (Quinn gt_al,, 1982).

The purpose of the freezing medium is to maintain the ova and
embryos during handling. Therefore, a medium that supports the de-
velopment of the embryo is suitable as a medium for cryopreservation
of the embryo (Whittingham, 1980). No medium has been found to be in-

compatible with the cryopreservation procedure.

Cryoprotectants

 

Cryoprotectants are compounds which offer protection to organisms
as they are exposed to extremely lbw temperatures. The cryoprotec-
tants can be grouped into two categories: those that penetrate the
cell and those that do not penetrate the cell. The former group
offers protecttion from osmotic damage caused by slow cooling. This
is accomplished by preventing the concentrating of the extracellular
solutes during cooling and, thus, preventing cellular dehydration.

The nonpenetrating group of cryoprotectants prevent intracellular ice
damage caused by rapid cooling. This is accomplished by the creation
of osmotic forces which cause cellular dehydration (Merryman, 1963).

The exact mechanisms of action of the cryoprotectants are not known.

21
The cryoprotectants which have been helpful in the cryopreser-
vation of mammalian ova and embryos are summarized in Tables 1 through
7. They are all penetrating agents, protecting against slow cooling
damage. Nonpenetrating agents, such as sucrose and polyvinylpyroli—
dine do not protect mammalian embryos during cryopreservation (Wilmut,
1972; Wilmut gt_al,, 1973). This is probably due to the utilization

of slow cooling for cryopreservation.

Nucleation Temperature

 

The temperature at which extracellular ice formation is induced
in the freezing medium as the temperature of the medium is being
lowered is the nucleation or seeding temperature. Nucleation is
usually induced at 1°C to 2°C below the freezing point of the medium
(Whittingham, 1977). Nucleation can be accomplished in various ways:
(1) touching the surface of the suspending medium with ice crystals
contained in a Pasteur pipette or wire cooled in liquid nitrogen, (2)
touching the outside of the freezing container with dry ice or a cold
metal bar, or (3) tapping the samples (Whittingham, 1980). If nucle-
ation is not accomplished the suspending medium may supercool and ice
formation will occur spontaneously at a much reduced temperature
(Whittingham, 1977).

Supercooling of the medium has been shown to be detrimental to
the survival of mammalian embryos during cryopreservation. Neglecting
the nucleation of the suspending medium led to a decrease in survival
of sheep embryos (Willadsen, 1977). Furthermore, seeding at tempera-
tures below normal, -10°C and -12°C, was lethal to mouse embryos

(Miyamoto and Ishibashi, 1981; Whittingham, 1977).

22

Spontaneous ice formation of supercooled fluids causes a rise in
temperature of the fluid due to the released latent heat during the
phase change. In the case of supercooling during cryopreservation,
the suspending medium temperature is elevated during spontaneous ice
formation. This rise in the temperature of the freezing medium occurs
while the external temperature is being lowered. The thermal equili-
bration of the freezing medium with the external temperature causes
the embryos to be cooled at rates much faster than that observed.

This increase in cooling rate could lead to decreased survival of the
embryos. Adjustment of the cooling rate during spontaneous nucleation,
however, does not explain the total decrease in embryo survival. The
process of supercooling may have altered the permeability of oocyte
plasma membrane which would also lead to a decrease in survival

(Whittingham, 1977).

Assessment of Viability

 

The assessment of the viability of mammalian embryos after freez-
ing and thawing can be made by several methods: (1) morphological
observation, (2) use of vital dyes, (3) histological observations, (4)
metabolic assessment, (5) fertilization of oocytes, (6) development in_
31339, (7) development after xenogenous culture, and, (8) transfer of
the embryo to recipient females (Whittingham, 1978b). The techniques
used to assess viability for cryopreserved mammalian ova and embryos
are summarized in Tables 1 through 7. All the assays of viability
have their validity, but the ultimate purpose of the cryopreservation
of mammalian ova and embryos is the fertilization and subsequent

transfer of the embryos to yield live offspring.

23

Successful cryopreservation of unfertilized ova has been accom-
plished in the mouse (Tsumoda et_al,, 1975; Whittingham, 1977; Par-
kening and Chang, 1977), hamster (Tsunoda et_al,, 1975; Parkening and
Chang, 1977; Flemming 23.21:: 1979; Quinn et_al,, 1982), and rat
(Kasai et_al,, 1979; Parkening and Chang, 1977). In_yitrg_fertiliza-
tion of cryopreserved ova has been reported. Little has been done to
compare the fertilization rate of these cryopreserved ova with non-
frozen controls. However, Flemming et_al, (1979) has reported that
cryopreserved zona pellucida-free hamster ova bind homologous and
heterologous sperm at the same rate as the controls.

Transfer of frozen-thawed mouse embryos (Whittingham and Ander-
son, 1976), rabbit embryos (Maurer and Haseman, 1976; Whittingham and
Adams, 1976), and cow embryos (Tables 5 and 7) has yielded pregnancy
rates lower than nonfrozen controls. Ultrastructural examination of
cryopreserved mouse embryos shows no morphological explanation for
this phenomenon. The metabolic processes of mouse embryos may have
been slowed by cryopreservation because culture of cryopreserved mouse
embryos for 24 hours prior to transfer increased the pregnancy rates
compared to that of nonfrozen controls (Whittingham and Anderson,

1976). Culture of cow embryos after cryopreservation was not benefi-

cial to their transfer (Willadsen et_al,, 1978; Renard et_al,, 1981).

Xenogenous Fertilization

 

The oviduct of the pseudopregnant rabbit has been shown to
support the development of the embryos of many different mammalian

species. A list of species whose embryos have been shown to develop

24
in the rabbit oviduct can be found in Table 8. Umbaugh in 1949 demon—
strated that the rabbit oviduct can support not only the development
of mammalian embryos, but also the fertilization of bovine ova. Those
species whose ova have been fertilized in rabbit oviduct can be found
in Table 9. The rabbit is not the only species which can support
fertilization of heterologous gametes in its oviduct. The oviduct of
the ewe (Sreenan, 1970) and of the gilt (Shea__ttal., 1976; Bedirian
§t_al,, 1975) can support the fertilization of bovine embryos. Fur-
thermore, the mouse oviduct can support the fertilization of hamster
ova while the hamster oviduct can support the fertilization of mouse
and rabbit ova. Finally, the rat oviduct can support the fertiliza-
tion of mouse and hamster gametes (Saling and Bedford, 1981). This
fertilization of ova of one species in the oviduct of another species

is termed xenogenous fertilization.

25

TABLE 8

The Culture of Mammalian Ova in the Rabbit Oviduct

 

Embryo Donor

Reported Development

Investigator

 

Mouse

Rat

Ferret

Snowshoe Hare

Bovine

Ovine

Equine

2 cell to Blastocyst
Early Blastocyst to
Hatched Blastocyst

2-4 cell to 8-12 cell

Blastocyst to Expanded
Blastocysts
2 cell to Blastocyst

Early Cleavage to
Blastocyst

Early Cleavage to
Blastocyst

32 cell to Hatched
blastocyst

Brinster and Ten Broek,
1969

Yoshinaga and Adams, 1967

Chang, 1966
Chang et_al,, 1971

Chang, 1965

Sreenan and Scanlon, 1968
Hafez and Sugie, 1963
Lawson et_al,, 1972
Sreenan 93.21:, 1968
Adams §t_al,, 1968

Averill et_al,, 1955
Adams et_a1., 1961
Hunter eti§1,, 1962
Adams et_al,, 1968
Lawson et_al,, 1972

Allen et_al:, 1976

 

26

TABLE 9

Xenogenous Fertilization of Mammalian Ova
in the Rabbit Oviduct

 

 

Species Investigators
Mouse Saline and Bedford, 1981
Hamster Demayo et_a1:, 1980

Pig Hirst et_al,, 1981

Cow Umbaugh, 1949

Trounson et 21,, 1977

Sreenan, T970

Hirst at al,, 1981
Squirrel Monkey Demayo gt_al,, 1980

Baboon Kuehl, 1983

 

MATERIALS AND METHODS

Animal Care

 

Squirrel monkeys of the Bolivian and Guyanan types were obtained
from Primate Imports Corp. (Port Washington, NY) and South American
Primates (Miami, FL), respectively. They were housed in groups of
six, in stainless steel, flush-type, cages from October through June.
During the summer, June through September, they were housed outdoors,
in groups of 50, in 4 outdoor colonies (Jarosz and Dukelow, 1976).
The animals were fed High Protein Monkey ChowR, Jumbo 5047 (Ralston
Purina Co., St. Louis, MO), water ag_libitum and fruit as a diet
supplement. While being housed indoors they were exposed to 12h:12h
lightzdark (0600 hrs to 1800 hrs light) cycle using fluorescent
lighting.

The golden hamsters were obtained from the State of Michigan
Health Laboratories. The hamsters were housed in groups of six in
plastic cages with stainless steel tops with ground corn cobs as
bedding (San-I-CellR, Paxton Processing Co., Paxton, IL). Wayne.
Laboratory Animal Diet (Allied Mills Inc., Chicago, IL) and water was
fed ad libitum. The animals were exposed to a light/dark cycle of
14:10 hrs (0600 hr to 1800 hr lights on).

(Rabbits were housed singly in stainless steel cages with ground

corn cobs as bedding. The rabbits were fed with Laboratory Rabbit

27

28
ChowR (Ralston Purina Co.) and water ag_libitum. Rabbits were not

maintained under a specific light/dark cycle.

Cryopreservation

 

The procedure for cryopreservation consists of: ovum collection,
addition of the cryoprotectant, nucleation of the medium, slow cool-
ing, thawing, removal of the cryoprotectant and assessment of the
ovum's viability.

Medium; The media used to supply a safe environment for the
hamster ovum during cryopreservation was either Dulbeccos phosphate
buffered saline (PBS) (Gibco Laboratories, Grand Island, NY) or a
modification of the squirrel monkey ig_yitrg_fertilization medium used
by Kuehl and Dukelow (1979), TC-199. The basic contents for this
medium are listed in Table 10. In order to facilitate the removal of
cumulous cells from the hamster ova during ovum collection, the collec-
tion medium was supplemented with 1 mg/ml hyaluronidase (Sigma Corp.,
St. Louis, MO). Squirrel monkey ova were manipulated in the medium as
described in Table 10. All media were sterilized by filtration
through a 0.45 pm Millipore filter and stored in a sterile Vacutainer
at 4°C.

Ovum Collection: Hamster ova were obtained from oviducts of

 

animals superovulated with an intraperitoneal injection of 30 IU
pregnant mare serum (PMS, FolligonR; Intervet Laboratories; Bar Hill,
Cambridge, Great Britain) between 0900 hrs and 1200 hrs followed 56 to
64 hrs later with an I.P. injection of 30 IU human chorionic gonado-

tropin, HCG (APLR; Ayerst Laboratories Inc., NY) (Yanagimachi and

29

TABLE 10

The Composition of the Supplemented TC-199 Medium
used for Gamete Manipulation

 

 

 

Ingredient Amount Source
TC-199] 80% GIBCO Laboratories
Grand Island, N.Y.
GG Free Fetal Bovine 20% GIBCO Laboratories
Serum2 _Grand Island, N.Y.
Sodium Pyruvate 115.2 ug/ml Sigma Chemical Co.
St. Louis, MO
Gentamicin 0.1 mg/ml Schering Corp.
Kenilworth, N.J.
Penicillin-Streptomycin 100 units and North American Biological
100 ug/ml Miami, FL
Hyalarunidase3 1 mg/ml Sigma Chemical Co.
St. Louis, MO
Heparin4 1 unit/m1 The Upjohn Co.
Kalamazoo, MI
1

2Heated at 56°C for 30 minutes.
3

Medium 199 with 25 mM HEPES buffer, Earle's salts and L-Glutamine.

Added when collecting hamster ova to remove camalus cells.

4Added to prevent blood clotting when collecting monkey ova.

30
Chang, 1964). These hamsters were sacrificed, by cervical disloca-
tion, 14 to 16 hrs after HCG administration. The oviducts were re-
moved, dissected free of fat and flushed from the fimbrie with 0.3 m1
of medium. Mature ova were counted and held in an ice bath until
preservation.

Squirrel monkey ova were collected by laparoscopic aspiration of
follicles (Dukelow and Ariga, 1976) from females after administering a
regimen of gonadotropins to induce ovulation (Dukelow, 1970). This
ovulatory regime of gonadotropins consisted of injecting I.M. for four
days 1 mg follicle stimulating hormone, FSH (Bruns Biotec, Oakland,
CA) with an intramuscular injection of 250 I.U. HCG on the fourth day
(Dukelow, 1979). During the summer months, July to October, five days
of I.M. injections of 1 mg FSH were administered due to the seasona-
lity of the squirrel monkey (Harrison and Dukelow, 1973). The squir-
rel monkeys were then laparoscoped 12 to 18 hrs after the administra-
tion of HCG. Follicles were then aspirated with a 25 gauge, 5/8"
needle into 0.1 ml medium. The follicular contents were deposited
into an 8 chamber tissue culture chamber slide (Lab-Tek, Miles Labora-
tories Inc., Naperville, IL) and incubated in a 37°C, moist atmosphere
with 5% C02 in air until cryopreservation.

Cryoprotectant: The cryoprotectant used in this study was

 

dimethylsulfoxide (DMSO; Baker Chemical Co., Phillipsburg, PA). A
stock solution of 3 M or 4 M DMSO was dissolved in either Dulbecco's
phosphate buffered saline or supplemented TC—l99 and was stored at

4°C.

31

Squirrel monkey or hamster ova were placed in 0.1 m1 of media in
a 2 m1 (1 cm x 7 cm) glass ampule (Wheaton Scientific, Millville, NJ).
The ampules containing hamster ova were then placed in an ice bath
while those ampules containing squirrel monkey ova were placed in an
ice bath or held at room temperature. An equal volume (0.1 m1) of
either 3 M or 4 M DMSO was added, in one step, to the ampules to bring
the final concentration of DMSO to either 1.5 M or 2 M. The DMSO was
added at the same temperature as that of the medium containing the
ova. In one trial monkey ova were placed directly in 0.2 ml of 3 M
DMSO. After the addition of DMSO the ampules were sealed using a
methane-oxygen hand sealing machine, Model HSI (Cozzoli Machine Co.,
Plainfield, NJ). The ova were now ready for freezing.

Nucleation of the Medium: The sealed ampules, containing ova,
were placed in a 95% ethanol bath which had been cooled to a tempera-
ture of -5°C to -7°C. Nucleation or seeding was accomplished by
touching the side of the ampule with forceps which had been immersed
in liquid nitrogen. Once iCe formation was initiated the ampules were
placed in the cooling apparatus.

Slow Cooling: The apparatus used for slow cooling is shown in

 

Figure 1. An unsilvered 900 m1 dewar flask (9 cm x 30 cm) (Ace Glass
Inc., Vineland, NJ), was filled with 350 ml of 95% ethanol. This
dewar flask was of 6 Torr vacuum. The unsilvered dewar flask was
placed in a 4.5 liter silvered dewar flask which had been filled with
liquid nitrogen. The cooling rate of the ethanol bath was determined
by the least squares regression analysis of the decline in the tem-

perature of the bath with time. The cooling rate was a function of:

32

 

Figure l. The slow cooling apparatus used in the cryopreservation
of hamster and squirrel monkey ova.

33
the volume of ethanol in the unsilvered dewar flask, the depth of
immersion of the usilvered dewar flask in the liquid nitrogen bath and
the vacuum of the unsilvered flask. The depth of immersion of the
unsilvered dewar flask in the liquid nitrogen bath was regulated by a
laboratory jack. The cooling rates achieved for the cooling apparatus
ranged from 0.l9°C/min to 0.35°C/min.

Ampules containing hamster ova, which had been seeded, were
placed in the cooling apparatus at temperatures of -10°C, -20°C or
30°C. The ampules were removed from the cooling apparatUs and plunged
in liquid nitrogen after they had been cooled to either -40°C, -50°C,
-60°C, -70°C or -80°C. This 3x5 factorial was used to determine the
optimum temperature range for slow cooling of hamster ova. Ampules
containing squirrel monkey ova were placed in the cooling apparatus at
the seeding temperature and cooled to -60 or -80°C before plunging
into liquid nitrogen. The storage time for the ova ranged from 1 week
to 3 months

Thawing: There were three methods used to thaw the cold stored
ampules, tested:

1) Ampules were placed in a 600 m1 beaker filled with 200 m1 of
95% ethanol which had been cooled to -110°C. The ethanol bath was
then allowed to equilibrate with room temperature. Least squares
regression analysis of the change in temperature of the ethanol bath
showed a warming rate of 1 to 4°C/min.

2) Ampules were placed in a styrofoam cup and allowed to warm.
Observation of the time required for melting of the contents of the

ampule gave an estimate of warming rates of 17.6°C/min - 27.6°C/min.

34
3) Ampules were placed in an ice bath. A warming rate cf 92°C/
min was estimated by the same method previously given.

Removal of the Cryoprotectant: Once the contents of the ampule

 

had thawed the DMSO was removed by seria dilution at 4°C. This was
accomplished by the addition of 0.2 ml, 0.2 ml, 0.4 m1 and 1.0 m1 of
freezing medium at 1 minute intervals.

Assessment of Viability: Viability of cryopreserved ova was

 

assessed by: morphological normality, the ovum's ability to exclude
trypan blue, and the ovum's ability to incorporate fluorescein diace-
tate (FDA).

The criteria for morphological normality was the appearance of a
light, nongranular and translucent vitellus (Quinn et_al,, 1982) and
the presence of an intact zona pellucida. This subjective analysis of
viability led to the need for a more objective determination of via-
bility. Hamster ova were incubated in 25 pl of 0.2% trypan blue for 1
min and washed in 25 pl of medium. If the ooplasm was free of trypan
blue the ova was judged viable. Squirrel monkey ova were placed in a
depression slide containing 25 ul of 15 DM FDA in PBS. After holding
the ova for l min, the ova were examined under a fluorescence micro-
scope (Leitz BG12 and BG38 exciter filter and a K510 long pass barrier
filter (The Microscope Co., New Castle, PA). If fluorescence was

observed the ova was classified as viable.

Xenogenous Fertilization Procedure

 

The procedure for xenogenous fertilization consists of: ovum

collection, sperm collection, deposition of gametes in the rabbit

35
oviduct and embryo recovery. Ovum collection has been discussed in a

previous section.

Sperm Collection: Hamster sperm was collected from the epidi—

 

dymi of mature males. Hamsters were sacrificed, by cervical dislo-
cation, and their epididymi were dissected. The cauda epididymus was
minced in 1 ml of medium. A 0.05 ml aliquot of this solution was then
diluted five times and a sample of this solution was evaluated under
the light microscope for motility and structural normality.

Semen, obtained by electroejaculation of unanesthetized male
squirrel monkeys, was collected in 0.5 m1 of medium. A sample of this
solution was evaluated for motility and structural normality under the
light microscope.

Hamster and squirrel monkey sperm suspensions were held in a 37°C
water bath until deposited in a rabbit oviduct within 1/2 to 1 hour.
Twenty-four hours after collection, the sperm concentration was deter-
mined with a hemocytometer.

Rabbit Surgery: Adult female rabbits (New Zealand White) were

 

given 100 I.U. HCG, I.V., to induce pseudopregnancy (Harper, 1963).
On the day of surgery, each rabbit was anesthetized with 60 mg/2.25 kg
body weight of sodium pentobarbital (NembutalR; Abbott Laboratories,
North Chicago, IL) followed by ether inhalation to maintain a surgical
plane of anesthesia. The reproductive tract was then exposed through
a 7 cm mid-ventral incision. Ova, in 5 pl aliquots were deposited
using a MicropetterR (SMI, Scientific Manufacturing Industries,
Emeryville, CA) into the fimbriated end of the ampulla. Depending on

the number of ova, l to 4 aliquots were deposited. Viable frozen

36
thawed ova were placed in one oviduct and nonfrozen control ova were
placed in the opposite oviduct.

After deposition of ova 0.05 ml of nonspecific sperm solution was
deposited. Sperm were deposited using a 0.25 ml tuberculin syringe,
fitted with a 20 gauge, 1.5“ needle to which 5 cm polystyrene tubing
(0.034 inches id, 0.050 inches od; Clay Adams, Parsippany, NJ) was
affixed. After insemination the uterotubal junction was ligated with
00 gut suture and the abdominal incision closed.

Embryo Recovery: Each rabbit was killed by cervical dislocation
24 hrs later and the reproductive tract was removed. Using a 5m1
syringe with blunted 25 gauge, 5/8" needle, medium (2 ml) and air (1.5
ml) was flushed through the oviduct from the uterine end. The ovi-
ductal contents were collected in a watch glass and under a dissecting
microscope recovered embryos were observed. Fertilization was judged
to have occurred if 2 polar bodies and 2 pronuclei were observed or if

cleavage had occurred.

Statistical Analysis

 

A11 percentage data, analyzed by parametric statistical analysis,
was tranformed using the arc sine X transformation. The effects
of medium, concentration of DMSO and warming rate on the viability of
cryopreserved hamster ova were analyzed using the Student's t-test.
The optimum temperature range for slow cooling of hamster ova was
analyzed by 2-way analysis of variance.

Parametric statistical analysis was not used on the analysis of

factors affecting the cryopreservation of squirrel monkey ova. The

37
low number of monkey ova in each ampule prevented the assumption of
continuity of the percentage data needed for parametric statistical
analysis. Chi square analysis and Fishers Exact test was used to

analyze the squirrel monkey data (Zar, 1974).

RESULTS

A total of 4,908 hamster ova were cooled to -l96°C and stored for
1 day to 3 months. After thawing, 3,267 ova (66.6%) were recovered
and 2,842 (87.0%) of the recovered ova were judged to be viable by
their ability to exclude trypan blue. A total of 363 squirrel monkey
ova were frozen and 342 (94.2%) ova were reocvered after storage. Of
the recovered ova, 146 (42.7%) were judged viable by their ability to
incorporate fluorescein diacetate.

Hamster ova were cooled at rates of 0.19-O.25°C/min to a tempera-
ture of -80°C. The ova were then plunged into liquid nitrogen and
stored. Recovery of these ova was accomplished by slow warming at
rates of 1 to 4°C/min. The effects of storage medium, PBS gs, TC-

199 on the cryopreservation of hamster ova is shown in Figure 2. No
significant difference was observed on the viability of hamster ova
cryopreserved in PBS or TC-l99 (t3 = 1.53) as judged by the ovum's
exclusion of trypan blue. The ova were then cultured for 3 hr at 37°C
in a 5% C02, in air atmosphere with 100% humidity. As can be seen
from Figure 2, no significant difference was observed, after 3 hr
culture, in the viability of the hamster ova with the two media (t3 =
0.76). Since TC-199 is the medium used in the xenogenous fertiliza-
tion of hamster and squirrel monkey gametes, this medium was utilized

in all future cryopreservation trials.

38

39

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Thawing of hamster ova at 1-2°C/min or 92°C/min had no signifi-
cant effect on the viability of cryopreserved hamster ova (t2 = 1.56)
and no difference was observed after the culture of the ova for 3 hrs
(t2 = 3.79). These results are shown in Figure 3.

Increasing the concentration of DMSO from 1.5 M to 2.0 M had no
significant effect on the viability of cryopreserved hamster ova
immediately after thawing (t8 = 1.32). No difference in viability was
observed with these two treatments after a 3 hour culture of the ova
(t8 = 1.60). These results are shown in Figure 4.

The temperature range for which hamster ova must be slow cooled,
to yield maximum viability, was investigated. Hamster ova were
rapidly cooled to -10°C, -20°C or -30°C after the seeding of the
ampules. The ampules were then slow cooled to -40°C, -50°C, -60°C,
-70°C or -80°C before they were plunged into liquid nitrogen. The
results of this 3x5 factorial experiment are shown in Figure 5. The
analysis of variance of this data, as shown in Table 11, shows no
significant effect caused by the temperature at which slow cooling is
initiated and no significant effect caused by the temperature at which
slow cooling is terminated. There was no interaction between the two
main effects.

Squirrel monkey ova were cooled slowly to -80°C in 1.5 M DMSO-
TC-l99 and stored in liquid nitrogen. Survival upon thawing was
significantly decreased when compared to nonfrozen controls (x3 =
70.6) as shown in Figure 6. When the concentration of DMSO was
increased to 2 M there was a significant increase in the viability of

2

squirrel monkey ova when compared to ova frozen in 1.5 M DMSO (x2 =

10.1), as is shown in Figure 6.

 

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-80°C before plunging into liquid nitrogen.
plished by rapid thawing at =92°C/min.

Recovery was accom-

44

 

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Preliminary trials indicated that the decrease in viability after
the cryopreservation of squirrel monkey ova may be due to the tem-
perature at which DMSO is added, 0°C. The addition of DMSO at room
temperature, 20°C, significantly increased the viability of squirrel
monkey ova when frozen in 1.5 M and 2 M DMSO. When compared to simi-
lar concentrations of DMSO added at 0°C, Figure 7 (x? = 25.5 and 8.4,
respectively). This increase in viability was seen immediately after
recovery and after the recovered ova were cultured for 3 hours (x? =
2.0 and 8.7, respectively) as seen in Figure 8. DMSO was added to
squirrel monkey ova at 20°C, prior to slow cooling, in all the follow-
ing trials.

DMSO, when used as a cryoprotectant, at concentrations of 1.5 M,
2 M or 3 M was equally effective at producing viable squirrel monkey
ova. No significant effect, due to these concentrations of DMSO, was
observed immediately after thawing (x3 = 0.4) or after the recovered
ova were cultured for 3 hours (x; = 1.1). These results are shown in
Figure 9. (Data for 1.5 M and 2.0 M DMSO were taken from Figure 8.)

Thawing rates of l-2°C/min, l7.6-27.6°C/min or <92°C/min showed
no significant effect on the viability of squirrel monkey ova after
freezing. No difference was observed immediately upon recovery (X; =
2.7) or after the ova were cultured for 3 hours (x; = 2.8). These
results are shown in Figure 10.

The fertility of the frozen hamster and squirrel monkey ova is
shown in Table 12. Hamster ova, when frozen under optimal conditions,

showed a significant decrease (x; = 13.1) in their ability to be

 

 

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52
fertilized, by homologous sperm, in the rabbit oviduct. No cleavage
of frozen hamster ova was observed after xenogenous fertilization.
Squirrel monkey ova, when frozen under optimal conditions, showed no
difference, when compared to nonfrozen controls, in their ability to
be fertilized by homologous sperm in the rabbit oviduct (x? = 0.1).
The fertility of cryopreserved squirrel monkey ova was also

tested by their ability to be fertilized in_yitrg. These results are
shown in Table 13. No difference in maturation rate or fertilization
rate was observed between control and frozen ova. This was determined

by use of the Fishers Exact test.

53

 

 

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DISCUSSION

Hamster ova are very elastic in their requirements for successful
frozen storage. Quinn §t_al. (1982) showed that hamster ova survived
freezing when they were slow cooled to -40°C, plunged into liquid
nitrogen, and recovered by rapid thawing (=500°C/min) or when they
were slow cooled to -80°C, plunged into liquid nitrogen, and recovered
by slow thawing (=8°C/min). Viability was judged only by the appear—
ance of normal morphology and the ability of zona pellucida free
hamster ova to bind homologous or heterologous sperm. However, Quinn
et_al, did not show that other combinations of cooling and thawing
rates would have any effect on the viability of frozen hamster ova.

In this present report, a high proportion of hamster ova survived
slow (=1-2°C/min) or rapid (=92°C/min) thawing after being slowly
I cooled to -80°C and stored in liquid nitrogen. Therefore, both
thawing rates allowed proper equilibration of solution between the
ovum and extracellular fluid during thawing.

Slow cooling from seeding temperature to -80°C before storage in
liquid nitrogen was not mandatory for survival of hamster ova.

Hamster ova survived freezing equally well if they were slow cooled
from -30°C to -40°C or if they were slow cooled from -10°C to -80°C

before plunging into liquid nitrogen. From this data, it can be

54

55
inferred that the temperature range at which the necessary dehydration
of hamster ova occurs, is between 30°C and 40°C.

Leibo et_al, (1978) noted that the temperature at which intra-
cellular ice formation occurs in mouse ova cooled rapidly in 1.0 M
DMSO was —45°C. This formation of intracellular ice was correlated
with ovum lethality. Furthermore, Rall gt_al. (1983) noted that the
lethal event of rapid freezing occurred a few degrees (l-2°C) above
the temperature at which intracellular ice was observed forming. The
present data agree with these findings. Hamster ova must be slow
cooled to at least —40°C or there will be insufficient dehydration in
the ova and ice formation will occur at the nucleation temperature.

The choice of =92°C/min as the thawing rate to recover ova in the
determination of the optimum slow cooling temperature range may have
obscured the true temperature at which proper dehydration must occur.
Rapid thawing prevents the regrowth of ice crystals that are lethal,
upon the slow thawing of improperly dehydrated cells (Rall §t_gl.,
1980). Choosing 92°C/min buffered against the growth of ice crystals
in cells in which insufficient dehydration had occurred. Furthermore,
this thawing rate allowed sufficient time for fluid equilibration when
cells, which had been cooled to -80°C, were thawed. Therefore, this
thawing rate may allow more leeway in the requirements for hamster ova
cryopreservation.

In contrast, squirrel monkey ova are not as flexible in their
requirements for cryopreservation. Monkey ova slow cooled to -80°C
in 1.5 M DMSO and stored at -196°C did not show the high survival rate

observed with the hamster ova. Increasing the concentration of DMSO

56
from 1.5 M to 2.0 M increased the survival of monkey ova. The need
for a higher concentration of DMSO for increased survival indicated
that DMSO was improperly penetrating the ova and insuring proper
protection. Increasing the temperature, at which the DMSO was added,
from 0°C to 20°C allowed the proper penetration of DMSO into the
monkey ova to insure proper protection. This need for the addition of
DMSO at a temperature above 0°C has been observed for rat oocytes
(Kasai et_al,, 1979), sheep embryos (Moore and Bolton, 1976), and cow
embryos (Willadsen, 1976). Although reports show that mouse embryos
also require DMSO to be added at temperatures higher than 0°C before
freezing (Parkening §t_al,, 1976; Wilmat, 1972), the addition of DMSO
at 0°C before freezing is suitable for the storage of mouse embryos
(Whittingham et_al,, 1972; Leibo et_al,, 1974; Whittingham, 1975).

The viability of hamster ova was determined by the ability of the
ovum to exclude trypan blue. Trypan blue measures the integrity of
the plasma membrane. However, it may overestimate viability because
the plasma membrane is not the sole factor controlling viability of a
cell (Tennat, 1964; Dolan, 1965). In this case, trypan blue did not
prove to be an index of fertilizability of frozen hamster ova.
Frozen—thawed hamster ova did not fertilize xenogenously, as well as
the controls. Although previous reports have demonstrated that
frozen-thawed zona pellucida free hamster ova were able to bind homo—
logous sperm, as well as the controls (Flemming et_al,, 1979; Quinn et
al,, 1982), the process of fertilization requires the penetration of
the zona pellucida by sperm. Damage to the zona pellucida during

freezing and thawing is not detected by the vital dye, trypan blue.

57
Therefore, this undetected damage to the zona pellucida may account
for the lowered fertility.

Frozen-thawed squirrel monkey ova, which were jn_yjtrg_and xeno-
genously fertilized, gave results similar to those of the controls.
The viability assay used to determine squirrel monkey ovum viability
was FDA incorporation. This vital dye tests membrane integrity and
the presence of esterases (Rotman and Papermaster, 1966). It has been
used to test the viability of frozen-thawed HeLa S-C cells (McGrath et
31., 1975), mouse embryos (Mohr and Trounson, 1980), and squirrel
monkey ova and embryos (Chan gt_al., 1982).

The difference in fertility of frozen-thawed squirrel monkey and
hamster.ova may not be due to differences in the estimation of viabi-
lity. FDA incorporation has the same limitations as trypan blue in
that it only estimates two of the many factors necessary for cell
survival (Dolan, 1965). Furthermore, FDA has not been observed to be
a more conservative estimator of viability than trypan blue. In fact,
FDA provides estimates of squirrel monkey ova viability similar to the
estimates of hamster ova viability obtained with trypan blue (Hutz,
1983).

The differences in the fertility of frozen hamster and squirrel
monkey ova could lie in the differences in the zona pellucida of the
respective ova. The zona pellucida of the squirrel monkey ova could
tolerate freezing better than the zona pellucida of hamster ova. This
would not be detected by either of the vital dyes. Also, the differ-
ences could lie in the inability of the hamster ova to tolerate mani-

pulation. Hamster embryos are very sensitive to manipulation in

 

58
culture. The slightest manipulation can place developing hamster
embryo into unexplained refractoriness to development (Whittingham and
Bavister, 1974). The additional manipulation involved in the freezing

of the hamster ova may be the cause of refractoriness in development.

 

SUMMARY AND CONCLUSIONS

The aim of this present study was to develop a system for the
cryopresrvation of hamster and squirrel monkey ova. This system would
be used to create a readily accessible pool of ova for the production
of embryos by jg_yitrg and xenogenous fertilization. To achieve this
end, hamster and squirrel monkey ova were placed in media containing
various concentrations of DMSO. The ova were then cooled to various
subzero temperatures before being stored in liquid nitrogen. Ova were
recovered from cold storage by thawing at l-4°C/min, l7.6-27.6°C/min,
or 92°C/min. Viability of the thawed hamster and squirrel monkey ova
was determined by the ovum's ability to exclude trypan blue and to
incorporate FDA, respectively. After freezing by the optimum proce—
dure, hamster ova and squirrel monkey ova were xenogenously fertilized
by placing the ova into the oviducts of pseudopregnant rabbits with
homologous sperm. Frozen squirrel monkey ova were also fertilized in
yitrg. The following conclusions can be made from this study:

1) Cryopreservation of hamster ova was achieved by using
either PBS or TC-199 as the freezing medium.

2) The viability of frozen hamster ova was not affected by
increasing the concentration of DMSO from 1.5 M to 2.0 M or if re-
covery of the frozen ova was accomplished by thawing at l-2°C/min or

92°C/min.

59

 

60

3) The optimum temperature range for which hamster ova must be
cooled slowly was very broad. The beginning of the slow cooling of
hamster ova at —10°C, -20°C, or -30°C and the termination of the slow
cooling at -40°C to -80°C did not alter the viability of frozen ham—
ster ova.

4) Frozen hamster ova showed a decreased ability to be ferti-
lized in the oviducts of pseudopregnant rabbits.

5) The temperature at which DMSO is added prior to cryopreser—
vation is vital for successful freezing of squirrel monkey ova.
Addition of DMSO at 20°C, in lieu of 0°C, significantly increased the
viability of the squirrel monkey ova.

6) The viability of frozen squirrel monkey ova was not affected
by increasing the concentration of DMSO from 1.5 M to 3.0 M added at
20°C.

7) Increasing the thawing rate of frozen squirrel monkey ova
from l-4°C/min to either l7.6—27.6°C/min or 92°C/min did not alter
viability after freezing.

8) Frozen squirrel monkey ova showed no difference in their

ability to be fertilized xenogenously or in_vitro.

BIBLIOGRAPHY

BIBLIOGRAPHY

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distance transport of sheep ova. Proc. 4th Cong. Anim. Reprod.
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Adams, C.E., Moor, R.M., and Rowson, L.E.A. Survival of cow and
sheep eggs in the rabbit oviduct. Proc. 6th Int. Cong. on Anim.
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Allen, W.R., Stewart, F., Trounson, 0.0., Tishner, M., and Bielanski,
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Bank, H. and Maurer, R.R. Survival of frozen rabbit embryos. Exp.
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Bedirian, K.N., Shea, B.F., and Baker, R.D. Fertilization of bovine
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Bilton, R.J. and Moore, N.W. In vitro culture, storage and transfer
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Bilton, R.J. and Moore, N.W. Successful transport of frozen cattle
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Bon Durant, R.H., Anderson, G.B., Boland, M., Cupps, P.T. and Hughes,
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Bon Durant, R.H., Anderson, G.B., Boland, M.P., Cupps, P.T. and
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Bouyssou, B. and Chapin, 0. Two step freezing of cattle blastocysts

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61

 

62

Bouyssou, B. and Chapin, 0. Two step freezing of cattle blastocysts
in french straws. Theriogenology 11, 80 (1982b).

Brinster, R.L. and Ten Broeck, J.T. Blastocyst development of mouse
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63

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64

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APPENDIX A

Publications by the Author

 

Papers:

DeMayo, F.J., Mizoguchi, H., and Dukelow, W.R. Fertilization of
squirrel monkey and hamster ova in the rabbit oviduct (xeno-
genous fertilization. Science 255; 1468-1469 (1980).

Hirst, P.J., DeMayo, F.J., and Dukelow, W.R. Xenogenous fertiliza-
tion of laboratory and domestic animals in the oviducts of the
pseudopregnant rabbit. Theriogenology 15; 67-75 (1981).

Sacco, A.G., Yurewicz, E.C., Subramanian, M.G., and DeMayo, F.J.
Zona pellucida composition: Species cross reactivity and con-
traceptive potential of antiserum to a purified pig zona antigen
(ppza). Biol. Reprod. 25, 997-1008 (1982).

 

Sacco, A.G., Subramanian, M.G., Yurewicz, E.C., DeMayo, F.J., and
Dukelow, W.R. Heteroimmunization of squirrel monkeys (Saimiri
sciureus) with a purified porcine zona antigen (ppza): Immune
response and biologic activity of antiserum. Fertil. Steril.
55; 350—358 (1983).

DeMayo, F.J., Hutz, R.J., and Dukelow, W.R. Cryopreservation of
squirrel monkey ova. Proceedings of the IXth Congress of the
International Primatological Society, Atlanta, GA (1982) (sub—
mitted).

Dukelow, W.R., Chan, P.J., Hutz, R.J., DeMayo, F.J., Dooley, V.D., and
Rawlins, R.G. Early development of the in vitro fertilized pre-
implantation primate embryo. J. Exp. 20617 in press) (1983).

Dukelow, W.R., Chan, P.J., Hutz, R.J., DeMayo, F.J., Dooley, V.D.,
and Rawlins, R.G. Early development of the 15_vitro fertilized
preimplantation primate embryo. In Proc. Kroc. Foundation Conf.,
Santa Ynez, CA, March 7-11, 1983, W.H. Stone and C. Markert, eds.,
in press.

 

Hutz, R.J., DeMayo, F.J., and Dukelow, W.R. The use of vital dyes to
assess embryonic viability in the hamster (Mesocricetus auratus).
Cell Tissue Res. (Submitted) (1983).

 

Hutz, R.J., DeMayo, F.J., Chan, P.J., and Dukelow, W.R. 2-Deoxy-D-
glucose uptake of embryos fertilized 15_vitro. J. Cell. Physiol.
(Submitted) (1983).

DeMayo, F.J., Chan, P.C., and Dukelow, W.R. The fertilization of
cryopreserved squirrel monkey ova. Science (Submitted) (1983).

DeMayo, F.J. and Dukelow, W.R. The effect of the temperature of the
addition of DMSO on the cryopreservability of squirrel monkey
follicular oocytes. J. Reprod. Fert. (Submitted) (1983).

DeMayo, F.J. and Dukelow, W.R. The effect of altering the tempera-
ture range of slow cooling on the cryopreservability of hamster
ova. Gamete Res. (Submitted) (1983).

Abstracts:

DeMayo, F.J., Mizoguchi, H., and Dukelow, W.R. Xenogenous fertiliza-
tion of hamster and squirrel monkey ova in the oviduct of the
pseudopregnant rabbit. 13th Annual Meeting of the Society for
the Study of Reproduction, Ann Arbor, MI (1980).

DeMayo, F.J., Chan, P.J., and Dukelow, W.R. Nonsurgical (laparo-
scopic) ovum and embryo recovery in the squirrel monkey and mink.
Blst Annual Session of the American Association for Laboratory
Animal Science, Indianapolis, IN (1980).

Hirst, P.J., DeMayo, F.J., and Dukelow, W.R. Xenogenous fertiliza-
tion of nonhuman primate, laboratory and domestic animals. 3lst
Annual Session of the American Association for Laboratory Animal
Science, Indianapolis, IN (1980).

Dukelow, W.R., Chan, P.J., DeMayo, F.J., and Ridha, M.T. Alterna-
tives to natural fertilization in primates. American Society of
Primatologists, Winston-Salem, NC (1980).

Asakawa, T., Ghosh, M., DeMayo, F.J., Chan, P.J., Ridha, M.T., Hutz,
R.J., Dooley, V.D., and Dukelow, W.R. Reproductive test system
involving in vitro and xenogenous fertilization. Proceedings of
the CenterTFOr Environmental Toxicology, East Lansing, MI (1981).

Dukelow, W.R., andDeMayo, F.J. Laparoscopic applications to ferti-
lization studies in the squirrel monkey. American Society of
Primatologists, San Antonio, TX (1981).

DeMayo, F.J. and Dukelow, W.R. Xenogenous fertilization of squirrel
monkey ova with varying sperm concentrations and stages of
pseudopregnancy. American Society of Primatologists, San Anto-
nio, TX (1981).

 

 

Sacco, A.G., Subramanian, M.G., Yurewicz, E.C., Dukelow, W.R., and
DeMayo, F.J. Meteroimmunization of squirrel monkey (Saimiri
sciureus) with a purified porcine zona antigen (ppza): Immuno
response and biologic activity of antiserum. American Fertility
Society, Las Vegas, NV (1982).

DeMayo, F.J. Cryopreservation of squirrel monkey ova. International
Primatological Society, Atlanta, GA (1982).

DeMayo, F.J. Cryopreservation and xenogenous fertilization of ham-
ster and squirrel monkey ova. Michigan Academy of Science.
Ypsilanti, MI (1983).

Dooley, V.D., Hutz, R.J., Chan, P.J., DeMayo, F.J., and Dukelow, W.R.
Biochemical evaluation of ovum and embryo viability 15_vitro.

Ameripan Association for the Advancement of Science, Detroit, MI
1983 .

Hutz, R.J., DeMayo, F.J., Chan, P.J., and Dukelow, W.R. Glucose uti-
lization by early primate embryos fertilized 15_vitro. Pro-
ceedings Society for the Study of Reproduction, Cleveland, OH
(August, 1983).

 

DeMayo, F.J., Chan, P.J., and Dukelow, W.R. The effects of DMSO
addition on the cryopreservation and fertilization of squirrel

monkey ova. American Society of Primatologists, East Lansing, MI
(1983).

DeMayo, F.J. and Dukelow, W.R. Xenogenous fertilization of cryopre-
served squirrel monkey oocytes. American Association for Labora-
tory Animal Science, San Antonio, TX (1983).

 

NAME:

BORN:

FORMAL EDUCATION:

DEGREES RECEIVED:

APPENDIX B

Vita

 

Francesco John DeMayo

December 24, 1957
Staten Island, New York

Cornell University
Ithaca, New York, 1975-1979

Michigan State University
East Lansing, Michigan, 1979-present

Bachelor of Science (1979)
Cornell University
Ithaca, New York

Master of Science (1981)
Michigan State University
East Lansing, Michigan

 

 

    

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