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Uterine, oviductal, and conceptus responses to heat shock

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Uterine, oviductal, and conceptus responses to heat shock
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Malayer, Jerry Rhea, 1957-
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English
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xv, 302 leaves : ill., photos ; 29 cm.

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Subjects / Keywords:
Cattle ( jstor )
Embryos ( jstor )
Endometrium ( jstor )
Gels ( jstor )
Heat shock proteins ( jstor )
Heat stress disorders ( jstor )
Pregnancy ( jstor )
Proteins ( jstor )
Secretion ( jstor )
Shock heating ( jstor )
Animal Science thesis Ph. D ( lcsh )
Dissertations, Academic -- Animal Science -- UF ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1990.
Bibliography:
Includes bibliographical references (leaves 263-301).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jerry Rhea Malayer.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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UTERINE, OVIDUCTAL AND CONCEPTS RESPONSES
TO HEAT SHOCK


















BY

JERRY RHEA MALAYER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1990













ACKNOWLEDGMENTS


I would like to express my appreciation and gratitude to my

major professor, Dr. Peter J. Hansen, for encouragement and

support during my time at the University of Florida. His

enthusiasm and encouragement were essential to my successful

completion of this degree. I want to express my gratitude to Dr.

William W. Thatcher, who provided significant guidance and

support. I also want to thank the members of my supervisory

committee, Dr. Fuller W. Bazer, Dr. William C. Buhi, and Dr.

Daniel C. Sharp, III.

I feel very fortunate to have been associated with an

outstanding group of scientists and to have been exposed to a

program of research and graduate education which has few equals.

I shall always try to live up to the standards of excellence in

research displayed by persons in the reproductive biology group

at the University of Florida.

I want to thank everyone who contributed to the research

described in this dissertation. Dr. Jim Putney, Dr. Tim Gross,

John Pollard, Matt Lucy, Frank Michel, Boon Low, Dr. Claire

Plante, Dr. Steven Helmer, Marie Leslie, David Stephensen, and

Leslie Smith all made important contributions. I also want to






express appreciation to everyone in the Dairy Science Department

for making it a very pleasant place to work.

Last, but most importantly, I want to express my gratitude

to my wife, Cindy, for making many sacrifices so that I could

pursue my career goals.


iii













TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS ......................................... ii

LIST OF TABLES ................. ..................... vii

LIST OF FIGURES .................. ... ..... .......... x

ABSTRACT .................... .. ......... .......... xiv

CHAPTERS

1 REVIEW OF LITERATURE .................... 1

Introduction ........... .................... 1
Maintenance of Homeothermy ............... 4
Effects of Hyperthermia on Reproduction:
Effects at the Reproductive
Tract Level ......... ................... 32
Effects of Hyperthermia on Cells .......... 48
Objectives .... ........................... 71

2 DIFFERENCES BETWEEN BRAHMAN AND HOLSTEIN
COWS IN ALTERATIONS OF PROTEIN SECRETION
BY OVIDUCTS AND UTERINE ENDOMETRIUM
INDUCED BY HEAT SHOCK ..................... 75

Introduction .................... ......... 75
Materials and Methods .................... 76
Results .................................... 85
Discussion ...................... ......... 100

3 EFFECT OF DAY OF THE ESTROUS CYCLE, SIDE
OF THE REPRODUCTIVE TRACT AND HEAT SHOCK
ON IN VITRO PROTEIN SECRETION BY BOVINE
ENDOMETRIUM .............................. 106

Introduction ............................. 106
Materials and Methods .................... 108
Results .................................... 114
Discussion ............................... 123







4 SECRETION OF PROTEINS BY CULTURED BOVINE
OVIDUCTS COLLECTED FROM ESTRUS THROUGH
EARLY DIESTRUS ........................... 132

Introduction .............................. 132
Materials and Methods ....................... 133
Results ............................... ... 138
Discussion ................................. 146

5 HEAT STRESS-INDUCED ALTERATIONS IN
SYNTHESIS AND SECRETION OF PROTEIN
AND PROSTAGLANDIN BY CULTURED BOVINE
CONCEPTUSES AND UTERINE ENDOMETRIUM
COLLECTED AT DAY 17 OF PREGNANCY ......... 152

Introduction ............. ............... 152
Materials and Methods .................... 153
Results .................................... 161
Discussion ............................... 173

6 EFFECT OF IN VITRO HEAT SHOCK UPON
SYNTHESIS AND SECRETION OF
PROSTAGLANDINS AND PROTEIN BY
UTERINE AND PLACENTAL TISSUES OF
THE SHEEP ............................... 177

Introduction ............................ 177
Materials and Methods .................... 179
Results ....................................... 183
Discussion ............................... 189

7 REGULATION OF HEAT SHOCK-INDUCED
ALTERATIONS IN RELEASE OF
PROSTAGLANDINS BY UTERINE
ENDOMETRIUM OF COWS ..................... 199

Introduction ............................. 199
Materials and Methods .................... 201
Results .................. ................ 207
Discussion ............. .................... 212

8 MODULATION BY ALANINE AND TAURINE OF
HEAT SHOCK-INDUCED KILLING OF BOVINE
LYMPHOCYTES AND MOUSE PREIMPLANTATION
EMBRYOS .................................. 216

Introduction .......................... 216
Materials and Methods .................... 218
Results ...................... ............. 221
Discussion ................................. 235








9 EFFECTS OF EXOGENOUS ALANINE
UPON EMBRYONIC SURVIVAL DURING
MATERNAL HYPERTHERMIA AT TWO
STAGES OF PREIMPLANTATION
DEVELOPMENT .............................


Introduction .........
Materials and Methods
Results ..............
Discussion ...........


10 GENERAL DISCUSSION ......................

LITERATURE CITED ...................................

BIOGRAPHICAL SKETCH ...............................


242

242
244
247
252

258

263

302


....................
.... .... .... ....
....................
....................









LIST OF TABLES


Table Page

3-1. In-vitro synthesis and secretion
of radiolabeled, macromolecular
[3H]leucine by endometrial explants
obtained during early stages of the
estrous cycle ............................ .... 116

3-2. Amounts of individual radiolabeled
proteins secreted by cultured
endometrial explants from early
stages of the estrous cycle and
isolated from electrophoretic gels .......... 117

3-3. Effect of incubation temperature
upon secretion of individual
radiolabeled proteins by cultured
endometrial explants obtained
during eary stages of the estrous
cycle ............................................ 120

3-4. Side x temperature interactions
affecting secretion of individual
proteins from cultured endometrial
explants obtained during early
stages of the estrous cycle ................. 121

3-5. Day of cycle x side interactions
affecting secretion of individual
proteins from cultured endometrial
explants ............................ ......... 122

4-1. In vitro synthesis and secretion
of radiolabeled, macromolecular
[H]leucine by oviductal explants
obtained during early stages of
the estrous cycle ............................ 140

4-2. Amounts of individual radiolabeled
proteins secreted by cultured
oviductal explants from early
stages of the estrous cycle ................... 141


vii






5-1. Incorporation of [3H]leucine
into TCA-precipitable protein
in tissue and medium after
24 h of culture by conceptuses
and endometrium cultured at
homeothermic (39C) or heat-stress
(43*C) temperatures .......................... 162

5-2. Concentrations of prostaglandin
(PG) F and PGE2 in medium
supernatants collected after
24 h of culture of conceptuses
and endometrium at homeothermic
or heat-stress temperatures ................... 162

6-1. Probability values for effects
of individual sources of variation
upon secretion of PGE2, PGF and
['H] labeled protein and on
accumulation of tissue protein ............... 184

7-1. Prostaglandin F production
from cotyledonary prostaglandin
generating system incubated at
39C or 42*C in presence or
absence of endometrial prostaglandin
synthesis inhibitor ................ ..... .... 210

7-2. Release of PGF and PGE2 by uterine
and placental tissues collected at
Day 70 of pregnancy and incubated
at 39*C or 42C ................................ 210

8-1. Effects of alanine upon development
and eosin B staining of 4 to 8-cell
embryos 24 h after heat shock ................. 232

8-2. Effect alanine and taurine on
development and eosin B staining
of 4 to 8-cell embryos at 24 h
after heat shock ............................. 234

8-3. Effects of alanine and taurine on
development and eosin B staining
of 16-cell embryos 24 h after heat
shock .......................................... 236

9-1. Effect and ovulation of intraperitoneal
injection of D,L-alanine on litter size
following maternal hyperthermia .............. 249


viii






9-2. Effect of intraperitoneal injection of
D,L-alanine on embryos per CL following
maternal hyperthermia ........................ 250

9-3. Effect of intraperitoneal injection of
D,L-alanine on embryo mean weight of
individual concepts following maternal
hyperthermia ......................... ....... 251













LIST OF FIGURES


Figure Page

2-1. Secretion of radiolabeled,
nondialyzable macromolecules into
culture medium by oviductal explants ......... 87

2-2. Incorporation of [3H]leucine into
TCA-precipitable macromolecules
in tissue homogenates ........................ 88

2-3. Incorporation of [3H]leucine into
non-dialyzable macromolecules in
culture supernatants and incorporation
of [3H]leucine into TCA-precipitable
macromolecules by explants of oviductal
tissue from crossbred cows .................... 91

2-4. Secretion of nondialyzable
[3H]leucine-labeled macromolecules by
endometrial explants in the first 0 to 30,
30 to 60 and 60 to 90 min after onset
of heat shock ................................. 92

2-5. Incorporation of [3H]leucine into
TCA-precipitable macromolecules
in tissue of endometrial explants ............. 94

2-6. Electrophoretic analysis of oviductal
secretary proteins ............................ 95

2-7. Electrophoretic analysis of endometrial
secretary proteins ........................... 97

2-8, Electrophoretic analysis of endometrial
tissue proteins ............................. 98

2-9. Western blot analysis of the appearance
of inducible heat shock protein in
representative samples obtained 2.5 h
after the onset of heat shock treatment ....... 99







3-1. Representative fluorographs of
two-dimensional SDS-PAGE of
secretary proteins from bovine
endometrial explants obtained at
Days (a) 0 (estrus), (b) 2, (c) 5
and (d) 8 of the estrous cycle ................. 125

3-2. Representative fluorographs of
one-dimensional SDS-PAGE of
tissue proteins solubilized from
bovine endometrial explants
incubated at (a) 39 C or (b) 43 C ............. 126

3-3. Western blotting of bovine heat-shock
proteins ...................... ....... ..... ... 127

4-1. Representative fluorographs of 2-D
SDS-PAGE of secretary proteins from
cultured bovine oviducts obtained at
A) Day 0 (estrus), B) Day 2, C) Day 5
and D) Day 8 of the estrous cycle .............. 147

4-2. Representative fluorographs of 1-D
SDS-PAGE of de novo synthesized
proteins in culture supernatants
and tissue homogenates from cultured
bovine oviducts .............................. 148

5-1. Incorporation of [3H]leucine into
TCA-precipitable macromolecules in
culture medium by endometrial and
concepts tissues and incubated at
homeothermic or heat shock temperature ........ 164

5-2. Two-dimensional polyacrylamide gel
electrophoresis of proteins present
in endometrial and concepts tissues .......... 165

5-3. One-dimensional polyacrylamide gel
electrophoresis of concepts secretary
proteins ....................................... 166

5-4. One-dimensional polyacrylamide gel
electrophoresis of endometrial
secretary proteins ........................... 167

5-5. Electrophoretic profile of
[ H]leucine-labeled polypeptides
accumulated in concepts
culture medium after 24 h of
culture ...................................... 170







5-6. Immunoblotting of bovine trophoblast
protein-1 (bTP-1) released by
conceptuses during 24 h of culture ............ 171

5-7. Release of prostaglandins (PGF and PGE2)
into culture medium by endometrium
and conceptuses ................. ............ 172

6-1. Release of PGE2 from ovine tissue
explants of maternal (intercaruncular
and caruncular endometrium) and
fetal (cotyledon and chorioallantois)
tissues collected at Day 100 or Day
140 of pregnancy ............................. 186

6-2. Release of PGF from ovine tissue
explants of maternal (intercaruncular
and caruncular endometrium) and fetal
(cotyledon and chorioallantois) tissues
collected at Day 100 or Day 140 of
pregnancy ................. ............ ...... 187

6-3. Release of PGE2 (upper panels) and PGF
(lower panels) from ovine tissue
explants of maternal tissues from the
nongravid and gravid uterine horns
collected at Day 140 of pregnancy ............. 188

6-4. Secretion of [3H]leucine-labeled
macromolecules from ovine tissue
explants of maternal (intercaruncular
and caruncular endometrium) and fetal
(cotyledon and chorioallantois) tissues
collected at Day 100 or Day 140 of pregnancy .... 190

6-5. Incorporation of [3H]leucine into
macromolecules present in tissue
homogenates of ovine tissue explants
of maternal (intercaruncular and
caruncular endometrium) and fetal
(cotyledon and chorioallantois)
tissues collected at Day 100 or
Day 140 of pregnancy ........................... 191

6-6. Secretion and tissue incorporation
of [3H]leucine-labeled
macromolecules from ovine
tissue explants of maternal
tissues from the nongravid and
gravid uterine horns collected
at Day 140 of pregnancy ....................... 192


xii






6-7. Representative fluorographs of
[ H]leucine-labeled macromolecules
in ovine uterine and placental
tissues resolved by l-D SDS PAGE ............. 194

7-1. Effect of PAF and incubation temperature
on secretion of PGF and PGE2 by bovine
endometrial explants ...................... 209

7-2. Dose-response of PGF secretion
at 39*C and 42*C to increasing
concentrations of PAF ........................ 211

8-1. Lymphocyte survival after 1 h
heat shock at 45"C ......................... 223

8-2. Dose-response effects of
alanine and taurine ......................... 224

8-3. Effect of D-alanine upon
lymphocyte survival ......................... 226

8-4. Effect of L-alanine upon
lymphocyte survival during
prolonged heat shock in vitro ............... 227

8-5. Effect of heat shock and
L-alanine upon mouse embryo
development in vitro ....................... 229

8-6. Effect of L-alanine upon development
and viability during heat shock in
vitro ..... .................... ........... 230

9-1. Incorporation of D,L-l- 4C]alanine
into tissues following intraperitoneal
injection .... .............................. 253


xiii












Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


UTERINE, OVIDUCTAL AND CONCEPTS RESPONSES TO HEAT SHOCK

by

Jerry Rhea Malayer

May 1990


Chairman: Peter J. Hansen
Major Department: Animal Science



Direct effects of elevated temperature on maternal repro-

ductive tract tissues and conceptuses were examined in vitro.

Protein secretion by bovine endometrial and oviductal explants

varied with day of estrous cycle and secretion rate increased in

response to elevated temperature at estrus, but not during the

remainder of the estrous cycle and early pregnancy. Protein

secretion by explants of ovine utero-placental tissues in late

pregnancy was not affected by elevated temperature. All bovine

and ovine tissues examined produced heat-shock proteins identi-

fied by SDS-PAGE and immunological methods.

Secretion of 7 peptides was reduced by heat shock only in

explants from the uterine horn ipsilateral to the corpus luteum.

Protein secretion by explants of the ipsilateral oviduct was more

sensitive to heat shock. Endometrial and oviductal tissue
xiv






responses of thermotolerant Brahman cattle were compared to

tissues from more heat-sensitive Holsteins. Variation in

responses of tissues to heat shock was consistent with greater

thermal tolerance of Bos indicus cattle.

Heat shock disrupted events critical to luteal maintenance

at day 17 of pregnancy in the cow, increasing endometrial

secretion of luteolytic PGF and depressing the concepts signal

for luteal maintenance. Increased PGF secretion was not due to

incapacity of regulatory proteins or increased cyclooxygenase

activity. Heat shock increased PGF secretion at day 17 of the

estrous cycle and pregnancy, but had no effect on PGF release

from other tissues examined. Heat shock depressed PGE2 release

from caruncular and intercaruncular endometrium collected from

pregnant ewes.

Using murine embryos in vitro, heat shock was found to block

development and cause increased mortality. This could be

partially blocked by alanine and taurine. In vivo heat shock of

pregnant mice at times before or after development of embryonic

capacity to produce heat-shock proteins in response to heat

stress resulted in equally reduced numbers of embryos per corpus

luteum. There was no benefit of intraperitoneal injection of

alanine although alanine was sequestered in the oviduct in

relatively high amounts per milligram of tissue.

In conclusion, direct thermal effects on maternal and

concepts tissues may act in addition to maternal systemic

perturbations during hyperthermia.













CHAPTER 1
REVIEW OF THE LITERATURE


Introduction

Efficiency of reproduction in animals is sensitive to various

environmental factors including temperature, nutrient avail-

ability, photoperiod and social interactions. Pregnancy rates

are greatly reduced during periods of elevated environmental

temperature in various species, including cattle (Stott and

Williams, 1962; Postenetal., 1962; Badinga et al., 1985), sheep

(Dutt et al., 1959; Dutt, 1963), pigs (Warnick et al., 1965;

Edwards et al., 1968) and rabbits (Ulberg and Sheean, 1973;

Wolfenson and Blum, 1988). Adverse effects of elevated ambient

temperature on fertility may be caused by effects on various key

events of reproduction in mammals. There are negative effects

on gamete development (Bellve, 1973; Lenz et al., 1983; Baum-

gartner and Chrisman, 1988; Putneyet al., 1989a) and on survival

of the early cleavage-stage embryo during the period after

fertilization (Dutt et al., 1959; Dunlap and Vincent, 1971;

Elliott and Ulberg, 1971; Ulberg and Sheean, 1973; Gwazdauskas

et al., 1973; Putney et al., 1988a). Adverse effects of high

temperature can also be seen during later stages of preimplan-

tation development (Omtvedt et al., 1971; Monty, 1984; Biggers

et al., 1987; Geisert et al., 1988; Wettemann et al., 1988),






2

during embryonic organogenesis (Trujano and Wrathall, 1985;

Mirkes, 1987) and during mid- to late-gestation (Alexander and

Williams, 1971; Omtvedt et al., 1971; Collier et al., 1982; Lewis

et al., 1984).

The effects of climate and season upon productivity and pro-

lificacy of animals were observed by ancient peoples. No doubt

the survival of many groups of early people depended on an

understanding of the seasonal changes in their environment and

of the life-cycles of the animals upon which they depended. Much

knowledge of the Greek classical period has come down to us

through the writings of historians and philosophers and persons

such as Hippocrates (ca. 400 B.C.), who wrote:

For knowing the changes of the seasons, the risings and
settings of stars, how each of them takes place, he will be
able to know beforehand what sort of year is going to
ensue.... And if it shall be thought that these things
belong rather to meteorology, it will be admitted, on second
thoughts, that astronomy contributes not a little, but a
very great deal, indeed, to medicine.
A city that is exposed to hot winds... and the following
diseases are peculiar to the district: in the first place,
the women are sickly and subject to excessive menstruation;
then many are unfruitful from disease, and not from nature,
and they have frequent miscarriages....
but such parts of the country as lie intermediate hebsm
the heat and cold are best supplied with fruits and trees...
the cattle also which are reared there are vigorous,
particularly prolific, and bring up young of the fairest
description.... In: On Airs, Waters, and Places. Great
Books of the Western World, W. Benton (ed.). Vol. 10,
Encyclopedia Britannica, Inc., Chicago, pp. 9-18, 1952.

Aristotle was an astute observer of nature whose writings

on a vast array of topics ranging from logic and politics to life

and death (including treatises on animal reproduction and

meteorology) influenced science for over a thousand years.






3

Aristotle recognized the periodicity of sexual activity of

domestic animals; he wrote, for example, that the cow would

accept the bull every 21 days. He described the cervical mucus

of the cow in estrus and related that to the optimal breeding

period. He ascribed this mucus to menstrual discharge, on the

assumption that all female animals were alike in this respect.

The concept of heat was integral to Aristotle's view of the

underlying mechanisms of life:

In a similar fashion as the fish move their gills,
respiring animals with rapid action raise and let fall the
chest according as the breath is admitted or expelled. If
air is limited in amount and unchanged they are suffocated,
for either medium, owing to contact with the blood, rapidly
becomes hot. The heat of the blood counteracts refrigera-
tion and, when respiring animals can no longer move the lung
or aquatic animals their gills, whether owing to disease or
old age, their death ensues.
The source of life is lost to its possessors when the
heat with which it is bound up is no longer tempered by
cooling, for as I have often remarked, it is consumed by
itself. In: On Life and Death. Great Books of the Western
World, W. Benton (ed.). Vol. 8, Encyclopedia Britannica,
Inc., Chicago, pp. 714-726, 1952.

Aristotle (ca. 330 B.C.)

The concept of heat as kinetic energy arose in the mid-19th

century when Browne recognized the constant motion of particles

in fluid media (Cossins and Bowler, 1987), thereafter termed

"Brownian motion". About the same time, Ludwig Boltzman con-

ceived of heat as a random motion of atoms (Bronowski, 1973).

Thermal energy is now defined as the energy of motion of the

atoms and molecules of which matter is composed (Cossins and

Bowler, 1987).






4

It is the input of energy, in the form of increased atomic and

molecular motion, into a thermodynamically-balanced biological

system, and the changes brought about by such an input, that will

be the focus of this review. Several levels of integration will

be considered, beginning in general terms with maintenance of

homeothermy in the whole animal including mechanisms of heat

exchange, thermoregulation and responses to hyperthermia. Next,

thermal effects at the tissue level, specifically tissues

related to reproductive function, will be considered. Finally,

thermal effects upon cellular functions will be discussed

including thermal effects on dynamics of their constituent

macromolecules.


Maintenance of Homeothermy

We have defined heat as the kinetic energy of atoms and mole-

cules; temperature describes the intensity of the kinetic motion

of the atoms and molecules of a system. Temperature has no

physical units and must be measured by reference to an arbitrary

scale. Thus temperature is useful only to predict the direction

of heat flux along a thermal gradient.

Cowles (1962) classified vertebrate animals according to

characteristics of their body temperature (Tb) regulation.

Poikilotherms, so called cold-blooded animals, are those such as

reptiles and amphibians whose Tb is labile and varies with

changes in ambient temperature (Ta) ; it is important to note that

while Tb is labile, Ta and Tb are not equal. Homeotherms have







5

evolved mechanisms to maintain a relatively constant Tb even

though Ta might vary. Birds and mammals are the principal

examples of such animals in nature. The source of heat used to

establish Tb is the basis of a parallel classification. Ecto-

therms depend upon heat sources external to the body, such as

solar radiation, air or water temperature, to set Tb. Endo-

therms, on the other hand, are animals whose Tb is based on energy

derived from cellular metabolism. Many ectotherms have evolved

strategies for balancing low metabolic heat with tight control

of heat exchange with the environment to maintain Tb within a

fairly narrow range. Cowles (1962) emphasized that most, if not

all, ectothermic animals have specific thermal preferences.

Dreisig (1984) described behavior patterns of ectotherms as

alternative shuttling between microclimates, i.e., alternative

sun to shade shuttling, designed to achieve preferred Tb. As the

Ta increases during the day, there is a basking-activity phase in

which preferred Tb is attained. This is followed by a control

phase in which maintenance of Tb is accomplished through behav-

ioral responses such as basking, alterations of activity level

and sun to shade shuttling.

Endotherms have a relatively higher level of metabolic heat

production coupled with mechanisms for its conservation, which

results in maintenance of Tb greater than Ta. Mammals and birds

have developed complex mechanisms for regulation of heat

exchange with the environment to maintain a constant internal

temperature.








Heat Exchange

Animals exchange heat with the environment in several ways.

Three modes of energy transfer depend upon thermal gradients and

can therefore be sensed by a thermometer (and are thus referred

to as sensible heat flow) are conduction, convection and radia-

tion. Two other routes of energy transfer are evaporation and

condensation. These occur along a vapor-pressure gradient, not

a thermal gradient, and are called insensible or latent heat

flows. As water molecules move from the liquid to the gaseous

state, their kinetic energy of motion does not increase, thus

temperature does not change. Energy is released through

breakdown of the noncovalent bonds which hold the water molecules

together.

Radiant-energy transfer is the result of conversion of heat

energy into electromagnetic-wave energy by the emitter, passage

of these waves (requiring no medium) through space and reconver-

sion of electromagnetic-wave energy into heat energy in the

absorber. This transfer is dependent upon several physical

characteristics of the emitter and absorber bodies; their

emissivity, reflectivity and absorbtivity. The net radiant-

thermal flux can be expressed mathematically by describing these

physical characteristics of emitter and absorber as well as

surface area of the animal, the surface and ambient environmental

temperatures, and an expression called the Stefan-Boltzman

constant which accounts for the radiant emissive power density

(a feature of the surface temperature of the emitter).






7

Conductive-heat exchange is transfer of heat through a medium

without material movement or transfer. This transfer is based

upon movement of higher energy molecules into collisions with

lower energy molecules; when they collide some of the energy is

transferred. The net conductive-thermal flux can be described

mathematically using the conductive surface area, the thermal

conductivity of the substance (which is dependent on the density

of molecules in the conducting material), and the environmental

temperature at the surfaces of the conducting substances.

Heat exchange by convection occurs by movement of streams of

molecules from a warm to a cool place. Convective heat flux is

modelled using convective-surface area, ambient temperature and

the convection coefficient which describes the thermal conduc-

tivity and thicknesses of boundary layers on surfaces where

convective movement of the medium cannot reach.

Animals may lose heat by evaporation of water from the skin

and respiratory tract surfaces. Evaporation occurs when the

molecules of a liquid acquire sufficient kinetic energy to

overcome cohesive forces in the liquid. The removal of energy

from the liquid in the form of breakdown of the noncovalent

interactions holding the water molecules together results in

heat loss. The evaporative heat flux is dependent upon the

latent heat of water at the surface temperature of the liquid,

the surface area of the liquid, the evaporation diffusion

coefficient (which is based on air velocity at the evaporative

surface), and the vapor-pressure gradient.








Regulation of Body Temperature

Homeotherms regulate their Tb within a relatively narrow

range called the thermal-comfort zone or zone of thermo-

neutrality (Curtis, 1981; Bligh, 1984). Within this range of

temperature, the animal does not undergo temperature-induced

changes in metabolic rate and does not need to expend energy to

maintain body temperature. Regulation of core temperature

within the thermal-comfort zone is accomplished primarily by

autonomic control of the tone of the cutaneous vasculature and

by behavioral means such as changes in posture to alter heat

exchange with the environment (Bligh, 1984). Thermoregulation

is accomplished through feedback and feedforward interactions

between thermal receptors and thermal effectors. Integration

between these receptors and effectors occurs in the thermo-

regulatory center of the hypothalamus (Curtis, 1981; Stitt,

1983; Bligh, 1984; Cossins and Bowler, 1987).

The Set Point

Regulation of thermal-energy flux requires a high degree of

precision in order to control errors which might result in

drifting away from the preferred Tb. This has led to the concept

of a homeostatic regulator of Tb. The so-called thermal "set

point" is maintained by assessing input of thermal energy, both

directly by temperature-sensitive neurons in the central nervous

system and via neural input from thermal receptors in the

periphery (Simon, 1981; Curtis, 1981; Stitt, 1983; Bligh, 1984;

Cossins and Bowler, 1987), and adjusting heat-loss or heat-gain







9

effectors accordingly. The set point is the physiological state

at which the sum of all inhibitory and stimulatory inputs cancel

each other out (Bligh, 1984).

Theories of set-point homeostasis depend mechanistically on

the concept that Q1, for firing rate in the neurons of the

hypothalamic thermoregulatory center differs for heat and cold

and that several populations of neurons are present, some

sensitive to cold, some sensitive to heat. The Qo1, or the van't

Hoff effect, describes the change in rate of biological processes

with changing temperature and can be represented by the expres-

sion

Q10 = kTo+ 10/ kTo
where kTo = reaction velocity at initial temperature,

and kTO 10 = reaction velocity after 10 C increase.

In general, the Q10 for biological systems in the range of

temperature common to mammals is 2, although this prediction is

not linear over a wide range of temperature (Cossins and Bowler,

1987). Temperature-sensitive neurons in the hypothalamus and

spinal cord of mammals have been demonstrated to alter their

activity in response to local warming and cooling (Baldwin and

Ingram, 1966; Whittow and Findlay, 1968; Forsling et al., 1976) .

Cattle respond to hypothalamic warming by panting (Whittow and

Findlay, 1968), and this response varies directly with Ta, a

result in accordance with the idea of both central and peripheral

neural inputs. Hypothalamic warming also induces changes in

thermoregulatory behavior in pigs (Carlisle and Ingram, 1973)






10
and sheep (Whittow and Findlay, 1968). Studies in humans

(Benzinger, 1969), dogs (Chatonnet, 1983) and pigs (Baldwin and

Ingram, 1966) have shown that responses to altered brain

temperature vary directly with skin temperature, an indication

of an integration of both peripheral and central sites of thermal

responsiveness. In a similar manner, cooling the preoptic area

of pigs during elevation of Ta blocks endocrine responses to

hyperthermia (Forsling et al., 1976) and alters thermoregulatory

behavior (Baldwin and Ingram, 1966), and integration with

peripheral receptors modulated thermoregulatory responses to

hypothalamic cooling (Baldwin and Ingram, 1966). Baldwin and

Parrott (1984) placed electrodes in the dorsal preoptic area of

sheep and examined the effect of electrical stimulation on

thermoregulatory responses of conscious animals. Electrical

stimulation elicited a concurrent thermoregulatory response

characterized by increased respiration rate, vasodilation of

peripheral blood vessels and lowered Tb. Stimulation of the

dorsal preoptic area inhibited shivering when ewes were exposed

to 5C. After the ewes had been shorn, resulting in exposure of

cold receptors in the skin, thermoregulatory response to

electrical stimulation at 5C was reduced; a further indication

of the integration of peripheral and central response elements.

Large changes in the temperature of the hypothalamus and spinal

cord of active birds have been recorded without activation of

thermoregulatory mechanisms however (Simon, 1981) .







11

Catecholamines (adrenaline, noradrenaline) and 5-hydroxy-

tryptamine can all induce change in Tb when injected into

cerebral ventricles of the cat (Feldberg and Myers, 1963).

Experiments of this type led to the concept of a neurochemical

set point (Feldberg and Myers, 1963). However, results of a

number of experiments of this type involving injection of

monoamines into cerebral ventricles of several species were

inconsistent. By comparing the neurotransmitter-induced changes

at different Ta in order to challenge the thermoregulatory

system, it was determined that the exogenous neurotransmitters

inhibited whichever heat-flux system was being driven at the

time. At low Ta, 5-hydroxytryptamine or noradrenaline injected

into cerebral ventricles of sheep caused lower heat production

and lower rectal temperature (Bligh, 1979). At high Ta, nora-

drenaline injection caused reduced respiration rates and

increased Tb (Bligh, 1979). Denervation of the medullary

adrenal, a major source of catecholamines, abolished thermoregu-

latory responses to hypothalamic warming in cattle (Robertshaw

and Whittow, 1966), demonstrating the importance of catecho-

lamines in thermoregulation.

Hyperthermia

As Ta increases toward the upper critical temperature, i.e.,

the temperature at which metabolic rate begins to increase,

mechanisms to facilitate heat loss are initiated. This point

will vary among animals of a species. Kibler (1957) found that

metabolic rate of Brahman heifers exposed to elevated ambient






12

temperature (80F) was lower than that of Shorthorn heifers.

Reduction of tissue insulation by vasodilation and increasing

surface area through changes in posture are the earliest

manifestations of heat-loss mechanisms (Curtis, 1981). These

are attempts to increase conductive- and convective-heat

exchange with the environment. Additionally, behavioral changes

such as seeking shade (Seath and Miller, 1946; Robertshaw, 1986)

and reduced daytime activity (Payne et al., 1951) help to

increase radiant-heat loss. The upper critical temperature is

defined as the point at which metabolic energy production begins

to increase. Alternatively, Nichelmann (1983) described studies

in birds and mammals demonstrating that the relationship between

Ta and the metabolic energy level of animals is a parabola; the

vertex of the parabola is referred to as thermoneutral tempera-

ture. In domestic animals, where even slight diversion of

metabolic energy away from productive activity manifests itself

in lowered productivity, such as in the lactating dairy cow

(Thatcher and Collier, 1982), thermoneutral temperature is a

useful concept.

As Ta continues to rise and thermal gradients reduce heat

exchange away from the animal, active heat-loss mechanisms must

be initiated. Sweating and panting are modes of evaporative

heat loss which require no thermal gradient and thus are critical

when thermal gradients favor heat flux toward the animal. These

active heat-dissipation mechanisms require the expenditure of

energy which slightly increases the metabolic heat load of the







13

animal in addition to the larger Q10 increase in metabolic heat

generation (Whittow and Findlay, 1968; Ames et al., 1971). As

the Ta continues to rise, a point is reached where the ability to

dissipate heat via evaporative means is maximal. As the upper

critical temperature threshold is exceeded, metabolic heat

production exceeds the capacity of the animal to dissipate its

heat load and it becomes hyperthermic. The resulting rise in Tb

results in a further rise in metabolic rate measurable as

increased 02 consumption (Whittow and Findlay, 1968; Ames et al.,

1971) a result of the QIo effect. The increase in metabolic rate

results in a further increase in heat production which further

increases metabolic rate; the resulting positive feedback effect

sequence has been called spiralling hyperthermy (Curtis, 1981) .

Physical and Chemical Means to Maintain Homeothermy

The physiological responses of cattle to hot environments

have been the subject of many reviews over the past 30 years

(Yeck, 1959; Bianca, 1965; Thatcher, 1974; Thatcher and Collier,

1982, 1986; Gwazdauskas, 1985; Beede and Collier, 1986), an

indication of the tremendous impact of hyperthermia upon

productivity in cattle. Maintenance of homeothermy is given such

a high priority that other physiological systems are challenged

(Thatcher and Collier, 1982), and this homeokinesis results in

depression of many productive traits such as growth, lactation

and reproduction. The prioritization of physiological systems,

though detrimental in terms of agricultural production, is a

characteristic with evolutionary advantages for mammals.






14

Homeokinesis to regulate Tb in response to increasing Ta can

be divided into two main categories; physical and chemical.

Physical adjustments are attempts to promote conductive,

convective and evaporative heat flux. There is dilation of

cutaneous arterial-venous anastomoses to reduce tissue and body-

cover insulation and carry heat from the body core to the

periphery, thus promoting a thermal gradient. Additionally,

increased respiration rate and thermal sweating promote evapora-

tive heat flux. Chemical adjustments are attempts to reduce

generation of metabolic heat and to support the physical

adjustments. Nutritional changes as well as alterations in water

turnover and intake, electrolyte balance, and osmoregulation

occur when Tb increases; many of these changes are integrated by

alterations in endocrine activity.

Cardiovascular changes

At rest, cutaneous blood flow accounts for a very small

fraction of cardiac output (Hertzman, 1959). Increased skin

temperature results in increased cardiac output (Bianca and

Findlay, 1962; Ingram and Whittow, 1963; Whittow, 1965; Marple

et al., 1974), primarily a result of increased heart rate because

stroke volume decreases (Whittow, 1965). Elevated temperature

also results in dilation of cutaneous arterial-venous anas-

tomoses and capillaries (Whittow, 1965), and increased cutaneous

blood flow (Hales, 1983; Hales et al., 1984). Peripheral

vascular tone is under sympathetic neuronal control, and regula-

tion occurs chiefly in the periphery (Brown and Harrison, 1981).






15

Movement of blood from the body core to the skin results in

increased skin temperature, creating a sensible heat-loss

gradient that allows thermal energy to move toward the environ-

ment and away from the animal. In particular, blood flow to the

extremities, i.e., the legs, tail, ears, snout and dewlap in

cattle, increases during periods of elevated temperature

(Whittow, 1962), while abdominal and thoracic blood flow remain

relatively constant. Alteration of blood flow patterns affects

organ perfusion, such is the case in heat-stress-induced

decrease in uterine blood flow (Gwazdauskas et al., 1973;

Alexander and Williams, 1971; Brown and Harrison, 1981).

Seasonal changes in blood volume occur in humans (Yoshimura,

1958), and warmer seasons are characterized by increased blood

volume in animals (Dale et al., 1956; Bianca, 1957; Whittow,

1965).

Changes in respiration rate

An important response to elevated temperature is increased

respiration rate. Respiration rate may increase from 30 to 300%

in pigs, sheep, goats and cattle (Riek and Lee, 1948; Appleman

and Delouch, 1958; Judge et al., 1973; Baldwin and Parrott,

1984). Initially, increased respiration rate is characterized

by rapid, shallow breathing resulting in increased respiratory

volume (liters per min) and lower tidal volume (liters per

breath), while not affecting alveolar gas exchange (Bianca and

Findlay, 1962; McDowell et al., 1969). The purpose of this

panting is increased evaporative heat flux from the respiratory






16

tract (Whittow and Findlay, 1968; Ames et al., 1971). Findlay

(1956) determined that a rectal temperature of 40.5C was the

threshold for increased respiration rate in calves. At severe

hyperthermia, the respiratory pattern may change to a lower-

frequency, higher-tidal-volume pattern called second-stage

breathing (Bianca and Findlay, 1962). During second-stage

breathing, the potential for blood alkalosis occurs (Brown and

Harrison, 1981) as the increased alveolar ventilation rate

results in decreased blood pCO2 and increased blood pH (Dale

and Brody, 1954; Bianca and Findlay, 1962; Judge et al., 1973;

Brown and Harrison, 1981).

One consequence of increased respiratory rate is increased

thermal energy production due to utilization of metabolic

energy. Oxygen consumption of hyperthermic cattle (Tb = 41.5"C)

increased 67% (Whittow and Findlay, 1968). The increase in 02

consumption is not linear with increasing rectal temperature,

however (Whittow and Findlay, 1968). By observing this and other

aspects of increased 02 consumption, Whittow and Findlay (1968)

and Ames et al. (1971) determined that panting in the cow and

sheep accounted for only a small portion (about 10%) of the

increase in metabolic energy utilization. Ames et al. (1971)

concluded that most of the increase in 02 consumption above the

upper critical temperature was due to the Q10 effect.

Changes in physiological fluids

Water intake of animals exposed to elevated temperature

increases as both volume and frequency of drinking increase






17

(Appleman and Delouch, 1958; Johnson and Yeck, 1964; Kelley et

al., 1967; McDowell et al., 1969; Gengler et al., 1970; Wettemann

et al., 1988). Influx of cool water provides immediate relief

from hyperthermia as heat can be exchanged down a thermal

gradient from the body to the contents of the gut, whereas intake

of warm water elicits no such immediate effect (Bianca, 1964).

Total body water content increases in water buffaloes and

Friesian cattle during the summer (Kamal and Seif, 1969). Blood

volume increases in cattle exposed to elevated temperature (Dale

et al., 1956; Bianca, 1957; Whittow, 1965); however, when water

intake is restricted during hyperthermia, osmolality of the

blood serum increases (El-Nouty et al., 1980) Indirect evidence

of increased blood volume, in the absence of a pathological

state, is a reduction of the volume of packed red blood cells per

unit volume of whole blood, i.e., the hematocrit. Dale et al.

(1956) measured both blood volume, using dye-dilution methods,

and hematocrit; blood volume increased and hematocrit decreased

in heat-stressed heifers. The hematocrit is lower during

hyperthermia in pigs (Forsling et al., 1976) goats (Appleman and

Delouch, 1958), heifers (Dale et al., 1956) and lactating cows

(McDowell et al., 1969).

Skin evaporation increases during exposure to elevated

temperature (Riek and Lee, 1948; Joshi et al., 1968; McDowell et

al., 1969; Ames et al., 1971) and there is increased salivation

and licking (Riek and Lee, 1948). Animals such as sheep, goats,

horses and cattle have sweat glands associated with the hair






18

follicles called epitrichial sweat glands (Allen and Bligh,

1969). These glands are surrounded by a layer of myoepithelium

which is stimulated to contract by epinephrine and norepi-

nephrine; the glands actively secrete fluid in response to

increasing skin temperature as well as in response to adrenergic

stimulation, though no direct innervation has been demonstrated

(Joshi et al., 1968; Allen and Bligh, 1969). Sweating can occur

due to increased glandular secretion rate of fluid to the skin

surface (bulk flow) or due to contractions of myoepithelial cells

to expel fluid (Allen and Bligh, 1969). Allen and Bligh (1969)

examined patterns of sweating in several species of domestic

animals. Sheep and goats do not sweat continuously, but rather

in bursts, apparently in response to stimulation of the myoepi-

thelium. This stimulation was mimicked with injections of

adrenaline (Allen and Bligh, 1969). Cattle exhibit continuous

sweating with periodic increases in discharge, also a result of

stimulation of myoepithelial contractions (Joshi et al., 1968;

Allen and Bligh, 1969). The skin secretions of cattle contain

sodium, potassium, magnesium, chloride, phosphate, lactate and

nitrogen (Joshi et al., 1968; Jenkinson and Mabon, 1973; Singh

and Newton, 1978) in concentrations greater than in blood plasma.

Electrolytes are actively transported into the duct lumen

creating an osmotic gradient which causes water to move into the

lumen; as the fluid moves through the duct on its way to the skin

surface, the electrolytes may be recovered from the fluid and

conserved (Guyton, 1981). In contrast to humans which secrete







19

sweat high in sodium (Guyton, 1981), the secretions of cattle

contain 3 to 5 times as much potassium as sodium (Johnson, 1970).

Water is conserved by increased reabsorption in the kidney

as a result of increased antidiuretic hormone (ADH) concentra-

tion (Forsling et al., 1976; El-Nouty et al., 1980). Cattle

retain water during periods of heat stress, but in contrast to

nonruminants, urinary sodium concentration and urine osmolality

do not change significantly, this is a result of low aldosterone

concentrations (El-Nouty et al., 1980). During dehydration,

aldosterone concentrations do rise in cattle (El-Nouty et al.,

1980) During heat stress in nonruminants, the serum aldosterone

concentration is high and stimulates reabsorption of sodium from

the urine (Guyton, 1981). This further concentrates the urine,

which already has a low volume due to increased ADH activity.

The nonruminant utilizes sodium in the skin secretions and is

predisposed to conserve the body store for this purpose; the

extracellular concentration of sodium increases during warm

seasons (Yoshimura, 1958). The ruminant, on the other hand,

utilizes greater amount of potassium in the skin secretions

(Johnson, 1970) and sodium conservation is less critical.

Seasonal changes in water content and electrolyte concentra-

tions in body fluids occur in humans. Blood concentrations of

serum protein, sodium, chloride and potassium are lower in summer

(June, July and August) than in winter (December, January,

February) in humans (Yoshimura, 1958). The sodium to potassium

ratio is higher in summer as is total blood volume, serum volume






20

and total sodium in extracellular fluid (Yoshimura, 1958), the

result of a sodium-conserving mechanism.

Changes in nutritional patterns

Feed intake is reduced in response to elevated temperature

in cattle, leading to reduced heat generation during ruminal

fermentation and to lower body metabolism in cattle (Johnson and

Yeck, 1964; McDowell et al., 1969; Gengler et al., 1970; Baile

and Forbes, 1974), goats (Appleman and Delouch, 1958) and sheep

(Cartwright and Thwaites, 1976). Dry matter intake begins to

decline in lactating cattle at ambient temperature of about 25*

to 27"C (Beede and Collier, 1986), possibly as a result of

depression of the hypothalamic appetite center (Baile and

Forbes, 1974). Alternatively, reduced gut motility and rate of

feed passage might lead to depressed appetite (Beede and Collier,

1986) and alteration of blood flow patterns away from the

alimentary tract may reduce nutrient absorption. When feed

intake of cattle was controlled by intraruminal infusion, the

ratios of volatile fatty acids were altered by elevated tempera-

ture as acetic acid content of the rumen increased and propionate

concentration decreased (Kelley et al., 1967). When feed intake

declines, so do fatty acid concentrations (Gengler et al., 1970) .

In lactating cattle, the decrease in digestible energy intake was

exceeded by the decrease in milk energy output, indicating that

maintenance requirements had increased during heat stress

(McDowell et al., 1969). Increased maintenance requirements

coupled with reduced intake of digestible energy and altered







21

volatile fatty acid concentrations leads to lowered productivity

of lactating or growing cattle.

Changes in endocrine function

Short-term exposure to elevated temperature results in

increased concentrations of catecholamines in the blood of

cattle (Robertshaw and Whittow, 1966; Alvarez and Johnson,

1973). Removing the neural link between the hypothalamus and the

adrenal medulla abolishes this increase in catecholamines and

induction of heat-loss responses during hypothalamic warming

(Robertshaw and Whittow, 1966). Increased concentrations of

catecholamines result in increased 02 consumption in cattle

(Whittow and Findlay, 1968).

Many endocrine changes during hyperthermia are consistent

with reduction of activities which might generate excess

metabolic heat. Thyroid activity is reduced during hyperthermia

(Brooks et al., 1962; Bianca, 1965; Thompson, 1973; El-Nouty et

al., 1976; Collier et al., 1982) as a result of depressed release

of thyroid-stimulating hormone from the anterior pituitary

(Krulich et al., 1976). Ruminal heating in heifers results in

lower thyroid activity and metabolic heat production, as

measured by indirect calorimetry (Yousef et al., 1968). Reduced

thyroid activity results in lowered 02 consumption as metabolic

rate declines (Guyton, 1981).

Short-term heat exposure causes increased secretion of growth

hormone (GH) in cattle (Mitra and Johnson, 1972) possibly through

neural stimulation of the hypothalamus as a result of increased






22

skin temperature. The rise in GH concentration in the blood

preceded the rise in rectal temperature; additionally, the

decline of GH at the end of the 4 h heat exposure preceded the

decline in rectal temperature (Mitra and Johnson, 1972). Long-

term exposure to elevated temperature causes a depression in GH

secretion (Mitra et al., 1972). Heat stress increases turnover

rate of GH in pigs following an initial, transient rise in plasma

concentrations of GH (Marple et al., 1972). Depressed GH during

thermal stress may represent a mechanism to prevent hyperthermia

because injection of GH during hyperthermia in cattle resulted

in increased thyroid activity, increased 02 consumption, and

increased heart and respiration rate (Yousef and Johnson, 1966) .

In addition, blood concentrations of insulin are depressed by

heat stress (Kamal et al., 1970; Thompson, 1973).

Episodic and estradiol-induced surges of luteinizing hormone

(LH) secretion in ovariectomized ewes were not affected by

hyperthermia (Schillo et al., 1978) and there was a slight

depression in basal serum-concentrations of LH. Elevated

temperature depressed basal and peak LH concentrations in cyclic

heifers (Madan and Johnson, 1973; Miller and Alliston, 1973).

Frequency of LH peaks also was reported to decline in the summer

in cyclic cows (McNatty et al., 1984). Vaught et al. (1977) and

Gwazdauskas et al. (1981) found no effect of summer heat stress

on LH secretion in lactating cows or cyclic heifers, respec-

tively, however. Hyperthermia depresses LH secretion in hens







23

through depression of hypothalamic LH-releasing hormone

(Donoghue et al., 1989).

Hyperthermia results in increased prolactin secretion in

heifers (Wettemann and Tucker, 1974) and ovariectomized ewes

(Schillo et al., 1978). Serum prolactin concentrations are

elevated during the summer (Koprowski and Tucker, 1973) and the

prolactin release following injections of thyroid-stimulating

hormone is greater in heat-stressed cattle (Wettemann and

Tucker, 1974; Roman-Ponce et al., 1981). Schillo et al. (1978)

found that the hyperthermia-induced rise in prolactin secretion

preceded the rise in rectal temperature, indicating that neural

stimulation, perhaps as a result of increased skin temperature,

is responsible for the increased secretion of prolactin.

Short-term hyperthermia results in increased concentrations

of circulating glucocorticoids secreted from the zona fasiculata

and zona reticularis of the adrenal cortex in response to

adrenocorticotropic hormone (ACTH) in lactating and nonlactating

cyclic cows (Christison and Johnson, 1972; Alvarez and Johnson,

1973), dogs (Chowers et al., 1966) and pigs (Marple et al., 1972;

1974) Local heating of the preoptic area, exposure to elevated

temperature and injection of pyrogens (lipopolysaccharide) all

caused increased cortisol release from the adrenal gland in dogs

(Chowers et al., 1966) Cortisol release in response to exposure

to elevated temperature preceded the rise in rectal temperature

(Chowers et al., 1966). Chronic hyperthermia causes depression

of glucocorticoid secretion. Glucocorticoid concentrations






24

after 24 h of heat stress were below pretreatment values in

nonlactating cows (Christison and Johnson, 1972; Alvarez and

Johnson, 1973; Abilay and Johnson, 1975), lactating cows (Stott

and Robinson, 1970) and pigs (Marple et al., 1972). Gluco-

corticoid release in response to ACTH injection is lower in heat-

stressed cattle (Gwazdauskas et al., 1981; Roman-Ponce et al.,

1981). In pigs, the clearance rate of ACTH is decreased during

hyperthermia and although ACTH levels may rise, glucocorticoid

secretion is depressed (Marple et al., 1974). Chronic stress

elicits specific responses to heat stress such as reduction of

calorigenic hormones, i.e., glucocorticoids, thyroxine, GH and

insulin.

The excretion of water by the kidney is controlled primarily

by antidiuretic hormone (ADH) (Houpt, 1977). Antidiuretic

hormone is released from the hypothalamus in response to

increases in plasma osmolarity and exerts its effect on the cells

lining the collecting tubules of the kidney to stimulate

reabsorption of water from the urine (Houpt, 1977). The result

of increased water reabsorption is decreased plasma osmolarity.

Secretion of ADH is elevated in cattle during periods of exposure

to elevated temperature (El-Nouty et al., 1980).

Sodium excretion is regulated by aldosterone (Houpt, 1977).

Aldosterone is secreted by the cells of the zona glomerulosa of

the adrenal cortex in response to stimulation by products of

renin metabolism, i.e., angiotensin, which is activated by low

sodium concentrations in serum (Houpt, 1977). Aldosterone acts







25

in the kidney at the level of the distal tubule to stimulate

reabsorption of sodium from the urine. If ADH is present, the

osmotic gradient across the tubule (a result of the [Na+]

difference) causes increased water reabsorption out of the lumen

of the collecting duct (Houpt, 1977). During hyperthermia,

aldosterone is depressed in ruminants if water availability is

adequate (El-Nouty et al., 1980). If water intake is limited,

there is a rise in aldosterone secretion (El-Nouty et al., 1980).

In humans there is sodium loss during periods of elevated

temperature, in skin secretions as well as urinary excretion,

resulting in lower serum concentrations of sodium; this results

in increased ADH and aldosterone secretion and ultimately in

lower urine output (Guyton, 1981). In ruminants there is greater

loss of potassium in skin secretions than of sodium (Johnson,

1970); sodium concentrations in the serum do not decrease and

aldosterone secretion remains low. As a result, reabsorption of

urinary sodium remains low. During dehydration associated with

heat stress in cattle, ADH remains elevated and aldosterone

concentration increases slightly to cause decreased urinary

output (El-Nouty et al., 1980).

Concentrations of ovarian steroids have been reported to be

affected by elevated temperature. Gwazdauskas et al. (1981)

reported that estradiol concentrations were depressed following

induced luteolysis in heat-stressed cows. Vaught et al. (1977)

found progesterone concentrations of lactating cows were






26

elevated during the summer, but there was no increase in non-

lactating cows. McNatty et al. (1984) found, however, that

progesterone concentrations were higher in cows during the

winter. Miller and Alliston (1973) also reported effects of heat

stress on progesterone concentrations in cyclic heifers.

Maximal serum concentrations were reached two days earlier in the

estrous cycle of heat stressed animals and had begun to decline

before peak concentrations were established in the control

heifers. A potential source of increased progesterone con-

centration is increased adrenal synthesis (Stott and Robinson,

1970). Injection of ACTH during heat stress in cattle causes

increased serum progesterone concentrations to appear in

addition to increased concentrations of glucocorticoids (Abilay

et al., 1975). Depressed ACTH turnover during heat stress

coupled with a reduction in adrenal 17a-hydroxylase activity,

adaptations designed to depress glucocorticoid secretion, might

result in increased progesterone secretion from the adrenal

cortex. Increased concentrations of progesterone caused by heat

stress in ewes are not sufficient to suppress pituitary release

of LH, however (Sheikheldin et al., 1988).

Changes in behavior patterns

Animals can relieve some effects of hyperthermia through

alteration of behavior patterns. In a manner similar to that

adopted by ectothermic animals, homeotherms may seek shade or

decrease daytime activity in favor of nighttime activity (Seath

and Miller, 1946; Robertshaw, 1986). Holstein cattle on the








island of Fiji did 67% of their grazing at night (Payne et al.,

1951), contrasted with about 16% in more temperate climates

(Seath and Miller, 1946). Animals may also utilize changes in

posture to increase convective surface area (Curtis, 1981) and

increase evaporative heat flux by licking and salivating (Riek

and Lee, 1948). Sexual behavior is altered during periods of

elevated ambient temperature, shortening the period of estrus

in cattle (Hall et al., 1959; Madan and Johnson, 1973; Monty et

al., 1974; Gwazdauskas et al., 1981). Alteration of estrous

behavior may be due to lower concentrations of plasma estradiol

(Gwazdauskas et al., 1981) which, along with progesterone, is

responsible for alterations of neuronal activity in areas of the

central nervous system resulting in disinhibition of mating

behavior at the appropriate time (Parsons and Pfaff, 1985).

Bos Taurus Versus Bos Indicus Cattle

Bos indicus, or zebu, cattle evolved in tropical and subtrop-

ical regions of the earth and exhibit phenotypic characteristics

which provide advantages in the maintenance of homeothermy

during periods of elevated ambient temperature. Among these are

a high-density hair coat, with hair of short length, generally

of a light color and arranged to produce a sleek appearance

(Robertshaw, 1986). All of these characteristics contribute to

a reduced absorption of radiant energy (Finch, 1986). Zebu

cattle also have lower amounts of body-cover insulation because

fat distribution is localized to the inguinal region and the area

above the shoulders rather than covering the thoracic and






28

abdominal regions as in Bos taurus (Ledger, 1959). Storage of

fat in anticipation of periods of lowered food availability is

a characteristic of both Bos taurus and Bos indicus cattle. Zebu

cattle, however, evolved in regions where reduced food avail-

ability is generally correlated with reduced water availability

(Ledger, 1959). Taurean cattle, on the other hand, evolved in

temperate regions where food availability might decline, but

water supplies remain relatively constant. Thus an insulative

body fat cover, an advantage in temperate regions where seasonal

temperatures become low, is not a disadvantage for taurean cattle

during periods of elevated temperature because capacity for

evaporative heat flux without dehydration is not limiting

(Ledger, 1959) Zebu cattle have a higher density of epitricheal

sweat glands than taurean cattle (Robertshaw, 1986), although

rates of secretary output are similar (Joshi et al., 1968) Zebu

cattle also have a higher blood volume than Bos taurus cattle

(Howes et al., 1957), indicating increased water retention

capability. Bos indicus cattle also generate less metabolic heat

than Bos taurus (Kibler, 1957; Finch, 1986).

As compared to Bos taurus, Zebu cattle exhibit shorter

estrous periods, less intense estrous behavior, including almost

no homosexual behavior, more "quiet ovulations" with no detected

estrus and a shorter interval from estrus to ovulation (Plasse

et al., 1970; Randel, 1980; Randel, 1984). Randel (1980) found

lower levels of luteinizing hormone during the preovulatory

surge in Zebu heifers. The preovulatory surge is also of lower







29

amplitude and shorter duration than in taurean cattle (Randel,

1984). Corpora lutea of Zebu cattle are smaller and they

synthesize and release less progesterone than corpora lutea of

Bos taurus (Adeyemo and Heath, 1980; Rhodes et al., 1982;

Segerson et al., 1984). Synthesis of estradiol by ovarian

follicles is also lower in Zebu than taurean cattle (Randel,

1980; Segerson et al., 1984). In the study of Segerson et al.

(1984) comparing morphological aspects of the reproductive

tracts of Angus and Brahman (Zebu) cows, ovaries of Brahman cows

were less asymmetric than ovaries of Angus cows in terms of

differences between the ovary on which ovulation had occurred and

the ovary contralateral to it. Ovaries of Brahman cows had

greater overall weight, greater follicular fluid weight and, on

the ovary contralateral to the corpus luteum, larger numbers of

follicles. Angus cows had greater amounts of uterine luminal

protein at day 17 of pregnancy, a result of greater numbers of

endometrial glands and greater cell height of luminal epithelium

(Segerson et al., 1984).

Reproductive performance of Bos indicus cattle in tropical

and subtropical climates can exceed that of Bos taurus (Wilkins,

1986). There is seasonal variation in fertility of Bos indicus

in more temperate climates (Randel, 1984). This may account for

observations that reproductive performance of Zebu cattle is

poor in temperate climates (Jochle et al., 1973). In Zebu cattle

in winter, frequency of LH surges declines, responsiveness of the

CL to exogenous LH challenge is depressed and conception rates






30

are lower than in summer (Randel, 1984). Conception rates of Bos

taurus cattle, on the other hand, show seasonal variation with

lowest conception rates in summer (Salisbury et al., 1978),

even at latitudes as far from the equator as Minnesota where

conception rates in dairy cows decline by 11% in August (Udom-

prasert and Williamson, 1987).

One reason for increased resistance to heat stress-induced

infertility in Zebu cattle is the fact that they are better able

to maintain homeothermy in the face of higher ambient tempera-

tures. It is unclear whether a given increase in Tb causes a less

severe reduction in fertility in Zebu. Turner (1982) analyzed

the relationship between conception rates of crossbred cattle

of either Bos indicus or Bos taurus breeding under sub-tropical

conditions in northern Australia. It was concluded that the

depression in conception rate per unit increase in Tb did not

differ among breed types. However, the Y-intercepts of the

regression curves were greater for Zebu-crossbred cattle than

for Bos taurus indicating greater fertility under subtropical

conditions. While not statistically significant, the percentage

decrease in fertility per unit elevation of body temperature was

less for Zebu than Bos taurus cows.

Peacock et al. (1971) found pregnancy rates for Brahman

cattle (Zebu) to be 10% greater than for Shorthorn cattle in the

subtropical environment of Florida. A similar finding was

reported in a study of crossbreeding using Brahman, Angus and

Charolais sires (Peacock et al., 1977) during a March to July






31

breeding season. In both of these studies, a significant breed

of sire effect was seen for pregnancy rate as cows mated to

Brahman and Brahman crossbred sires had the highest conception

rates, perhaps indicating improved genetic potential for

survival of Brahman or Brahman-crossbred embryos during periods

of elevated temperature. Among all possible crossbreeding

combinations of Brahman, Angus and Charolais cows and bulls over

an 11 year period, the highest conception rates were attained in

Brahman x Brahman matings (84%) (Peacock et al., 1977). Preg-

nancy rates for cows mated to Brahman sires (82%), Angus sires

(74%) and Charolais sires (79%) were significantly different

(Peacock et al., 1977). There was no difference in breed of dam

as all three breeds had a conception rate of 78%. Taken together

with the data of Turner (1982), these results suggest the possi-

bility exists that Zebu cattle exhibit advantageous traits at the

level of the concepts and/or the female reproductive tract that

provide benefit during periods of elevated temperature.

Breed affects seasonal variation in fertility within Bos

taurus cattle. Stott (1961) reported that Jersey cows in Arizona

showed no seasonal depression in fertility whereas Guernseys and

Holsteins exhibited steep depressions. Individuals within a

breed may also vary in their thermal adaptiveness. El-Nouty et

al. (1976) found that thermal adaptability varied among Hereford

heifers and that animals exhibiting the greatest adaptability

also had the lowest levels of thyroxine secretion in response to

elevated temperature.








Effects of Hyperthermia on Reproduction: Effects at the
Reproductive Tract Level

While much attention has rightly been given to heat stress-

induced infertility as a problem in tropical, subtropical and

arid climates it is important to consider that heat stress is a

problem not unique to these areas. In Minnesota, an 11% decline

in fertility in dairy cows during the month of August has been

reported (Udomprasert and Williamson, 1987) and Erb (1940)

reported that conception rates of dairy cows in the Purdue

University herd were lowest in August. If, as pointed out by

Beede and Collier (1986), trends toward global warming continue

and higher environmental temperatures are realized, then heat

stress-induced infertility of domestic animals will become an

increasingly significant problem at latitudes further from the

equator.

Failure to maintain homeothermy results in depression of

fertility. Negative effects of hyperthermia upon establishment

and maintenance of pregnancy can be manifested at a number of key

stages, from mating to parturition. Hyperthermia during

gestation may also affect subsequent lactation (Collier et al.,

1982), thereby impacting upon the health and performance of the

neonate. Avenues for heat-induced effects include direct

thermal effects on gametes and on the preimplantation embryo

resulting directly in developmental abnormality. There may

direct effects of elevated temperature on oviductal and uterine

secretary function. Additionally, interactions between







33

concepts and maternal systems may be altered through changes in

uterine blood flow and alterations of uterine-endometrial

secretary and transport activity as the hyperthermic animal is

subject to various metabolic, endocrine, cardiovascular and

nutritional perturbations.

Uterine Blood Perfusion

Uterine blood perfusion is the ultimate source of luminal

nutrients, oxygen and water for the developing concepts.

Uterine blood flow is also responsible for dissipation of uterine

metabolic heat (Gwazdauskas et al., 1974). Vasoregulation in the

uterine vascular bed is controlled by catecholamines, par-

ticularly norepinephrine, which exert effects locally in the

uterus (Roman-Ponce et al., 1978a; Brown and Harrison, 1981) and

whose effect is modulated by concentrations of circulating

steroids (Ford, 1982). Estradiol-induced protein synthesis

blocks synthesis and release of the a-adrenergic-stimulator

norepinephrine, thus blocking vasoconstriction. Progesterone

enhances norepinephrine-induced vasoconstriction through lower

clearance rate of secreted norepinephrine (Ford, 1982).

Additionally, prostaglandins exert vasoregulatory effects in the

uterine vascular bed, also modulated by steroids. Prostaglandin

E exerts vasodilatory effects by blocking norepinephrine release

while F series prostaglandins exert vasoconstrictor effects

under appropriate conditions, i.e., when estradiol is low, by

potentiating norepinephrine release (Ford, 1982).






34

Heat stress causes reduction of uterine blood flow in cattle

(Roman-Ponce et al., 1978b) sheep (Oakes et al., 1976; Alexander

et al., 1987; Bell et al., 1987) and rabbits (Leduc et al.,

1972), though apparently not in swine (Wettemann et al., 1988).

The cause remains uncertain, although endocrine alterations

associated with heat stress in cattle, such as elevated catechol-

amine concentrations (Whittow and Findlay, 1968; Alvarez and

Johnson, 1973) and decreased estradiol: progesterone ratio

(Gwasdauskas et al., 1981; Vaught et al., 1977; Roman-Ponce et

al., 1981), are consistent with increased vasoconstrictor

effects in the uterine vascular bed. Elevated concentrations

of progesterone depress estradiol-induced increases in uterine

blood flow in ovariectomized sheep and cattle (Caton et al.,

1974; Roman-Ponce et al., 1978a,b). In hepatocytes in vitro,

heat shock causes transient inhibition of the estrogen-mediated

increase in trancription of the vitellogenin gene with a

concomitant decrease in the number of nuclear estrogen-receptors

(Wolffe et al., 1984). Such an alteration of receptors in

addition to the inhibitory effects of elevated progesterone

might explain the refractoriness of uterine blood flow to

estradiol injection.

Elevated environmental temperature is associated with

reduced uterine blood flow and increased uterine luminal

temperature (Gwazdauskas et al., 1973; Thatcher, 1974). The

critical nature of uterine blood perfusion for dissipating

uterine heat is demonstrated by the fact that uterine temperature






35

increases more rapidly than arterial blood temperature as the

uterine blood flow diminishes (Gwazdauskas et al., 1973).

Increased uterine luminal temperature around the time of

insemination is associated with depressed conception rates in

cattle (Gwazdauskas et al., 1973; Thatcher, 1974). The role of

uterine blood flow during later stages of pregnancy and its

effect on fetal growth is discussed later.

Gamete Development

In the dairy industry where artificial insemination is

prevalent, the effect of heat stress upon the male gamete has not

been greatly considered with respect to embryonic mortality.

Exposure of spermatozoa to elevated temperature in vitro

(Burfening and Ulberg, 1968), in utero (Howarth et al., 1965) or

while in the epididymas (Bellve, 1972) results in no adverse

effect on fertilization but subsequent embryonic survival is

greatly reduced. Chromosomal abnormalities induced by heat

shock and carried over into embryonic development are the most

likely cause (Ulberg and Sheean, 1973; Waldbeiser and Chrisman,

1986). Lenz et al. (1983) found that bovine spermatozoa exposed

to 40C in vitro had lower viability and reduced potential for

capacitation. Alterations of uterine and/or oviductal factors

responsible for interactions with spermatozoa (Sutton et al.,

1984b), as part of the process of sperm transport (Hunter et al.,

1983) and capacitation (Bedford, 1969; Bedford, 1970), as well

as direct thermal effects on the gametes, are potential causes






36

of fertilization failure or subsequent embryonic loss due to

maternal heat stress.

In vivo heat-stress in the 2 or 3 days prior to ovulation is

associated with low conception rates in cattle (Stott and

Williams, 1962; Ingraham et al., 1974). A number of studies make

this association (Dutt et al., 1959; Stott and Williams, 1962;

Ingraham et al., 1974) but cannot relate the effect temporally

to the complex series of events (Moor and Gandolfi, 1987;

Wassarman, 1988) that occur prior to ovulation of a fully

competent oocyte.

Fertilization rate in vivo is probably not affected in sheep

(Dutt, 1963), cattle (Monty, 1984; Putney et al., 1989a) or pigs

(Warnick et al., 1965; Edwards et al., 1968) following hyper-

thermia on the day of ovulation. Putney et al. (1989a) subjected

superovulated heifers to elevated temperature for 10 h, begin-

ning at the onset of estrus. Heifers were inseminated 15 and 20

h after onset of estrus and reproductive tracts were flushed at

7 days after estrus to recover embryos. While heat-stress caused

more retarded and abnormal embryos to be present, there was no

difference in ovulation rate or numbers of fertilized ovum

recovered, compared to unstressed superovulated controls. In

vitro, the numbers of bovine oocytes that make the transition

from meiotic prophase I to metaphase II is reduced by elevated

temperature (40C) as is the fertilization rate (Lenz et al.,

1983).







37

In mice, heat stress of females prior to ovulation does not

affect fertilization rate but subsequent embryonic survival is

significantly reduced (Bellve, 1972; Baumgartner and Chrisman,

1988) This is associated with an increased incidence of chromo-

somal non-dysjunction and resultant polyploidy in the embryos

(Baumgartner and Chrisman, 1988). Non-dysjunction may result

from altered tubulin synthesis, which is depressed as a result

of heat shock (Thomas et al., 1982).

Exposure of gilts to elevated temperature for several days

prior to breeding affects neither ovulation rate nor fertiliza-

tion rate (Warnick et al., 1965; Edwards et al., 1968) As in the

case of the spermatozoa, alterations of oviductal factors

responsible for interactions with the gamete or zygote (Shapiro

et al., 1974; Kapur and Johnson, 1985; Leveille et al., 1987) as

well as direct thermal effects on the gamete or zygote, are

potential causes of failure of fertilization or loss of embryonic

viability.

In the sexually mature adult, the ovaries contain pools of

oocytes arrested since fetal development in prophase of the first

meiotic division. Fully grown oocytes resume meiosis and are

ovulated during each reproductive cycle. After the preovulatory

surge of LH, oocytes progress from meiotic prophase I to meta-

phase II in a process called oocyte maturation (Wassarman, 1988) .

This event is characterized by germinal vesicle breakdown,

condensation of the chromatin, spindle formation in preparation

for diakinesis, and extrusion of the first polar body (Moor and






38

Gandolfi, 1987; Wassarman, 1988). In preparation for spindle

formation, nearly two percent of the total protein in the oocyte

is tubulin (Wassarman, 1988) Tubulin synthesis in heat-shocked

cells is depressed (Thomas et al., 1982). High levels of cyclic

AMP in the oocyte inhibit maturation (Eppig and Downs, 1988),

these levels may be maintained through regulation by calcium-

calmodulin dependent processes, activation of adenylate cyclase

and inhibition of phosphodiesterase in the oocyte (Eppig and

Downs, 1988; Wassarman, 1988), effects which are reversed by LH

resulting in the trigger for maturation immediately prior to

ovulation (Wassarman, 1988). Elements important for meiotic

maturation such as synthesis of cytoskeletal tubulin (Thomas et

al., 1982), calcium-calmodulin activation (Weigant et al., 1985;

Stevenson et al., 1986) and patterns of steroid hormone (Roman-

Ponce et al., 1981) and LH secretion (Madan and Johnson, 1973;

Miller and Alliston, 1973) are all potentially altered by

elevated temperature.

Preimplantation Embryonic Development as Affected by
Hyperthermia

Heat shock during the zygote to morula stages

After sperm-egg fusion and fusion of the male and female

pronuclei, the initial cell cycle of embryonic development

begins and the first cell division occurs about 12 h later

(Pederson, 1988). Embryonic development is dependent upon

temporal coordination of a number of processes, many of which,

such as cell-cycle regulation, may be disrupted by hyperthermia.






39

Subtle disruptions may result in carry-over effects of hyper-

thermia that cause embryonic death several days or weeks after

the stress has passed (Elliott et al., 1968; Wildt et al., 1975).

Mouse embryos exposed in vivo to elevated temperature during the

first cell division suffer an increased incidence of post-

implantation death (Elliott et al., 1968). Likewise, many

porcine embryos exposed to high temperatures during placento-

genesis (days 14 to 25) appear normal at day 25, but have

degenerated by day 42 of pregnancy (Wildt et al., 1975). Through

the initial cell divisions (actual number varies among species)

following fertilization, the embryo is dependent upon mater-

nally-derived mRNA which was synthesized during the period of

oocyte growth preceding meiotic maturation and ovulation

(Wassarman, 1988). Activation of the embryonic genome occurs at

the 2-cell stage in the mouse (Bolton et al., 1984; Bensaude et

al., 1984) and 8 to 16-cell stage in the cow (King et al., 1985;

Camous et al., 1986; Frei et al., 1989). Protein synthesis by

the sheep embryo is very active in the first two cycles of cell

division followed by a 95% reduction in the third cycle (Crosby

et al., 1988). In the fourth and fifth rounds of cell division,

16 and 32-cell stages, protein synthesis increases once again

as a result of activation of the embryonic genome.

The early embryo is highly sensitive to hyperthermia and a

large reduction in embryonic survival, as much as 80% loss,

follows heat stress in the first days after fertilization in

cattle (Dunlap and Vincent, 1972; Stott and Wiersma, 1976; Putney






40

et al., 1989), sheep (Dutt et al., 1959; Alliston and Ulberg,

1961; Dutt, 1963), mice (Elliott and Ulberg, 1971; Ulberg and

Sheean, 1973), rabbits (Ulberg and Sheean, 1973) and pigs

(Warnick et al., 1965; Tompkins et al., 1967; Edwards et al.,

1968; Wildt et al., 1975). Gwazdauskas et al. (1973) found the

greatest correlation between environmental temperature and

conception rate on the day of insemination and on the day after

insemination.

Effects of hyperthermia in all the species listed above

occurred during the period of the first one to three cleavage

divisions when the embryos are present in the oviduct. The

embryo remains in the oviduct for 90 h in cattle, 72 h in sheep,

50 h in pigs (Hafez, 1974), and 68 to 77 h in mice (Biggers et

al., 1971). Adverse effects of hyperthermia have generally been

attributed to direct thermal effects upon the embryo rather than

alteration of oviductal-embryonic interactions. There is good

evidence for direct effects of elevated temperature on embryonic

development in sheep (Alliston and Ulberg, 1961), rabbits

(Alliston and Ulberg, 1965), mice (Elliott et al., 1968; Wittig

et al., 1983) and cattle (Putney et al., 1988a).

Nonetheless, it is possible that a portion of the effect of

elevated temperature could be due to alterations of oviductal

function. The volume of oviductal secretions are diminished by

heat stress in rabbits (Thorne et al., 1980). The importance of

the oviductal environment for early embryonic development has

been illustrated in experiments demonstrating superior in vitro







41

development of embryos cultured in the presence of oviductal

cells (Gandolfi and Moore, 1987; Eyestone et al., 1987; Rexroad

and Powell, 1988) or oviduct-conditioned culture medium (Kalt-

wasser et al., 1987). It may reasonably be assumed that some of

the effects of the oviduct are mediated by secretary proteins.

Proteins secreted by the oviduct have been implicated in a number

of functional roles, including binding to zona pellucida

(Shapiro et al., 1974; Kapur and Johnson, 1985; Leveille et al.,

1987) and spermatozoa (Sutton et al., 1984b) sperm capacitation

(Bedford, 1970) and inhibition of antibody-complement-mediated

immune function (Oliphant et al., 1984b). Stage-specific

changes in total protein secretion rate by bovine oviducts during

the estrous cycle (Geisert et al., 1987; Killian et al., 1987)

and appearance of individual stage-specific proteins from

oviducts of the rabbit (Oliphant et al., 1984a) pig (Buhi, 1985;

Buhi et al., 1989), cow (Geisert et al., 1987), sheep (Sutton et

al., 1984a), mouse (Kapur and Johnson, 1985), rat (Wang and

Brooks 1986), baboon (Fazleabas and Verhage, 1986), and human

(Verhage et al., 1988) all point to the potentially critical

nature of oviductal function around the time of fertilization and

during early pregnancy.

The capacity, or lack of capacity, of maternal mRNA-directed

protein synthesis to respond to stressors such as elevated

temperature may be a key to the susceptibility of the early

embryo to hyperthermia. It is unclear whether heat sensitivity

is temporally related to genomic activation; although in the






42

mouse embryo, RNA production is more sensitive to heat shock at

the time of genomic activation (2-cell stage) than at any other

time (Bellve, 1976) Probably as a consequence of maternal mRNA-

directed protein synthesis, embryos are apparently not able to

synthesize heat-shock proteins until the early blastocyst stage

(Wittig et al., 1983; Morange et al., 1984; Heikkila and Schultz,

1984) although mouse embryos are capable of constitutive (i.e.,

non-temperature regulated) synthesis of members of the 70-kDa

heat-shock protein family at the two-cell stage (Bensaude et al.,

1986; Howlett, 1986).

The Blastocyst and Heat Shock

The embryo remains susceptible to adverse effects of elevated

temperature throughout the preimplantation period as a number of

events critical to embryonic development occur. Following the

early rounds of cell division, the blastomeres form junctional

complexes (Ducibella et al., 1975) and become very tightly

associated. Compaction of the blastomeres occurs at 8-cells in

the mouse (Johnson and Pratt, 1983), 16-cells in the sheep

(Crosby et al., 1988) and 32-cells in the cow (Barnes et al.,

1987). The cell-cell adhesion required for compaction is

calcium-dependent (Ogou et al., 1982). Following compaction,

the embryo is referred to as a morula. The next major event of

embryonic development is blastocoele formation. Fluid transport

in the trophectoderm driven by newly synthesized Na-K+ ATPase

(Benos and Biggers, 1981; Overstrom et al., 1989) pumps water

into the center of the tightly associated cells and a hollow







43

cavity, the blastocoele, is formed and expanded. Early differen-

tiation events result in asynchronous rates of cell division and

positioning of cells whose developmental fates begin to be

established as restriction of potency begins to occur, generally

around the 32-cell stage (Pederson, 1988). Inner and outer cell

compartments form and the trophectoderm and inner cell mass

become distinguishable. The blastocyst at this stage contains

16 cells in hamsters and pigs, 32 cells in mice, 64 cells in sheep

and 128 cells in the rabbit (Pederson, 1988). In addition, free

amino acid content of embryos increases steadily from the 2-cell

stage through blastocyst development (Sellens et al., 1981).

Both Na+-dependent and independent amino acid transport systems

are present in the early embryo, Na+-dependent transport develops

at the morula stage in mouse embryos (Kaye et al., 1982). These

transport systems have Km of 10-5 to 10-6 M (Kaye et al., 1982).

As the blastocyst grows, it sheds the zona pellucida and

expands within the uterine lumen to form contacts with the

uterine vasculature (species having invasive implantation) or

the endometrial epithelium (noninvasive) and establish interac-

tions with the maternal system. Successful establishment and

maintenance of pregnancy involves a close communication between

the mother and the developing concepts. The critical importance

of synchrony in the development of these two biological systems

as pregnancy progresses has been demonstrated by embryo transfer

studies in domestic animals (Rowson et al., 1972; Wilmut and

Sales, 1981; Wilmut et al., 1985). Steroid-induced alterations






44

in the reproductive tract during the early period after estrus

are critical to embryonic survival (Miller and Moore, 1976; Moore

et al., 1983; Wilmut et al., 1985) and the rate of embryonic

development in the sheep and cow can be altered by the uterine

environment (Wilmut and Sales, 1981; Wilmut et al., 1985; Garrett

et al., 1987). Presumably, many of the steroid-driven functions

of the uterus are mediated by secretary proteins. Stage-speci-

fic secretion of proteins by the bovine uterine endometrium has

been reported (Roberts and Parker, 1974, 1976; Bartol et al.,

1981a,b). Endometrial proteins play roles as transport mole-

cules (Buhi et al., 1982; Pentacost and Teng, 1987), protease

inhibitors (Fazleabas et al., 1982), lysosomal enzymes (Hansen

et al., 1985) and immunoregulatory molecules (Murray et al.,

1978; Hansen et al., 1989).

Heat stress 8 to 16 days after fertilization results in sig-

nificant embryonic loss in sheep (Dutt, 1963; Dutt and Jabora,

1976) and cattle (Stott and Williams, 1962; Wise et al., 1988).

In several cases, prolonged lifespan of the corpus luteum in

bred-but-not-pregnant cows has been reported. This may reflect

embryonic death during the period 8 to 16 days after fertiliza-

tion (Stott and Williams, 1962; Madan and Johnson, 1973; Wise et

al., 1988) as the embryo survived for a sufficient length of time

to signal its presence to the maternal system and block luteoly-

sis for a short time.

Rescue of the corpus luteum from luteolysis is a critical

physiological event that may be sensitive to heat stress in






45

cattle. Heat stress from 8 to 16 days after insemination caused

alteration of the uterine environment, including elevated

protein and electrolyte concentrations (Geisert et al., 1988),

reduced weight of corpora lutea and impaired concepts growth

(Geisert et al., 1988; Biggers et al., 1987). During this

period, the concepts secretes the antiluteolysin, bovine

trophoblast protein-i (bTP-1) (Helmer et al., 1987), an

interferon-like (Imakawa et al., 1989) protein that causes

suppression of uterine prostaglandin (PG) F secretion resulting

in luteal maintenance (Helmer et al., 1989; Thatcher et al.,

1989). Depression of PGF secretion by bTP-1 may be mediated by

an inhibitor of PG synthesis that appears in uterine endometrial

cells during pregnancy (Gross et al., 1988) and after exposure

to bTP-1 (Helmer et al., 1989). Conceptus weight was signifi-

cantly reduced when cows were exposed to elevated temperature

from Day 8 to 16 of gestation, although no depression was

observed for subsequent secretion of bTP-1 during in vitro

culture at 37 C (Geisert et al., 1988). Hyperthermia could also

directly alter PGF secretion. Basal and oxytocin-induced PGF

secretion from cultured uterine endometrium from cyclic and

pregnant cows at Day 17 after estrus was increased by exposure

to elevated temperature (Putney et al., 1988; Putney et al.,

1989). Further, nonpregnant cows or cows with retarded embryos

at Day 17 after estrus displayed increased PGF secretion in

response to oxytocin during heat stress in vivo (Putney et al.,

1989).








Heat Stress During Mid- and Late-Gestation

After hatching from the zona pellucida, the blastocyst

continues to expand through accumulation of water within the

extraembryonic membranes that arise from the embryonic gut.

These are the yolk sac, a transient structure, and the allantoic

sac which continues to expand and, in animals with epithelio-

chorial placentation, supports expansion of the chorioallantoic

membranes and their apposition with the maternal endometrium

(Bazer et al., 1981). Water and electrolyte transport by the

chorioallantois are critical to successful establishment of

maximally efficient placental exchange between the maternal and

concepts systems. The basis of water and electrolyte transport

is the establishment by Na'-K ATPase in the chorioallantois of

a [Na*] gradient between the allantoic fluid and maternal blood.

Both the active pumping of the ATPase to establish the gradient

and the relative leakiness of the membrane to permit passive

diffusion of sodium-coupled ions and water are affected by the

estrogen: progesterone ratio and by prolactin and placental

lactogen (Bazer et al., 1981). Alterations in steroid production

and perturbation of prolactin secretion in heat-stressed animals

could result in alterations of establishment and maintenance of

conditions necessary for these processes to occur.

Although the functional roles of many of the proteins that

appear in the uterine lumen during pregnancy remain undefined,

it may reasonably be assumed that uterine and placental secretary

proteins play important roles during pregnancy. These proteins







47

have been implicated as transport molecules (Buhi et al., 1982),

immunomodulators (Hansen et al., 1989), lysosomal enzymes

(Hansen et al., 1985), protease inhibitors (Fazleabas et al.,

1982) and hormones (Talamantes et al., 1980).

Heat stress during mid- to late- gestation causes reduced

fetal and placental weight in cattle and sheep (Alexander and

Williams, 1971; Collier et al., 1982) and reduced cotyledonary

release of estrone sulfate in cattle (Collier et al., 1982).

Retardation of fetal growth caused by heat stress likely results

from perturbation of diverse physiological events. Central to

this phenomenon is a reduction in uterine and placental blood

flow (Oakes et al., 1976; Bell et al., 1987; Alexander et al.,

1987), especially maternal placentomal blood flow in ruminants

(Alexander et al., 1987). Placental size is depressed by heat

stress (Head et al., 1981; Bell et al., 1987) and 02 and glucose

transport is reduced (Bell et al., 1987), possibly an indication

of alteration of the water and electrolyte transport described

by Bazer et al. (1981). Reduced fetal weight is a result of

reduced placental size and transport activity (Alexander and

Williams, 1971; Bell et al., 1987). The depression in maternal

feed intake associated with heat stress is not sufficient to

account for malnutreated condition of fetuses (Cartwright and

Thwaites, 1976; Brown et al., 1977).

The decrease in blood flow to the pregnant uterus and placenta

is actively regulated at the uterine vascular bed since






48

elevated body temperature also reduced response to pharmaco-

logical stimulation of uterine blood flow in ewes (Roman-Ponce

et al., 1978a) and cows (Roman-Ponce et al., 1978b). Elevated

catecholamine concentrations reduce uterine blood flow (Bell et

al., 1987). Increasing pH of the blood, a result of respiratory

alkalosis induced by second-stage breathing (Dale and Brody,

1954; Brown and Harrison, 1981), results in a 30% depression of

umbilical blood flow (Oakes et al., 1976). There is a possib-

ility that local vasoconstriction during heat stress is caused

in part by changes in PG secretion. Elevated temperature in

vitro can alter the patterns of PG secretion by tissues of the

reproductive tract of the cow (Putney et al., 1988b) and pig

(Wettemann et al., 1988; Gross et al., 1989). Prostaglandins

affect blood flow (Ford, 1982) through vasoconstrictive and

vasodilatory activities which are modulated by circulating

steroids (Ford, 1982; Vincent et al., 1986). Prostaglandins

produced by the growing blastocyst and placenta may affect per-

meability of the endometrial vascular bed (Kennedy, 1980; Lewis,

1989) and stimulate fluid and electrolyte transport across the

uterine epithelium (Biggers et al., 1978).


Effects of Hyperthermia on Cells

Heat stress-induced cell killing involves perturbation of

multiple systems within the cell, which shall be discussed in

turn. Among these are alterations in membranes, oxidation-

reduction balance, second messenger system function,






49

intracellular calcium mobilization, protein synthesis, protein

conformation and stability, enzyme activity, RNA splicing and

DNA repair. Eukaryotic organisms have evolved complex systems

for meeting challenges posed by their environment, these

homeostatic mechanisms include stabilization of membranes and

proteins, the elimination of deleterious products such as oxygen

radicals and reduction of RNA processing and translation as a

precaution against potential errors. Activation of these

various mechanisms through incremental exposure to stress leads

to a state of increased thermotolerance (Henle and Dethlefson,

1979; Li and Werb, 1982; Ashburner, 1982; Nover, 1984), though

the complete response pattern to achieve this tolerant status

remains unclear. Heat-induced cell death is probably the result

of multiple lesions acting at various sites (Jung, 1986).

Heat-shock responses of reproductive tract tissues and

developing conceptuses after activation of the embryonic genome

might be explained at the level of the cellular stress response

of individual cells. Mammalian cells exhibit a characteristic

response pattern to stress, including the production of a set of

proteins, called heat-shock proteins, that are believed to exert

stabilizing effects within the stressed cell (Welch et al.,

1987). This response is apparently not possible until the early

blastocyst stage in mouse and rabbit embryos (Wittig et al.,

1983; Morange et al., 1984; Heikkila and Schultz, 1984) although

embryos are capable of constitutive synthesis of members of the






50

70-kDa heat-shock protein family at the 2-cell stage (Bensaude

et al., 1986; Howlett, 1986).

Heat Shock and Morphological Changes

Heat stressed cells undergo characteristic changes in

morphology, particularly in the areas of the nucleus, nucleolus

and mitochondria. There is general disruption of the nuclear

matrix (Welch and Suhan, 1985) and increased protein content

(Armour et al.,1988). Nucleoli of heat-stressed cells appear to

be swollen and decondensed (Pelham, 1984; Welch and Suhan, 1985;

McConnell et al., 1987). Nucleoli and nuclei of heat stressed

cells contain rod-like, fibrous structures shown by Welch and

Suhan (1985) to be actin. Following heat shock, mitochondria of

rat fibroblasts are swollen and exhibit prominent cristae

(Lepock et al., 1982; Welch and Suhan, 1985). Alterations of

mitochondria result in loss of respiratory control and uncou-

pling of oxidative phosphorylation from electron transport

(Christianson and Kvamme, 1969; Lepock et al., 1982). Addition-

ally, mitochondria are relocalized in heat-stressed cells to the

area around the nucleus, possibly the result of collapse of

cytoskeletal elements (Shyy et al., 1989) which also results in

aggregation of other cytoplasmic structures such as endosomes

and lysosomes following heat shock (Welch and Suhan, 1985; Shyy

et al., 1989). Other cytoplasmic changes include fragmentation

or disappearance of the Golgi apparatus and endoplasmic retic-

ulum (Welch and Suhan, 1985).








Heat Shock and Cytoskeletal Changes

The current concept of subcellular structure is one involving

a three-dimensional skeletal matrix composed of at least three

distinct filamentous components. This network of filaments is

called the microtrabecular lattice. The components of the

lattice include microfilaments, intermediate filaments and

microtubules.

Of the three major networks of cytoskeletal elements in

cells, microfilaments, intermediate filaments and microtubules,

only the microtubules remain relatively unaffected by heat shock

(Welch and Suhan, 1985; Shyy et al., 1989) although tubulin

synthesis stops within a few hours during heat shock (Thomas et

al., 1982). Heat shock caused increased numbers of actin

filaments (stress fibers) to be present in rat fibroblasts (Welch

and Suhan, 1985) but caused disappearance of actin filaments in

mammary epithelial cells (Shyy et al., 1989). Within 30 minutes

after heat shock, however, the actin filament network had

reassembled.

The network of intermediate filaments has been shown to

collapse following heat shock resulting in aggregation of

cytoplasmic organelles around the nucleus (Biessman et al.,

1982; Welch and Suhan, 1985; Shyy et al., 1989). Drummond et al.

(1988) incubated cells in a calcium-free, EGTA-buffered medium

to demonstrate that the heat-induced changes in the intermediate

filament network were not due to increased intracellular

calcium.








Heat Shock and Alteration of Membranes

The fluid-mosaic model of membrane structure (Singer and

Nicholson, 1972) based on the phospholipid bilayer with asso-

ciated peripheral and transmembrane proteins remains the

accepted basis for understanding membrane function. The

capability of biological membranes to compartmentalize and

sequester proteins and nucleic acids is critical to life of all

organisms from unicellular prokaryotes to complex multicellular

organisms such as mammals.

The characteristics of membranes depend upon interactions

between lipid constituents and between lipids and proteins in the

membrane (Quinn, 1981). The lipid composition and relative

amount and type of proteins present in a membrane affect its

physical characteristics. At biological temperatures, membranes

exhibit a considerable degree of disorder and molecular mobility

(Lee and Chapman, 1987). Flexing of hydrocarbon chains by

rotation about carbon-carbon bonds, wobbling of the entire

phospholipid molecule along its longitudinal axis and lateral

displacement of molecules along the plane of the membrane all

occur (Quinn, 1981). Biological membranes are heterogeneous

systems of diverse composition and intermolecular interactions

can result in formation of microdomains within the plane of the

membrane as well as exhibition of different physical properties

in each monolayer of the membrane (Quinn, 1981).

The physical state of the membrane is affected by association

with molecules of water, called lyotropic mesomorphism, and by






53

temperature, called thermotropic mesomorphism (Quinn, 1981).

The transition of membranes from a rigid, gel-crystalline state

to a more disordered, liquid-crystalline arrangement occurs over

a narrow temperature range in an artificial homogeneous lipid

bilayer, but in heterogeneous biological membranes this transi-

tion occurs over a broader range of temperatures (Quinn, 1981;

Cossins and Raynard, 1987). In addition to the temperature and

hydration state, ions, sterols and proteins all affect membrane

structure and behavior (Cossins and Raynard, 1987). The major

sterol in animal cell membranes, cholesterol, reduces the

enthalpy at transition from gel-crystalline to liquid-crystal-

line and at high concentrations nearly eliminates the change in

phase as temperature changes (Lee and Chapman, 1987).

Alterations in cell membranes of poikilotherms and bacteria

in response to temperature has led to the hypothesis of homeo-

viscous adaptation (Cossins and Raynard, 1987). As temperature

changes, the composition of the membrane is altered through

shifts in the degree of saturation of the fatty acyl chains of

the membrane phospholipids (thereby reducing the flexing of the

hydrocarbon chains) and removal or insertion of sterols to

maintain consistent viscosity (Anderson et al., 1981; Cossins

and Raynard, 1987). Anderson et al. (1981) found that mammalian

cells grown at elevated temperature (41C) altered the choles-

terol: phospholipid molar ratio in their membranes. Such changes

in membranes of animal cells may not occur at very high tem-

peratures (42 to 44C) as no alterations in cholesterol:






54

phospholipid ratio occurred in membranes of cells incubated for

24 h at these temperatures (Anderson and Parker, 1982).

Elevated temperature increases basal membrane permeability

as determined by passive diffusion of Ke, increases permeability

to water, alters facilitated transport, i.e., glucose transport,

and decreased Na-K ATPase activity (Burdon and Cutmore, 1982;

Ellory and Hall, 1987). There is net loss of ion gradients as

leakage increases (Bowler, 1987). In the case of the glucose

transporter, it was determined that the rate of return of the

unloaded transporter to the outer membrane surface was increased

(Ellory and Hall, 1987). Elevated temperature also results in

aggregation of integral membrane proteins, possibly due to their

denaturation (Lepock et al., 1983; Arancia et al., 1986; Lee and

Chapman, 1987). Alterations of permeability lead to loss of ion

gradients, influx of Na and Ca, loss of membrane bioelectric

properties, failure of ion transport and impairment of facili-

tated transport mechanisms (Bowler, 1987). Additionally,

activity of receptors is impaired (Magun, 1981; Calderwood and

Hahn, 1983). Radiolabeled epidermal growth factor (EGF) bound

to cell-surface EGF receptors of Rat-1 cells with lower affinity

after heat shock (Magun, 1981). The slope of the Scatchard plot

as well as the abcissa intercept were different between heat-

shocked and control cells indicating both reduced affinity and

receptor number after heat shock (Magun, 1981). Additionally,

degradation of internalized 125I-EGF in the lysosomes was slower

in heat-shocked cells (Magun, 1981). Calderwood and Hahn (1983)







55

examined 125I-insulin binding to HA-1 cells and found that

receptor numbers were depressed by heat shock, but affinity was

not affected.

As previously mentioned, heat shock caused mitochondria of

rat fibroblasts to become swollen and exhibit prominent cristae

(Lepock et al., 1982; Welch and Suhan, 1985). Heat-induced

alterations of mitochondrial membranes result in loss of

respiratory control and uncoupling of oxidative phosphorylation

from electron transport (Christianson and Kvamme, 1969; Lepock

et al., 1982).

Heat Shock Alters DNA Transcription and RNA Processing

Heat shock and DNA

The thermodynamic stability of the DNA double-helix at

elevated temperature is simply based upon the relative adenine-

thymine: guanosine-cytosine ratio of the nucleic acids (Lewin,

1987). Higher concentrations of guanosine-cytosine base-pairs

with their three hydrogen bonds between each nucleotide pair are

more stable than adenine-thymine base-pairs which interact with

two hydrogen bonds (Lewin, 1987).

Heat shock terminates DNA synthesis (Ashburner, 1982; Warters

and Henle, 1982) and results in failure of DNA repair mechanisms

in the cell (Henle and Dethlefson, 1978; Warters and Roti Roti,

1982), possibly due to accumulation of excessive protein in the

nucleus (Armour et al., 1988). Warters et al. (1980) tested the

accessibility of DNA to nuclease attack following heat shock.

There were fewer sensitive sites in DNA fractions from heated






56

cells compared to controls, possibly due to accumulation of

proteins associated with the DNA (Warters et al., 1980). Failure

of unstressed cells to complete replication of DNA during the S

phase of the cell cycle results in increased strand breakage

during the next replication event (Laskey et al., 1989) an effect

seen in heat-shocked cells (Warters and Roti Roti, 1982).

Transcription of DNA is altered by heat shock as there is

preferential transcription of genes coding for heat-shock

proteins (Ashburner, 1982; Nover, 1984) under the influence of

specific heat-shock transcription factors (Chousterman et al.,

1987; Wu et al., 1987; Sorger and Pelham, 1988). Temperature-

dependent phosphorylation of a heat-shock transcription factor

has been reported (Sorger and Pelham, 1988) and Rougvie et al.

(1988) found that RNA polymerase was bound to the 5' end of an

uninduced heat-shock protein gene in Drosophila apparently

waiting for a signal to transcribe the gene. Exons of the c-myc

oncogene product turn on heat-shock gene promoters (Kingston et

al., 1984). Ananthan et al. (1985) were able to show that

denatured proteins introduced into the cell (Xenopus oocytes),

either by microinjection of denatured peptides or by insertion

of gene constructs coding fragments of peptides incapable of

normal folding, would induce the promoter region of the

Drosophila 70-kDa heat-shock protein (70-kDa promoter plus a

P-galactosidase reporter gene) to activate gene transcription.

The same region of DNA in the promoter is responsible for both






57

the response to denatured proteins and for response to heat shock

(Ananthan et al., 1985).

Heat shock and RNA

Posttranscriptional processing of heterogeneous nuclear RNA

is terminated by heat shock, apparently due to a direct effect of

heat-shock proteins (Yost and Lindquist, 1986). If heat-shock

protein synthesis was blocked with cycloheximide, RNA splicing

was not stopped during heat shock. Ribonucleoprotein particles,

called snRNPs, are the effectors of RNA splicing and are composed

of multiple subunits (Cech, 1986); snRNPs are disrupted by heat

shock in stress-susceptible cells but not in thermotolerant

cells (Bond, 1988).

Heat shock reduces the half-life of mRNA in the cytoplasm by

induction, through synthesis of 2' ,5' oligoadenylates, of latent

endoribonucleases in a manner similar to the actions of inter-

ferons in virus-infected cells (Chousterman et al., 1987). Heat-

shocked MDBK cells produced a soluble heat-shock induction

factor that was able to induce 2',5' oligoadenylate synthetase

activity in fresh, unstressed MDBK cells (Chousterman et al.,

1987). This soluble factor was not a member of any known class

of interferons however.

Heat Shock and the Cell Cycle

The cell cycle is the coordinated series of events by which

the cell accomplishes growth and replication. The rate of

passage through various stages varies with cell type, physio-

logical state, nutrient availability and through control by






58

other exogenous cues. The Go phase is considered a subphase of

G, and is a quiescent state during which the cell awaits an

external signal, such as a particular growth factorss, to

initiate active replication (Pardee, 1989). The G1 phase of the

cell cycle is the period during which the cell prepares for the

S phase. There is protein and histone synthesis and some DNA

synthesis (Pardee, 1989). The cell is induced to enter the S

phase by coordination of cytoplasmic signals (Laskey et al.,

1989). During the S phase, the entire DNA complement of the cell

must be replicated. This is accomplished through bidirectional

replication at multiple sites (Laskey et al., 1989).

Sensitivity of CHO cells to heat shock varies with stage of

the cell cycle. Maximal thermotolerance is manifested at G1,

while the progression through S and G2-M are particularly heat

sensitive (Read et al., 1983; Rice et al., 1984a,b). Stabiliz-

ation of nucleolar components during heat shock is a function

proposed for members of the 70-kDa family of heat-shock proteins

(Pelham, 1984). Association of 70-kDa heat-shock protein with

proteins in the nucleolus varies in a cell-cycle dependent

fashion (Milarski et al., 1989). In G2, three different mono-

clonal antibodies to 70-kDa heat-shock protein failed to bind to

nucleolar protein in heat-shocked cells, although cells in G2

responded to heat shock with increased heat-shock protein mRNA

and protein synthesis (Milarski et al., 1989). As the cells

proceeded from S through G2 to the next M phase, there was

appearance of antibody binding in the nucleolus followed by






59

disappearance of binding and reappearance again. Three dif-

ferent antibodies, each recognizing a different epitope of the

70-kDa heat-shock protein, gave three different patterns of

appearance and disappearance of nucleolar binding during G2

(Milarski et al., 1989). This variation is believed to be due to

association of the heat-shock protein with nucleolar components,

which block the epitope to antibody binding, and is not observed

at other stages of the cell cycle (Welch and Feramisco, 1984;

Milarski et al., 1989). Mitogen-induced lymphocytes produce

heat-shock proteins through translation of preexisting mRNA

(Colbert et al., 1987), and this occurs particularly during the

Go through G1 transition preceding DNA synthesis (Kazmarek et

al., 1987; Haire et al., 1988).

Heat Shock and Metabolic Alterations

Heat shock induces alterations of mitochondrial membranes

resulting in loss of respiratory control and uncoupling of

oxidative phosphorylation from electron transport (Christianson

and Kvamme, 1969; Lepock et al., 1982). Adenosine, adenylate

activity and concentrations of ATP in the cell are not affected

by heat shock until temperatures become relatively high, i.e.,

45 to 48*C (Calderwood et al., 1985).

Heat Shock and Second Messenger Systems

Heat shock causes increased turnover rates of phosphatidyl-

inositol trisphosphate in cells (Calderwood et al., 1987). The

rate of turnover induced in the cell is rapid enough to quickly

deplete the membrane store of polyphosphoinositides, indicating






60

high activity of phospholipases. The hydrolysis of polyphospho-

inositides is normally the result of specific activation of a

membrane-bound receptor. There is subsequent activation,

mediated by a G-protein, of specific phospholipases (Berridge,

1984). Release of diacylglycerol in the membrane and of inositol

trisphosphate into the cytosol results in activation of protein

kinases, including protein kinase C, and release of intracel-

lular Ca stores, probably from the endoplasmic reticulum

(Berridge, 1984). Heat shock may, therefore, perturb regulation

of intracellular Ca+ concentrations and Ca-dependent events in

the cell as well as the pattern of intracellular protein phospho-

rylation. The intracellular pattern of tyrosine phosphorylation

is altered by heat shock (Maher and Pasquale, 1989) as is the

phosphorylation state of several protein-synthesis initiation

factors (Duncan and Hershey, 1989).

During heat shock, and following an increase in inositol

trisphosphate (Calderwood et al., 1987), there is a large

increase in intracellular Ca concentration (Stevenson et al.,

1986; Calderwood et al., 1988). The complete impact of this

increase in Ca concentration in the heat-shocked cell is

unclear, although it is of critical importance. Inhibition of

calmodulin activity in the cell increases its sensitivity to heat

(Weigant et al., 1985) and incubating cells in media containing

either low or high Ca concentrations increases the heat sensit-

ivity of the cells (Malhotra et al., 1986). There is also influx

of Ca+ from extracellular sources as a result of membrane






61

leakage. Intracellular Ca+ overload results in activation of

protein kinases, phospholipases and proteinases to perturb

cellular homeostasis (Bowler, 1987).

Heat-Shock Proteins

Among the responses to heat shock is the alteration of protein

synthesis in the cell in favor of the increased transcription

and translation of small number of evolutionarilyconserved

proteins called heat-shock proteins. Enhanced production of

heat-shock proteins in response to cellular stress has been

observed in virtually every species of plant or animal examined

(Ashburner, 1982; Nover, 1984). Since increased production of

these proteins has been observed during treatment with a variety

of other cellular stressors such as hydrogen peroxide, ethanol,

and heavy metals, they have come to also be called stress

proteins (Welch et al., 1982). Heat-shock proteins were first

observed in the early 1960s as products of gene transcription

associated with the heat-induced alteration of chromosomal

puffing in the giant polytene chromosomes of the Drosophila

salivary gland (Ashburner, 1982). Chromosomal puffing was

recognized as a characteristic of areas of active gene transcrip-

tion, and heat shock became one of the first model systems for

study of gene transcription. Until quite recently, most of the

work on heat shock was related to mechanisms of gene expression

and little attention was given to functional aspects of the heat-

shock proteins.






62

Appearance of heat-shock proteins is correlated with the

acquisition of thermotolerance (Li and Werb, 1982; Landry et al.,

1982; Heikkila et al., 1985). Activation of various homeostatic

mechanisms of the cell through incremental exposure to stress

leads to a state of increased thermotolerance (Henle and

Dethlefson, 1978; Li and Werb, 1982; Ashburner, 1982; Nover,

1984), the complete response pattern to achieve this tolerant

status remains unclear, however. Temperature sensitivity and

resistance can be created by altering the ability of organisms

to generate heat-shock proteins (Craig and Jacobsen, 1984;

Riabowel et al., 1988; Landry et al., 1989). Riabowel et al.

(1988) inserted antibodies against the 70-kDa heat-shock protein

into fibroblasts by microinjection. Cells containing antibody

to heat-shock protein were more heat-sensitive than cells

injected with control antibody (goat-anti-chicken IgG). The

ability of another heat-shock protein, the human 27-kDa protein,

to confer thermotolerance was demonstrated by placing a gene

construct containing the gene for 27-kDa plus a constitutive

(always on) promoter into mouse cells (Landry et al., 1989).

Transfected cells became thermotolerant, cells transfected with

the promoter elements only did not (Landry et al., 1989).

Heat-shock proteins can confer thermotolerance, however it

remains to be determined whether heat-shock proteins are the

primary effector in thermotolerance or a secondary response

since the correlation between appearance of heat-shock proteins

and thermotolerance does not always hold true. In the studies of






63

Hall (1983), Hallberg (1986), Widelitz et al. (1986) and Easton

et al. (1987) a thermotolerant state was achieved in yeast,

tetrahymena, rat fibroblasts and adult salamanders, respec-

tively, in the absence of heat-shock protein synthesis.

Several heat-shock proteins have been identified in mammalian

cells including a low molecular weight group appearing at about

27 to 28-kDa, 32-kDa and 58-kDa which have yet to be studied

extensively. Little is known about these low molecular-weight

heat-shock proteins and a 110-kDa heat-shock protein other than

their existence in stressed cells and a few physical charac-

teristics (Welch et al., 1989). It is known, in the case of the

27-kDa protein, that thermotolerance is associated with expres-

sion (Landry et al., 1989). By far the most studied are the 70-

kDa group of heat-shock proteins and a related group of glucose-

regulated proteins. Another mammalian heat-shock protein that

has been well studied is the 90-kDa species.

The 70-kDa heat-shock proteins

The 70-kDa family of heat-shock proteins include at least

eight related proteins in Drosophila and yeast, and at least

five, and as many as ten, related proteins in mammals (Zakeri et

al., 1988). Some of these are constitutively produced and hence

called heat-shock cognate, while others are induced by heat

shock. Other members of this family of proteins include the dnaK

and groEL proteins of E. coli and the YG 100 and YG 102 proteins

of S. cerevisiae, mutants of which confer temperature sensi-

tivity (Craig and Jacobsen, 1984). Since it is difficult to






64

distinguish individual members of this family of proteins except

through nucleic acid hybridization experiments, they have been

generally called simply the 70-kDa heat-shock protein.

Several characteristics of 70-kDa proteins provided clues

to potential function in the cell. During stress, these proteins

accumulate in the nucleus and nucleolus of the cell (Welch and

Feramisco, 1984; Pelham, 1984; Lewis and Pelham, 1985).

Nucleolar morphology is altered during heat shock (Pelham, 1984;

Welch and Suhan, 1985), presumably due to denaturation and

aggregation of proteins and ribonucleoproteins caused by heat

shock (Pelham, 1984) Transfection of cells with a plasmid which

constitutively expressed 70-kDa increased the rate of nucleolar

recovery after heat shock (Pelham, 1984). The 70-kDa protein is

also an ATP-binding protein and Lewis and Pelham (1985) found

that the ability of deletion mutants of 70-kDa to migrate to the

nucleolus and aid in recovery was dependent on ATP-binding. They

proposed that ATP-driven cycles of binding and release of 70-kDa

cause solubilization aggregates of proteins and ribonucleopro-

teins that form during heat shock. Another member of the 70-kDa

family was shown to be active in the ATP-driven process of

removal of clathrin from coated vesicles (Chappel et al., 1986).

Another ATP-binding protein related to the 70-kDa family is

the glucose-regulated protein 78, also known as immunoglobulin

heavy chain binding protein or BiP. This protein is present in

the endoplasmic reticulum and binds to unfolded protein subunits

or to improperly folded proteins (Munro and Pelham, 1986).







65

Following proper assembly of proteins in the endoplasmic

reticulum, BiP is released by ATP. Another related protein, the

groEL protein of E.coli, binds to subunits of secretary proteins

in an ATP-dependent manner during their assembly in the endoplas-

mic reticulum and is essential for correct folding and assembly

(Bochkareva et al., 1988). Collectively these data have led to

the hypothesis that members of the 70-kDa heat-shock protein

family are engaged in maintenance of protein and ribonucleo-

protein folding and assembly both in unstressed and in heat-

stressed cells (Welch et al., 1989).

Constitutive expression implies some function during normal

cell function. Recently, evidence has been accumulated that 70-

kDa proteins are integral to protein translocation across

membranes (Chirico et al., 1988; Deshaies et al., 1988; Murakami

et al., 1988; Flynn et al., 1989). In these studies, protein

translocation into mitochondria (Deshaies et al., 1988; Murakami

et al., 1988) or into synthetic microsomes (Chirico et al., 1988)

was dependent upon the presence of 70-kDa protein as the addition

and depletion of the 70-kDa protein in the system enhanced and

depressed, respectively, the rate of translocation. Import of

proteins across membranes and into organelles such as mitochon-

dria requires a high degree of control; sequestration of protein

by membranes is essential to life. The hypothesis of Chirico et

al. (1988), Deshaies et al. (1988) and Murakami et al. (1988) is

that translocation of completely formed protein requires the






66

ATP-driven alteration of conformation through binding with 70-

kDa heat-shock protein. Flynn et al. (1989) demonstrated

specific and saturable binding of BiP to peptides. BiP bound to

both hydrophobic and charged peptide sequences perhaps indi-

cating steric, or topographic, recognition (Flynn et al., 1989).

The 70-kDa proteins have also been implicated in the mechanisms

of peptide recognition during lysosomal degradation of intra-

cellular protein as part of normal protein turnover in the cell

(Chiang et al., 1989), a finding related to the data of Ananthran

et al. (1985) discussed previously.

The 90-kDa heat-shock protein

The 90-kDa protein is abundant in most cells and in amplified

three- to five-fold following heat shock (Welch et al., 1989).

The 90-kDa protein associates with a number of tyrosine kinases

in the cell, particularly the oncogene product pp60src (Brugge et

al., 1981). Association of 90-kDa with the pp60src apparently

inactivates its tyrosine kinase activity until the protein has

been delivered to the cell membrane (Brugge et al., 1981).

The 90-kDa protein is associated with the nontransformed or

non-DNA-binding form of the glucocorticoid and progesterone

receptor (Catelli et al., 1985; Sanchez et al., 1987; Howard and

Distelhorst, 1988; Baulieu and Catelli, 1989). Hormone-depen-

dent and temperature-dependent transformation of the receptor

to the DNA-binding form are analogous events (Sanchez et al.,

1987). In both cases, the 90-kDa protein dissociates from the






67

receptor complex. It is believed that dissociation of the 90-

kDa protein from the receptor complex uncovers the DNA-binding

site of the receptor (Baulieu and Catelli, 1989). Ramachandran

et al. (1988) have shown that synthesis of the 90-kDa heat-shock

protein can be regulated by steroid hormones in reproductive

tissues. Uterine 90-kDa protein was depressed following

ovariectomy in mice, and estradiol injections caused a 4-fold

increase in the concentration of the protein; data were normal-

ized to total protein in cytosolic extract to account for

estradiol-induced increases in uterine wet weight and protein

concentration. This increase in 90-kDa protein was accompanied

by a similar increase in progesterone receptor concentrations

(Ramachandran et al., 1988).

Ubiquitin

Ubiquitin is a protein involved in the process of protein

turnover in normal cells, proteins are generally short-lived and

turnover and recycling of amino acids is a normal activity in

most cells (Rechsteiner, 1987). Ubiquitin recognizes and binds

to peptides that are destined for degradation in the cell and

presents them to specific proteases (Rechsteiner, 1987).

Ubiquitin is also a heat-shock protein (Bond and Schlesinger,

1985). Enhancement of protein turnover by ubiquitin-mediated

activity occurs after heat shock (Carlson et al., 1987).

Thermostabilizing Agents

Thermostabilizing agents offer the potential to pharma-

cologically reduce heat stress-induced embryonic death,






68

particularly during early development when embryos may have no

inherent capability to respond to heat stress.

Amino acids

Vidair and Dewey (1987) found that CHO cells in a nutrient-

deprived state were extremely sensitive to heat shock and that

several hydrophilic, neutral amino acids, including alanine,

would reduce this sensitivity. Henle et al. (1988) found that

alanine exerted this protective effect upon CHO cells in the

absence of any nutrient deprivation-induced hypersensitivity.

In those studies also, concentrations of alanine in the milli-

molar range were required, the effect was time dependent, and

D-alanine was equally effective (Henle et al., 1988), indicating

that incorporation into protein was not necessary. Sequential

addition and removal of the supplemental alanine around the time

of heat shock demonstrated that alanine was a thermoprotective

agent, i.e., it must be present during the heat shock, rather

than an inducer of thermotolerance whose effect remains for a

time after it is removed (Henle et al., 1988). Both Henle et al.

(1988) and Vidair and Dewey (1987) found that alanine thermo-

protection was evident within 5 min of alanine addition to cell

cultures.

Taurine has been implicated in a variety of protective roles

within cells (Wright et al., 1986) and certain cell types

actively take up taurine. Banks et al. (1989) have shown that

alveolar macrophage, which generate oxidants and are relatively

resistant to oxidant injury, have a specialized transport system






69

for taurine which is, in part, a carrier-mediated transport. In

human lymphocytes, 60% of the intracellular free amino acid pool

is taurine (Fukuda et al., 1982) and taurine will enhance

viability of lymphocytes in vitro in a dose-dependent manner

(Gaull et al., 1983) under homeothermic conditions. Taurine is

abundant in the mammalian reproductive tract (Lorincz et al.,

1968) and is taken up by preimplantation embryos (Kaye et al.,

1986), making it attractive to speculate that taurine has some

role in the early embryo, possibly as an antioxidant. Like

D-alanine, taurine is not incorporated into cellular protein.

The roles of these amino acids may lie in stabilization of

proteins and organelles or antioxidation in the heat-shocked

cell. Vidair and Dewey (1987) suggested that because amino acids

were required in such large amounts and were not involved in

formation of proteins, stabilization of cellular components such

as proteins by aggregation around the surface of the protein to

cover hydrophobic sites exposed during denaturation, and thereby

impede non-specific aggregation of proteins, was the likely role

of protective amino acids. This type of effect, i.e., non-

specific aggregation around a protein molecule, is similar to

that suggested for the 70-kDa heat-shock protein (Lewis and

Pelham, 1985).

Reduced glutathione

The intracellular oxidative-reductive state may be related

to heat shock since oxidative stress also induces heat-shock

proteins (Li and Werb, 1982; Lee et al., 1983; Drummond et al.,






70

1987) and heat shock induces increases in antioxidant enzymes

such as superoxide dismutase (Omar et al., 1987). Additionally,

cellular responses to hydrogen peroxide exposure are similar to

heat-shock responses and development of tolerance to these two

stressors are, to a large degree, interchangeable (Li and Werb,

1982; Spitz et al., 1987).

Glutathione and glutathione S-transferase play a critical

role antioxidation and detoxification in cells. Glutathione S-

transferase catalyzes the nucleophilic addition of the thiol

group of glutathione to electrophilic acceptors including

peroxides, thiocyanates and alkyl halides (Armstrong, 1987).

Depletion of intracellular glutathione was reported to

increase sensitivity of cells to heat shock (Mitchell et al.,

1983; Russo et al., 1984) although metabolic inhibitors used to

deplete and prevent new synthesis of glutathione may have created

toxic conditions. Lilly et al. (1986) used a mutant fibroblast

cell line deficient in glutathione production and determined

that these cells were competent to develop thermotolerance even

in the presence of very low glutathione levels. Mitchell et al.

(1983) did find that glutathione concentrations increased in

cells during heat shock and during the development of thermo-

tolerance.

Vitamin E

Vitamin E is a hydrophobic, peroxyl radical-trapping, chain

breaking antioxidant found in membranes and its principal

function is to protect cells from the effects of spontaneous






71

autooxidation (Burton et al., 1983). Treatment with vitamin E

in vitro protected cells from chemical-induced injury (Pascoe et

al., 1987).

Cycloheximide

This protein synthesis inhibitor protects cells from heat-

induced cell killing (Lee and Dewey, 1986; Armour et al., 1988).

A 95% inhibition of protein synthesis could be attained without

toxicity to cells (Yong and Lee, 1986) and the onset of inhibi-

tion corresponded to onset of thermal protection. Increased

nuclear and nucleolar protein accumulation was blocked by this

inhibition and failure of DNA repair was also blocked (Armour et

al., 1988) suggesting that nuclear and nucleolar functions were

less impaired by heat shock in the absence of intranuclear

protein accumulation.

Others

Other chemical agents have been shown to exert beneficial

effects in reducing death of stressed cells in vitro, including

1 M glycerol (Henle and Warters, 1982) and soybean trypsin

inibitor (Korbelik et al., 1988).


Objectives

Elevated environmental temperature results in depressed

fertility in female cattle and other species. This effect is

multifaceted, occurring at various times and at multiple

locations during the establishment and maintenance of pregnancy.

There are both direct effects of hyperthermia on the early embryo






72

and indirect effects on maternal blood flow, hormone concentra-

tions and metabolism. The objectives of the research described

in this dissertation were to identify and characterize direct

effects of heat shock on tissues of the maternal reproductive

tract and concepts to identify possible causes of hyperthermia-

induced infertility.

Synthesis and secretion of proteins and prostaglandins by

reproductive tract tissues are critical to early maternal-

conceptus interactions. Disruption of oviductal and endometrial

protein secretion by direct hyperthermia, in addition to changes

caused by endocrine imbalance, may disrupt early maternal

concepts interactions. Comparisons of tissue responses of Zebu

and Bos taurus cattle were made in order to determine whether

Zebu cattle exhibit greater homeothermic advantages at the

tissue level in response to direct hyperthermia. Cellular

responses to elevated temperature can result in thermotolerance.

Another objective was to determine whether reproductive tract

tissues respond to elevated temperature in a similar manner to

the many cell types that have been examined in vitro, i.e., with

production of heat-shock proteins.

Alterations in prostaglandin release by the uterine endo-

metrium at day 17 of pregnancy in the cow as a result of concepts

signalling allows maintenance of the CL and permits pregnancy to

continue beyond this time. Disruption of these events by direct

hyperthermic effects might result in failure of luteal main-

tenance. Direct effects of hyperthermia on prostaglandin






73

secretion by endometrium, including effects on regulation of

secretion, were examined in addition to direct effects of

elevated temperature on secretion of bTP-1 by day 17 conceptuses.

Events later in pregnancy are also affected by hyperthermia.

Heat stress during mid- and late-pregnancy reduces fetal and

placental weight and efficiency of placental function. Protein

and prostaglandin synthesis and secretion in response to in vitro

heat shock were examined in ovine uterine and placental tissues

late in pregnancy to determine whether alterations occur that

might impact fetal growth.

Thermostabilizing agents were tested in vitro to determine

the efficacy of exogenous treatments to alter responses to heat

stress. Bovine peripheral lymphocytes and preimplantation mouse

embryos were used to test heat shock-induced killing in the

presence and absence of these agents in vitro. One of these

agents, alanine, was tested in an in vivo experiment with early

pregnant mice to determine whether intraperitoneal injection

could improve embryonic survival during maternal hyperthermia.

Two stages of embryonic development were tested, before and after

development of the ability to produce heat-shock proteins in

response to stress, to determine whether embryonic capacity to

produce heat-shock proteins in response to hyperthermia might

effect embryonic survival.

Some of these experiments, those reported in Chapter 5, were

carried out in collaboration with D. James Putney and these






74

results also appear in his Ph.D. dissertation (University of

Florida, 1988).












CHAPTER 2
DIFFERENCES BETWEEN BRAHMAN AND HOLSTEIN COWS IN
ALTERATIONS OF PROTEIN SECRETION
BY OVIDUCTS AND UTERINE ENDOMETRIUM
INDUCED BY HEAT SHOCK


Introduction

Maintenance of homeothermy is critical to fertility in

cattle (Gwazdauskas et al., 1973; Turner, 1982) and the repro-

ductive performance of Zebu cows in tropical or sub-tropical

climates is superior to that for European-type cows (Peacock et

al., 1971, 1977; Turner, 1982). Though not statistically

significant, Turner (1982) found a tendency for fertility to

decline less in Zebu cows for each unit increase in body tempera-

ture than in Bos taurus cows. This suggests that Zebu cattle may

exhibit advantageous characteristics at the level of the

reproductive tract tissues during periods of hyperthermia. If

Zebu cattle are more resistant to heat stress at the cellular and

tissue level, the disruptive effects of heat shock on protein and

prostaglandin secretion might be less severe. The objectives of

the present study were to determine whether tissues of the

oviduct and uterine endometrium of Brahman (Bos indicus) and

Holstein (Bos taurus) cattle differ in the synthesis and

secretion of proteins, prostaglandins and DNA in response to in

vitro heat shock. Cyclic cows were sampled at estrus since this






76

represents the time of greatest susceptibility to heat stress in

terms of adverse effects upon conception rates (Dutt et al.,

1959; Dutt, 1963; Dunlap and Vincent, 1971; Gwazdauskas et al.,

1973).


Materials and Methods

Materials

Eagle's minimum essential medium (MEM) was modified by

supplementation with penicillin (100 units/ml), amphotericin B

(250 ng/ml), streptomycin (100 Ag/ml), insulin (.2 units/ml),

non-essential amino acids (1%, v/v) and glucose (5 mg/ml).

Medium was also supplemented with D-calcium pantothenate (100

gg/ml), choline chloride (100 Ag/ml), folic acid (100 Ag/ml),

i-inositol (200 Ag/ml), nicotinamide (100 Ag/ml), pyridoxal-HCl

(100 Mg/ml), riboflavin (10 gg/ml) and thiamine (100 /g/ml).

Content of L-leucine was limited to .1 times normal (5.15 mg/l)

to enhance uptake of L- [4,5-3H] leucine added to cultures. Medium

was filter sterilized (.22 Am) and stored at 4C. All medium

components were from GIBCO (Grand Island, NY).

Materials used in isoelectric focusing, sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and

Western blotting were as follows: tris (hydroxymethyl)amino-

methane (Tris) base, phenylmethylsulfonylfluoride (PMSF),

Nonidet P-40, and N,N,N',N'-tetramethyl ethylenediamine (TEMED)

were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium







77

salicylate, 2-mercaptoethanol, glycine, and ammonium peroxydi-

sulfate were purchased from Fisher Scientific (Orlando, FL).

Acrylamide, urea, dithiothreitol, sodium dodecyl sulfate (SDS),

and amido black 10B were purchased from Research Organics

(Cleveland, OH). Bis-acrylamide, gelatin, and Tween-20 were

purchased from Bio-Rad (Richmond, CA). Carrier ampholytes used

in isoelectric focusing were purchased from Serva (Heidelberg,

FRG). Lutalyse (dinaprost tromethamine) was obtained from The

Upjohn Co. (Kalamazoo, MI). Synchromate-B was purchased from

Ceva Laboratories (Overland Park, KS). L-[4,5-3H]leucine

(SA,-150 Ci/mmol) and [5,6,8,11,12,14,15-3H]PGF2a (SA,-180

Ci/mmole) were purchased from Amersham Corp. (Arlington

Heights, IL) and [methyl- H]thymidine (SA,~6.7 Ci/mmol) was from

New England Nuclear (Boston, MA) Radioinert PGF2, was purchased

from Sigma. Rabbit antiserum to PGF2a was kindly provided by Dr.

T. G. Kennedy at the University of Western Ontario. Enzygnost

serum progesterone kits were a gift from Hoechst-Roussel

Agri-Vet Company (Somerville, NJ). Antibody against a major

mammalian heat-shock protein was generously provided by Dr. W.

J. Welch of the University of California, San Francisco. The

mouse monoclonal antibody (C92) was specific for the inducible

72-kDa heat-shock protein produced by HeLa cells (Welch et al.,

1982). CHAPS (3-[3-cholamidopropyldimethylammonio]-l-pro-

pane-sulfonate) was purchased from Calbiochem (La Jolla, CA).

Horseradish peroxidase (HRP) -conjugated second antibody was from






78

Bio-Rad (Richmond, CA) as was the HRP color development reagent,

4-chloro-l-napthol.

Animals

In experiment 1, six purebred Brahman and five purebred

Holstein cows having active corpora lutea as determined by rectal

palpation received 25 mg of prostaglandin F2a (Lutalyse).

Subsequently cows were given a steroid regimen to ensure that all

cows, irrespective of breed, were exposed to a common hormonal

environment so as to avoid breed-specific variation in steroid

production (Adeyemo and Heath, 1980; Randel, 1980) which might

confound tissue responses to in vitro treatments. Cows received

a norgestomet ear implant (Synchromate-B) 11 d after Lutalyse.

Implants were removed after 14 d and all cows were injected

(i.m.) with 6 mg estradiol cypionate. This pharmacological dose

of a long-acting estrogen was administered to create an estrogen

dominated uterus, as would be the case when embryos are most

susceptible to heat stress. Cows were slaughtered 70 h later,

tissues collected as described below and serum obtained for

determination of progesterone.

Due to the potentially critical nature of events occurring

in the oviduct, the response of oviductal tissue to in vitro heat

shock was examined in a second experiment in order to further

characterize heat-shock induced changes. In this experiment,

oviducts were collected from four cows of various percentages of

Angus and Brahman breeding to examine the effects of in vitro

heat shock on de novo synthesis and secretion of protein by the






79

bovine oviduct. These cows were slaughtered on the day of

naturally occurring estrus and oviducts collected as described

below.

In both experiments, reproductive tracts were collected

following exsanguination and transferred to a sterile laminar

flow hood. The ovaries were examined to determine the site of

preovulatory or recently ovulated follicles. Oviducts were sub-

sequently identified as ipsilateral or contralateral with

respect to the site of ovulation, hereafter called the active

ovary. Entire oviducts ampullaa and isthmus) were trimmed free

of connective and vascular tissue, sliced longitudinally to

expose inner endosalphinx and cut into 2-3 mm3 cubes. Thus, each

cube contained endosalpinx, myosalpinx and serosal layers.

Explants were weighed and cultured as described below. In each

culture, cubes from both isthmus and ampullary regions were

mixed together so that both types of tissue were present.

Uterine horns were opened just above the external bifurcation.

Intercaruncular endometrial tissue from both uterine horns was

dissected free from myometrium and cut into 2-3 mm3 cubes.

Explant Culture

Tissue explants (500 mg) were placed in sterile plastic 100

mm Petri dishes and cultured in 15 ml of modified MEM under an

atmosphere of 50% N2, 47.5% 02 and 2.5% CO2 (v/v/v). Cultures were

maintained in the dark on rocking platforms. In experiment 1,

explants of oviductal tissue were cultured at homeothermic (10

h, 39C) or heat-shock (6 h, 39C; 4 h, 43*C) temperature. At 6






80

h, cultures were pulse-chase labeled. For 2 h, endometrium was

cultured in MEM containing 50 MCi L-[4,5-3H]leucine. At the end

of this period the medium was replaced with MEM containing 51.5

mg/liter L-leucine. Tissue and medium were harvested 2 h later.

Endometrial explants were cultured similarly except that pulse

labeling was performed for 0 to 30, 30 to 60 and 60 to 90 min

following the onset of heat-shock treatment followed by a 2 h

chase period. Pulse-chase labeling also was done for the first

0 to 15 min after the onset of heat shock and considered separa-

tely for statistical analysis. Additional endometrial cultures

were incubated for 24 h at homeothermic or heat-shock tempera-

ture in the presence of 50 MCi L-[4,5-3H]leucine or 25 MCi

[methyl-3H]thymidine.

In the second experiment, oviduct cultures were incubated

at either homeothermic (24 h, 39C) or heat-shock (6 h, 39C; 18

h, 43 C) temperature in the presence of 50 pCi L-[4,5-3H]leucine

added after 6 h. For each experiment, cultures were stopped by

the separation of tissue and medium during centrifugation (700

x g; 30 min; 4C). Tissue was immediately placed in ice-cold

solubilization buffer [50 mM Tris-HCl, pH 7.6, which contained

1 mM phenylmethylsulfonylfluoride, 1 mM EDTA and 2% (w/v) CHAPS]

and homogenized. Samples of tissue and medium were stored at

-20C until analyzed.

Protein Synthesis and Secretion

Secretion of de novo synthesized macromolecules into

culture medium by oviducts and endometrium was determined by






81

measuring incorporation of [3H]leucine into nondialyzable

macromolecules. Conditioned culture medium was dialyzed

extensively (three changes of 4 liters) against deionized water

using dialysis tubing with a 6,000-8,000 dalton exclusion limit.

Radioactivity in the retentate was determined by scintillation

spectrometry. Measurement of [3H]leucine incorporated into

macromolecules in solubilized tissue was done by trichloroacetic

acid (TCA) precipitation. Samples (50 gl) of solubilized tissue

were placed onto Whatman 3MM paper (previously saturated with 20%

TCA [w/v]) and allowed to dry. Proteins were precipitated onto

filter paper by serial washings with 20% TCA, 5% TCA and 95%

ethanol as described by Mans and Novelli (1961). Radioactivity

of precipitated protein was determined by scintillation spec-

trometry.

DNA Synthesis

Incorporation of [3H]thymidine into tissues was determined

by TCA precipitation of nucleic acids onto filter discs (Mani-

atis et al., 1982). Tissue was solubilized in 10 mM Tris which

contained 1 mM EDTA and centrifuged (2,500 x g, 30 min, 4 C) Two

volumes of ice-cold 95% (v/v) ethanol were added to supernatants

and samples were frozen (-200C) overnight. Samples were then

centrifuged (12,000 x g, 10 min, 4 C) and the pellet resuspended

in 200 il of 10 mM Tris containing 1 mM EDTA. An equal volume of

ice-cold 5% (w/v) TCA was then added and samples chilled on ice

for 15 min. An aliquot of each sample was blotted and dried onto

Whatman filters previously treated with 5% (w/v) TCA. Filters






82

were washed five times each with 10% (w/v) TCA and 95% (v/v)

ethanol. Presence of radiolabel was determined by scintillation

spectroscopy.

Measurement of Prostaqlandin F

Medium from cultures incubated for 24 h in the presence of

[H]leucine was assayed for PGF using a RIA procedure (Knicker-

bocker et al., 1986) modified to use an antibody characterized

by Kennedy (1985) that recognizes F series prostaglandins. The

assay was validated for use with MEM in this culture system as

described in Chapter 5.

Electrophoresis

One-dimensional polyacrylamide gel electrophoresis in the

presence of SDS (1-D SDS-PAGE) was performed using the buffer

system of Laemmli (1970). Tissue homogenates were dialyzed

extensively (three changes of 4 liters) against deionized water

using dialysis tubing with a 6,000-8,000 dalton exclusion limit.

Radioactivity in the retentate was determined by scintillation

spectrometry. Proteins were prepared in a solubilization buffer

containing 62.5 mM Tris, pH 6.8, 5% (w/v) SDS, sucrose and 5%

(v/v) 2-mercaptoethanol. Solubilized proteins were resolved on

12.5% (w/v) polyacrylamide gels in the presence of .05% (w/v)

SDS. Two-dimensional SDS polyacrylamide gel electrophoresis (2-D

SDS-PAGE) was performed on proteins secreted into the culture

medium using procedures of Roberts et al. (1984). Samples were

dissolved in 5 mM K2C03, pH 10.5, which contained 9.0 M urea, 2%

(v/v) NP-40 and .5% (w/v) dithiothreitol. Proteins were resolved






83

in the first dimension by isoelectric focusing in 4% (w/v)

polyacrylamide disk gels containing 250 mM N,N' diallyltartar-

diamide (DATD), 8.0 M urea, 2% (v/v) NP-40 and 5.1% (v/v)

ampholytes (pH 3 to 10, 5 to 7 and 9 to 11; 50:36:16 by vol.,

respectively). Disk gels were equilibrated with 60 mM Tris, pH

6.8, 1% SDS (w/v) and 1% (v/v) 2-mercaptoethanol, and subjected

to electrophoresis in the second dimension on 12.5% (w/v)

polyacrylamide gels in the presence of .5% (w/v) SDS. Equal

amounts of radiolabeled protein were loaded onto each gel to

determine qualitative differences due to treatments. Proteins

were fixed in the gels using acetic acid, ethanol and water

(7:40:53, by vol., respectively), and the gels were dried and

fluorographs prepared using sodium salicylate as described by

Roberts et al. (1984).

Western Blotting

Proteins from control and heat-shocked tissue were resolved

by 1-D SDS-PAGE on 7.5% (w/v) polyacrylamide gels. Gels were

equilibrated for 15 min in 25 mM Tris-HCl buffer (ph 6.8) which

contained 200 mM glycine and 20% (v/v) methanol, overlaid with

nitrocellulose membrane (BA85, .45 Im) and subjected to electro-

phoresis (200 mA, 24 h, 4C) toward the cathode. Following

electrophoretic transfer the protein binding sites on the

membranes were blocked by incubation with 3% (w/v) gelatin.

Membranes were then incubated with a monoclonal mouse IgG

specific for the inducible 72-kDa heat-shock protein. Duplicate

membranes were incubated with normal mouse serum to determine the






84

extent of nonspecific binding. Membranes were then incubated

with anti-mouse IgG conjugated to horseradish peroxidase.

Presence of the proteins was detected by incubating with .05%

(w/v) 4-chloro-l-napthol in 100 mM Tris containing 20% (v/v)

methanol and .02% (v/v) hydrogen peroxide.

Serum Proaesterone Determination

Progesterone concentrations in serum were determined

utilizing the Enzygnost serum progesterone kit of Hoechst-

Roussel Agri-Vet Company. This kit utilizes a competitive

enzyme-linked immunoassay for progesterone and alkaline phospha-

tase detection system in which color development is read at 490

nm. Standards included in the kit were used to prepare a

standard curve from 0 to 10 ng/nl. Absorbance at 490 nm of

unknown samples were compared to standard curve.

Statistical Analysis

Data were analyzed by least squares analysis of variance

using the General Linear Models procedure of the Statistical

Analysis System (SAS, 1985) Models used to analyze endometrial

data included effects of breed, cow nested within breed,

incubation temperature of cultures and, for pulse-chase studies,

time of labeling and the interactions of all these effects. To

analyze data from oviductal explants, the model included effects

of breed, cow nested within breed, side of the reproductive tract

relative to the active ovary, incubation temperature, and their

interactions.








Results

Animals

At the time of slaughter of cows in experiment 1, 4 of 6

Brahman cows and 3 of 5 Holstein cows had ovulated as evidenced

by the appearance of a recent ovulation point on the ovary and

the lack of large follicles. One Holstein cow had a serum

progesterone concentration of 5.5 ng/ml and was subsequently

dropped from the statistical analyses, leaving four Holstein

cows in the study. Reproductive tracts of remaining animals had

evidence of estrogenic influence, including highly vascularized,

edematous uteri having a high degree of muscle tone. Serum

progesterone concentrations at slaughter were not different

between breeds and averaged less than 1 ng/ml.

At slaughter, none of the crossbred cows in experiment 2 had

yet ovulated. All exhibited large preovulatory follicles. Uteri

were edematous and vascularized and exhibited a high degree of

muscle tone.

Synthesis and Secretion of r3H1-Labeled Proteins by Oviducts

Secretion of nondialyzable macromolecules into the culture

medium in experiment 1 was affected by a breed x side x tempera-

ture interaction (P < 0.04). This was the result of several

effects. At 39*C, secretion was similar between breeds and

between sides of the reproductive tract relative to the active

ovary. Incubation at 43 C, however, caused an elevation in the

release of secreted macromolecules by explants from the con-

tralateral side in both breeds. For tissue from the side




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INGEST IEID EYROZZ5MA_GSOVY2 INGEST_TIME 2017-07-13T21:27:45Z PACKAGE AA00003750_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



UTERINE, OVIDUCTAL AND CONCEPTUS RESPONSES
TO HEAT SHOCK
BY
JERRY RHEA MALAYER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1990

ACKNOWLEDGMENTS
I would like to express my appreciation and gratitude to my
major professor, Dr. Peter J. Hansen, for encouragement and
support during my time at the University of Florida. His
enthusiasm and encouragement were essential to my successful
completion of this degree. I want to express my gratitude to Dr.
William W. Thatcher, who provided significant guidance and
support. I also want to thank the members of my supervisory
committee, Dr. Fuller W. Bazer, Dr. William C. Buhi, and Dr.
Daniel C. Sharp, III.
I feel very fortunate to have been associated with an
outstanding group of scientists and to have been exposed to a
program of research and graduate education which has few eguals.
I shall always try to live up to the standards of excellence in
research displayed by persons in the reproductive biology group
at the University of Florida.
I want to thank everyone who contributed to the research
described in this dissertation. Dr. Jim Putney, Dr. Tim Gross,
John Pollard, Matt Lucy, Frank Michel, Boon Low, Dr. Claire
Plante, Dr. Steven Helmer, Marie Leslie, David Stephensen, and
Leslie Smith all made important contributions. I also want to
11

express appreciation to everyone in the Dairy Science Department
for making it a very pleasant place to work.
Last, but most importantly, I want to express my gratitude
to my wife, Cindy, for making many sacrifices so that I could
pursue my career goals.
in

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS Ü
LIST OF TABLES vii
LIST OF FIGURES X
ABSTRACT xiv
CHAPTERS
1 REVIEW OF LITERATURE 1
Introduction 1
Maintenance of Homeothermy 4
Effects of Hyperthermia on Reproduction:
Effects at the Reproductive
Tract Level 32
Effects of Hyperthermia on Cells 48
Objectives 71
2 DIFFERENCES BETWEEN BRAHMAN AND HOLSTEIN
COWS IN ALTERATIONS OF PROTEIN SECRETION
BY OVIDUCTS AND UTERINE ENDOMETRIUM
INDUCED BY HEAT SHOCK 7 5
Introduction 75
Materials and Methods 76
Results 85
Discussion 100
3 EFFECT OF DAY OF THE ESTROUS CYCLE, SIDE
OF THE REPRODUCTIVE TRACT AND HEAT SHOCK
ON IN VITRO PROTEIN SECRETION BY BOVINE
ENDOMETRIUM 106
Introduction 106
Materials and Methods 108
Results 114
Discussion 123
IV

4 SECRETION OF PROTEINS BY CULTURED BOVINE
OVIDUCTS COLLECTED FROM ESTRUS THROUGH
EARLY DIESTRUS 132
Introduction 132
Materials and Methods 133
Results 138
Discussion 146
5 HEAT STRESS-INDUCED ALTERATIONS IN
SYNTHESIS AND SECRETION OF PROTEIN
AND PROSTAGLANDIN BY CULTURED BOVINE
CONCEPTUSES AND UTERINE ENDOMETRIUM
COLLECTED AT DAY 17 OF PREGNANCY 152
Introduction 152
Materials and Methods 153
Results 161
Discussion 173
6 EFFECT OF IN VITRO HEAT SHOCK UPON
SYNTHESIS AND SECRETION OF
PROSTAGLANDINS AND PROTEIN BY
UTERINE AND PLACENTAL TISSUES OF
THE SHEEP 177
Introduction 177
Materials and Methods 179
Results 183
Discussion 189
7 REGULATION OF HEAT SHOCK-INDUCED
ALTERATIONS IN RELEASE OF
PROSTAGLANDINS BY UTERINE
ENDOMETRIUM OF COWS 199
Introduction 199
Materials and Methods 201
Results 207
Discussion 212
8 MODULATION BY ALANINE AND TAURINE OF
HEAT SHOCK-INDUCED KILLING OF BOVINE
LYMPHOCYTES AND MOUSE PREIMPLANTATION
EMBRYOS 216
Introduction 216
Materials and Methods 218
Results 221
Discussion 235
v

9 EFFECTS OF EXOGENOUS ALANINE
UPON EMBRYONIC SURVIVAL DURING
MATERNAL HYPERTHERMIA AT TWO
STAGES OF PREIMPLANTATION
DEVELOPMENT 242
Introduction 242
Materials and Methods 244
Results 247
Discussion 252
10 GENERAL DISCUSSION 258
LITERATURE CITED 263
BIOGRAPHICAL SKETCH 302
vi

LIST OF TABLES
Table Page
3-1. In-vitro synthesis and secretion
of radiolabeled, macromolecular
[ H]leucine by endometrial explants
obtained during early stages of the
estrous cycle 116
3-2. Amounts of individual radiolabeled
proteins secreted by cultured
endometrial explants from early
stages of the estrous cycle and
isolated from electrophoretic gels 117
3-3. Effect of incubation temperature
upon secretion of individual
radiolabeled proteins by cultured
endometrial explants obtained
during eary stages of the estrous
cycle 120
3-4. Side x temperature interactions
affecting secretion of individual
proteins from cultured endometrial
explants obtained during early
stages of the estrous cycle 121
3-5. Day of cycle x side interactions
affecting secretion of individual
proteins from cultured endometrial
explants 122
4-1. In vitro synthesis and secretion
of radiolabeled, macromolecular
[ H]leucine by oviductal explants
obtained during early stages of
the estrous cycle 140
4-2. Amounts of individual radiolabeled
proteins secreted by cultured
oviductal explants from early
stages of the estrous cycle 141
Vll

5-1. Incorporation of [3H]leucine
into TCA-precipitable protein
in tissue and medium after
24 h of culture by conceptuses
and endometrium cultured at
homeothermic (39°C) or heat-stress
(43°C) temperatures 162
5-2. Concentrations of prostaglandin
(PG) F and PGE2 in medium
supernatants collected after
24 h of culture of conceptuses
and endometrium at homeothermic
or heat-stress temperatures 162
6-1. Probability values for effects
of individual sources of variation
upon secretion of PGE2, PGF and
[3H]labeled protein and on
accumulation of tissue protein 184
7-1. Prostaglandin F production
from cotyledonary prostaglandin
generating system incubated at
39 °C or 42’C in presence or
absence of endometrial prostaglandin
synthesis inhibitor 210
7-2. Release of PGF and PGE2 by uterine
and placental tissues collected at
Day 70 of pregnancy and incubated
at 39 ° C or 42 ° C 210
8-1. Effects of alanine upon development
and eosin B staining of 4 to 8-cell
embryos 24 h after heat shock 232
8-2. Effect alanine and taurine on
development and eosin B staining
of 4 to 8-cell embryos at 24 h
after heat shock 234
8-3. Effects of alanine and taurine on
development and eosin B staining
of 16-cell embryos 24 h after heat
shock 236
9-1. Effect and ovulation of intraperitoneal
injection of D,L-alanine on litter size
following maternal hyperthermia
viii
249

9-2. Effect of intraperitoneal injection of
D,L-alanine on embryos per CL following
maternal hyperthermia 250
9-3, Effect of intraperitoneal injection of
D,L-alanine on embryo mean weight of
individual conceptus following maternal
hyperthermia 251
ix

LIST OF FIGURES
Figure Page
2-1. Secretion of radiolabeled,
nondialyzable macromolecules into
culture medium by oviductal explants 87
2-2. Incorporation of [3H]leucine into
TCA-precipitable macromolecules
in tissue homogenates 88
2-3. Incorporation of [3H]leucine into
non-dialyzable macromolecules in
culture supernatants and incorporation
of [3H]leucine into TCA-precipitable
macromolecules by explants of oviductal
tissue from crossbred cows 91
2-4. Secretion of nondialyzable
[3H]leucine-labeled macromolecules by
endometrial explants in the first 0 to 30,
30 to 60 and 60 to 90 min after onset
of heat shock 92
2-5. Incorporation of [3H]leucine into
TCA-precipitable macromolecules
in tissue of endometrial explants 94
2-6. Electrophoretic analysis of oviductal
secretory proteins 95
2-7. Electrophoretic analysis of endometrial
secretory proteins 97
2-8. Electrophoretic analysis of endometrial
tissue proteins 98
2-9. Western blot analysis of the appearance
of inducible heat shock protein in
representative samples obtained 2.5 h
after the onset of heat shock treatment 99
x

3-1. Representative fluorographs of
two-dimensional SDS-PAGE of
secretory proteins from bovine
endometrial explants obtained at
Days (a) 0 (estrus) , (b) 2, (c) 5
and (d) 8 of the estrous cycle 125
3-2. Representative fluorographs of
one-dimensional SDS-PAGE of
tissue proteins solubilized from
bovine endometrial explants
incubated at (a) 39°C or (b) 43°C 126
3-3. Western blotting of bovine heat-shock
proteins 127
4-1. Representative fluorographs of 2-D
SDS-PAGE of secretory proteins from
cultured bovine oviducts obtained at
A) Day 0 (estrus) , B) Day 2, C) Day 5
and D) Day 8 of the estrous cycle 147
4-2. Representative fluorographs of 1-D
SDS-PAGE of de novo synthesized
proteins in culture supernatants
and tissue homogenates from cultured
bovine oviducts 148
5-1. Incorporation of [3H]leucine into
TCA-precipitable macromolecules in
culture medium by endometrial and
conceptus tissues and incubated at
homeothermic or heat shock temperature 164
5-2. Two-dimensional polyacrylamide gel
electrophoresis of proteins present
in endometrial and conceptus tissues 165
5-3. One-dimensional polyacrylamide gel
electrophoresis of conceptus secretory
proteins 166
5-4. One-dimensional polyacrylamide gel
electrophoresis of endometrial
secretory proteins 167
5-5. Electrophoretic profile of
[3H]leucine-labeled polypeptides
accumulated in conceptus
culture medium after 24 h of
culture 17 0
xi

5-6. Immunoblotting of bovine trophoblast
protein-1 (bTP-1) released by
conceptuses during 24 h of culture 171
5-7. Release of prostaglandins (PGF and PGE2)
into culture medium by endometrium
and conceptuses 172
6-1. Release of PGE2 from ovine tissue
explants of maternal (intercaruncular
and caruncular endometrium) and
fetal (cotyledon and chorioallantois)
tissues collected at Day 100 or Day
140 of pregnancy 186
6-2. Release of PGF from ovine tissue
explants of maternal (intercaruncular
and caruncular endometrium) and fetal
(cotyledon and chorioallantois) tissues
collected at Day 100 or Day 140 of
pregnancy 187
6-3. Release of PGE2 (upper panels) and PGF
(lower panels) from ovine tissue
explants of maternal tissues from the
nongravid and gravid uterine horns
collected at Day 140 of pregnancy 188
6-4. Secretion of [3H]leucine-labeled
macromolecules from ovine tissue
explants of maternal (intercaruncular
and caruncular endometrium) and fetal
(cotyledon and chorioallantois) tissues
collected at Day 100 or Day 140 of pregnancy .... 190
6-5. Incorporation of [3H]leucine into
macromolecules present in tissue
homogenates of ovine tissue explants
of maternal (intercaruncular and
caruncular endometrium) and fetal
(cotyledon and chorioallantois)
tissues collected at Day 100 or
Day 140 of pregnancy 191
6-6. Secretion and tissue incorporation
of [3H]leucine-labeled
macromolecules from ovine
tissue explants of maternal
tissues from the nongravid and
gravid uterine horns collected
at Day 140 of pregnancy 192
Xll

6-7. Representative fluorographs of
[3H]leucine-labeled macromolecules
in ovine uterine and placental
tissues resolved by 1-D SDS PAGE 194
7-1. Effect of PAF and incubation temperature
on secretion of PGF and PGE2 by bovine
endometrial explants 209
7-2. Dose-response of PGF secretion
at 39 °C and 42 °C to increasing
concentrations of PAF 211
8-1. Lymphocyte survival after 1 h
heat shock at 45°C 223
8-2. Dose-response effects of
alanine and taurine 224
8-3. Effect of D-alanine upon
lymphocyte survival 226
8-4. Effect of L-alanine upon
lymphocyte survival during
prolonged heat shock in vitro 227
8-5. Effect of heat shock and
L-alanine upon mouse embryo
development in vitro 229
8-6. Effect of L-alanine upon development
and viability during heat shock in
vitro 230
9-1. Incorporation of D,L-[1-KC]alanine
into tissues following intraperitoneal
injection 253
Xlll

Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
UTERINE, OVIDUCTAL AND CONCEPTOS RESPONSES TO HEAT SHOCK
by
Jerry Rhea Malayer
May 1990
Chairman: Peter J. Hansen
Major Department: Animal Science
Direct effects of elevated temperature on maternal repro¬
ductive tract tissues and conceptuses were examined in vitro.
Protein secretion by bovine endometrial and oviductal explants
varied with day of estrous cycle and secretion rate increased in
response to elevated temperature at estrus, but not during the
remainder of the estrous cycle and early pregnancy. Protein
secretion by explants of ovine utero-placental tissues in late
pregnancy was not affected by elevated temperature. All bovine
and ovine tissues examined produced heat-shock proteins identi¬
fied by SDS-PAGE and immunological methods.
Secretion of 7 peptides was reduced by heat shock only in
explants from the uterine horn ipsilateral to the corpus luteum.
Protein secretion by explants of the ipsilateral oviduct was more
sensitive to heat shock. Endometrial and oviductal tissue
xiv

responses of thermotolerant Brahman cattle were compared to
tissues from more heat-sensitive Holsteins. Variation in
responses of tissues to heat shock was consistent with greater
thermal tolerance of Bos indicus cattle.
Heat shock disrupted events critical to luteal maintenance
at day 17 of pregnancy in the cow, increasing endometrial
secretion of luteolytic PGF and depressing the conceptus signal
for luteal maintenance. Increased PGF secretion was not due to
incapacity of regulatory proteins or increased cyclooxygenase
activity. Heat shock increased PGF secretion at day 17 of the
estrous cycle and pregnancy, but had no effect on PGF release
from other tissues examined. Heat shock depressed PGE2 release
from caruncular and intercaruncular endometrium collected from
pregnant ewes.
Using murine embryos in vitro, heat shock was found to block
development and cause increased mortality. This could be
partially blocked by alanine and taurine. In vivo heat shock of
pregnant mice at times before or after development of embryonic
capacity to produce heat-shock proteins in response to heat
stress resulted in egually reduced numbers of embryos per corpus
luteum. There was no benefit of intraperitoneal injection of
alanine although alanine was sequestered in the oviduct in
relatively high amounts per milligram of tissue.
In conclusion, direct thermal effects on maternal and
conceptus tissues may act in addition to maternal systemic
perturbations during hyperthermia.
xv

CHAPTER 1
REVIEW OF THE LITERATURE
Introduction
Efficiency of reproduction in animals is sensitive to various
environmental factors including temperature, nutrient avail¬
ability, photoperiod and social interactions. Pregnancy rates
are greatly reduced during periods of elevated environmental
temperature in various species, including cattle (Stott and
Williams, 19 62 ; Posten et al. , 1962 ; Bad inga et al. , 1985) , sheep
(Dutt et al., 1959; Dutt, 1963), pigs (Warnick et al., 1965;
Edwards et al. , 1968) and rabbits (Ulberg and Sheean, 1973 ;
Wolfenson and Blum, 1988). Adverse effects of elevated ambient
temperature on fertility may be caused by effects on various key
events of reproduction in mammals. There are negative effects
on gamete development (Bellvé, 1973; Lenz et al., 1983; Baum¬
gartner and Chrisman, 1988; Putney et al., 1989a) and on survival
of the early cleavage-stage embryo during the period after
fertilization (Dutt et al., 1959; Dunlap and Vincent, 1971;
Elliott and Ulberg, 1971; Ulberg and Sheean, 1973; Gwazdauskas
et al., 1973; Putney et al., 1988a). Adverse effects of high
temperature can also be seen during later stages of preimplan¬
tation development (Omtvedt et al., 1971; Monty, 1984; Biggers
et al., 1987; Geisert et al., 1988; Wettemann et al., 1988),
1

2
during embryonic organogenesis (Trujano and Wrathall, 1985;
Mirkes, 1987) and during mid- to late-gestation (Alexander and
Williams, 1971; Omtvedt et al., 1971; Collier et al., 1982; Lewis
et al., 1984) .
The effects of climate and season upon productivity and pro¬
lificacy of animals were observed by ancient peoples. No doubt
the survival of many groups of early people depended on an
understanding of the seasonal changes in their environment and
of the life-cycles of the animals upon which they depended. Much
knowledge of the Greek classical period has come down to us
through the writings of historians and philosophers and persons
such as Hippocrates (ca. 400 B.C.) , who wrote:
For knowing the changes of the seasons, the risings and
settings of stars, how each of them takes place, he will be
able to know beforehand what sort of year is going to
ensue.... And if it shall be thought that these things
belong rather to meteorology, it will be admitted, on second
thoughts, that astronomy contributes not a little, but a
very great deal, indeed, to medicine.
A city that is exposed to hot winds. . . .and the following
diseases are peculiar to the district: in the first place,
the women are sickly and subject to excessive menstruation;
then many are unfruitful from disease, and not from nature,
and they have frequent miscarriages....
but such parts of the country as lie intermediate bethBBi
the heat and cold are best supplied with fruits and trees. . .
the cattle also which are reared there are vigorous,
particularly prolific, and bring up young of the fairest
description.... In: On Airs, Waters, and Places. Great
Books of the Western World. W. Benton (ed.). Vol. 10,
Encyclopedia Britannica, Inc., Chicago, pp. 9-18, 1952.
Aristotle was an astute observer of nature whose writings
on a vast array of topics ranging from logic and politics to life
and death (including treatises on animal reproduction and
meteorology) influenced science for over a thousand years.

3
Aristotle recognized the periodicity of sexual activity of
domestic animals; he wrote, for example, that the cow would
accept the bull every 21 days. He described the cervical mucus
of the cow in estrus and related that to the optimal breeding
period. He ascribed this mucus to menstrual discharge, on the
assumption that all female animals were alike in this respect.
The concept of heat was integral to Aristotle's view of the
underlying mechanisms of life:
In a similar fashion as the fish move their gills,
respiring animals with rapid action raise and let fall the
chest according as the breath is admitted or expelled. If
air is limited in amount and unchanged they are suffocated,
for either medium, owing to contact with the blood, rapidly
becomes hot. The heat of the blood counteracts refrigera¬
tion and, when respiring animals can no longer move the lung
or aquatic animals their gills, whether owing to disease or
old age, their death ensues.
The source of life is lost to its possessors when the
heat with which it is bound up is no longer tempered by
cooling, for as I have often remarked, it is consumed by
itself. In: On Life and Death. Great Books of the Western
World. W. Benton (ed.). Vol. 8, Encyclopedia Britannica,
Inc., Chicago, pp. 714-726, 1952.
Aristotle (ca. 330 B.C.)
The concept of heat as kinetic energy arose in the mid-19th
century when Browne recognized the constant motion of particles
in fluid media (Cossins and Bowler, 1987), thereafter termed
"Brownian motion". About the same time, Ludwig Boltzman con¬
ceived of heat as a random motion of atoms (Bronowski, 1973).
Thermal energy is now defined as the energy of motion of the
atoms and molecules of which matter is composed (Cossins and
Bowler, 1987).

4
It is the input of energy, in the form of increased atomic and
molecular motion, into a thermodynamically-balanced biological
system, and the changes brought about by such an input, that will
be the focus of this review. Several levels of integration will
be considered, beginning in general terms with maintenance of
homeothermy in the whole animal including mechanisms of heat
exchange, thermoregulation and responses to hyperthermia. Next,
thermal effects at the tissue level, specifically tissues
related to reproductive function, will be considered. Finally,
thermal effects upon cellular functions will be discussed
including thermal effects on dynamics of their constituent
macromolecules.
Maintenance of Homeothermy
We have defined heat as the kinetic energy of atoms and mole¬
cules; temperature describes the intensity of the kinetic motion
of the atoms and molecules of a system. Temperature has no
physical units and must be measured by reference to an arbitrary
scale. Thus temperature is useful only to predict the direction
of heat flux along a thermal gradient.
Cowles (1962) classified vertebrate animals according to
characteristics of their body temperature (Tb) regulation.
Poikilotherms, so called cold-blooded animals, are those such as
reptiles and amphibians whose Tb is labile and varies with
changes in ambient temperature (Ta) ; it is important to note that
while Tb is labile, Ta and Tb are not equal. Homeotherms have

5
evolved mechanisms to maintain a relatively constant Tb even
though Ta might vary. Birds and mammals are the principal
examples of such animals in nature. The source of heat used to
establish Tb is the basis of a parallel classification. Ecto-
therms depend upon heat sources external to the body, such as
solar radiation, air or water temperature, to set Tb. Endo-
therms, on the other hand, are animals whose Tb is based on energy
derived from cellular metabolism. Many ectotherms have evolved
strategies for balancing low metabolic heat with tight control
of heat exchange with the environment to maintain Tb within a
fairly narrow range. Cowles (1962) emphasized that most, if not
all, ectothermic animals have specific thermal preferences.
Dreisig (1984) described behavior patterns of ectotherms as
alternative shuttling between microclimates, i.e., alternative
sun to shade shuttling, designed to achieve preferred Tb. As the
Ta increases during the day, there is a basking-activity phase in
which preferred Tb is attained. This is followed by a control
phase in which maintenance of Tb is accomplished through behav¬
ioral responses such as basking, alterations of activity level
and sun to shade shuttling.
Endotherms have a relatively higher level of metabolic heat
production coupled with mechanisms for its conservation, which
results in maintenance of Tb greater than Ta. Mammals and birds
have developed complex mechanisms for regulation of heat
exchange with the environment to maintain a constant internal
temperature.

6
Heat Exchange
Animals exchange heat with the environment in several ways.
Three modes of energy transfer depend upon thermal gradients and
can therefore be sensed by a thermometer (and are thus referred
to as sensible heat flow) are conduction, convection and radia¬
tion. Two other routes of energy transfer are evaporation and
condensation. These occur along a vapor-pressure gradient, not
a thermal gradient, and are called insensible or latent heat
flows. As water molecules move from the liquid to the gaseous
state, their kinetic energy of motion does not increase, thus
temperature does not change. Energy is released through
breakdown of the noncovalent bonds which hold the water molecules
together.
Radiant-energy transfer is the result of conversion of heat
energy into electromagnetic-wave energy by the emitter, passage
of these waves (requiring no medium) through space and reconver¬
sion of electromagnetic-wave energy into heat energy in the
absorber. This transfer is dependent upon several physical
characteristics of the emitter and absorber bodies; their
emissivity, reflectivity and absorbtivity. The net radiant-
thermal flux can be expressed mathematically by describing these
physical characteristics of emitter and absorber as well as
surface area of the animal, the surface and ambient environmental
temperatures, and an expression called the Stefan-Boltzman
constant which accounts for the radiant emissive power density
(a feature of the surface temperature of the emitter).

7
Conductive-heat exchange is transfer of heat through a medium
without material movement or transfer. This transfer is based
upon movement of higher energy molecules into collisions with
lower energy molecules; when they collide some of the energy is
transferred. The net conductive-thermal flux can be described
mathematically using the conductive surface area, the thermal
conductivity of the substance (which is dependent on the density
of molecules in the conducting material) , and the environmental
temperature at the surfaces of the conducting substances.
Heat exchange by convection occurs by movement of streams of
molecules from a warm to a cool place. Convective heat flux is
modelled using convective-surface area, ambient temperature and
the convection coefficient which describes the thermal conduc¬
tivity and thicknesses of boundary layers on surfaces where
convective movement of the medium cannot reach.
Animals may lose heat by evaporation of water from the skin
and respiratory tract surfaces. Evaporation occurs when the
molecules of a liquid acquire sufficient kinetic energy to
overcome cohesive forces in the liquid. The removal of energy
from the liquid in the form of breakdown of the noncovalent
interactions holding the water molecules together results in
heat loss. The evaporative heat flux is dependent upon the
latent heat of water at the surface temperature of the liquid,
the surface area of the liquid, the evaporation diffusion
coefficient (which is based on air velocity at the evaporative
surface), and the vapor-pressure gradient.

8
Regulation of Body Temperature
Homeotherms regulate their Tb within a relatively narrow
range called the thermal-comfort zone or zone of thermo¬
neutrality (Curtis, 1981; Bligh, 1984). Within this range of
temperature, the animal does not undergo temperature-induced
changes in metabolic rate and does not need to expend energy to
maintain body temperature. Regulation of core temperature
within the thermal-comfort zone is accomplished primarily by
autonomic control of the tone of the cutaneous vasculature and
by behavioral means such as changes in posture to alter heat
exchange with the environment (Bligh, 1984) . Thermoregulation
is accomplished through feedback and feedforward interactions
between thermal receptors and thermal effectors. Integration
between these receptors and effectors occurs in the thermo¬
regulatory center of the hypothalamus (Curtis, 1981; Stitt,
1983; Bligh, 1984; Cossins and Bowler, 1987).
The Set Point
Regulation of thermal-energy flux requires a high degree of
precision in order to control errors which might result in
drifting away from the preferred Tb. This has led to the concept
of a homeostatic regulator of Tb. The so-called thermal "set
point" is maintained by assessing input of thermal energy, both
directly by temperature-sensitive neurons in the central nervous
system and via neural input from thermal receptors in the
periphery (Simon, 1981; Curtis, 1981; Stitt, 1983; Bligh, 1984;
Cossins and Bowler, 1987) , and adjusting heat-loss or heat-gain

9
effectors accordingly. The set point is the physiological state
at which the sum of all inhibitory and stimulatory inputs cancel
each other out (Bligh, 1984) .
Theories of set-point homeostasis depend mechanistically on
the concept that Q10 for firing rate in the neurons of the
hypothalamic thermoregulatory center differs for heat and cold
and that several populations of neurons are present, some
sensitive to cold, some sensitive to heat. The Q10, or the van't
Hoff effect, describes the change in rate of biological processes
with changing temperature and can be represented by the expres¬
sion
Qio = ^to + 10/ ^To >
where kTo = reaction velocity at initial temperature,
and kTo+10 = reaction velocity after 10°C increase.
In general, the Q10 for biological systems in the range of
temperature common to mammals is 2, although this prediction is
not linear over a wide range of temperature (Cossins and Bowler,
1987). Temperature-sensitive neurons in the hypothalamus and
spinal cord of mammals have been demonstrated to alter their
activity in response to local warming and cooling (Baldwin and
Ingram, 1966 ; Whittow and Findlay, 1968; Forsling et al., 1976).
Cattle respond to hypothalamic warming by panting (Whittow and
Findlay, 1968), and this response varies directly with Ta, a
result in accordance with the idea of both central and peripheral
neural inputs. Hypothalamic warming also induces changes in
thermoregulatory behavior in pigs (Carlisle and Ingram, 1973)

10
and sheep (Whittow and Findlay, 1968) . Studies in humans
(Benzinger, 1969), dogs (Chatonnet, 1983) and pigs (Baldwin and
Ingram, 1966) have shown that responses to altered brain
temperature vary directly with skin temperature, an indication
of an integration of both peripheral and central sites of thermal
responsiveness. In a similar manner, cooling the preoptic area
of pigs during elevation of Ta blocks endocrine responses to
hyperthermia (Forsling et al., 1976) and alters thermoregulatory
behavior (Baldwin and Ingram, 1966), and integration with
peripheral receptors modulated thermoregulatory responses to
hypothalamic cooling (Baldwin and Ingram, 1966). Baldwin and
Parrott (1984) placed electrodes in the dorsal preoptic area of
sheep and examined the effect of electrical stimulation on
thermoregulatory responses of conscious animals. Electrical
stimulation elicited a concurrent thermoregulatory response
characterized by increased respiration rate, vasodilation of
peripheral blood vessels and lowered Tb. Stimulation of the
dorsal preoptic area inhibited shivering when ewes were exposed
to 5°C. After the ewes had been shorn, resulting in exposure of
cold receptors in the skin, thermoregulatory response to
electrical stimulation at 5'C was reduced; a further indication
of the integration of peripheral and central response elements.
Large changes in the temperature of the hypothalamus and spinal
cord of active birds have been recorded without activation of
thermoregulatory mechanisms however (Simon, 1981).

11
Catecholamines (adrenaline, noradrenaline) and 5-hydroxy-
tryptamine can all induce change in Tb when injected into
cerebral ventricles of the cat (Feldberg and Myers, 1963) .
Experiments of this type led to the concept of a neurochemical
set point (Feldberg and Myers, 1963). However, results of a
number of experiments of this type involving injection of
monoamines into cerebral ventricles of several species were
inconsistent. By comparing the neurotransmitter-induced changes
at different Ta in order to challenge the thermoregulatory
system, it was determined that the exogenous neurotransmitters
inhibited whichever heat-flux system was being driven at the
time. At low Ta, 5-hydroxytryptamine or noradrenaline injected
into cerebral ventricles of sheep caused lower heat production
and lower rectal temperature (Bligh, 1979). At high Ta, nora¬
drenaline injection caused reduced respiration rates and
increased Tb (Bligh, 1979) . Denervation of the medullary
adrenal, a major source of catecholamines, abolished thermoregu¬
latory responses to hypothalamic warming in cattle (Robertshaw
and Whittow, 1966), demonstrating the importance of catecho¬
lamines in thermoregulation.
Hyperthermia
As Ta increases toward the upper critical temperature, i.e. ,
the temperature at which metabolic rate begins to increase,
mechanisms to facilitate heat loss are initiated. This point
will vary among animals of a species. Kibler (1957) found that
metabolic rate of Brahman heifers exposed to elevated ambient

12
temperature (80°F) was lower than that of Shorthorn heifers.
Reduction of tissue insulation by vasodilation and increasing
surface area through changes in posture are the earliest
manifestations of heat-loss mechanisms (Curtis, 1981) . These
are attempts to increase conductive- and convective-heat
exchange with the environment. Additionally, behavioral changes
such as seeking shade (Seath and Miller, 1946; Robertshaw, 1986)
and reduced daytime activity (Payne et al., 1951) help to
increase radiant-heat loss. The upper critical temperature is
defined as the point at which metabolic energy production begins
to increase. Alternatively, Nichelmann (1983) described studies
in birds and mammals demonstrating that the relationship between
Ta and the metabolic energy level of animals is a parabola; the
vertex of the parabola is referred to as thermoneutral tempera¬
ture. In domestic animals, where even slight diversion of
metabolic energy away from productive activity manifests itself
in lowered productivity, such as in the lactating dairy cow
(Thatcher and Collier, 1982), thermoneutral temperature is a
useful concept.
As Ta continues to rise and thermal gradients reduce heat
exchange away from the animal, active heat-loss mechanisms must
be initiated. Sweating and panting are modes of evaporative
heat loss which require no thermal gradient and thus are critical
when thermal gradients favor heat flux toward the animal. These
active heat-dissipation mechanisms require the expenditure of
energy which slightly increases the metabolic heat load of the

13
animal in addition to the larger Q10 increase in metabolic heat
generation (Whittow and Findlay, 1968; Ames et al., 1971). As
the Ta continues to rise, a point is reached where the ability to
dissipate heat via evaporative means is maximal. As the upper
critical temperature threshold is exceeded, metabolic heat
production exceeds the capacity of the animal to dissipate its
heat load and it becomes hyperthermic. The resulting rise in Tb
results in a further rise in metabolic rate measurable as
increased 02 consumption (Whittow and Findlay, 1968; Ames et al. ,
1971) , a result of the Q10 effect. The increase in metabolic rate
results in a further increase in heat production which further
increases metabolic rate; the resulting positive feedback effect
sequence has been called spiralling hyperthermy (Curtis, 1981) .
Physical and Chemical Means to Maintain Homeothermv
The physiological responses of cattle to hot environments
have been the subject of many reviews over the past 30 years
(Yeck, 1959; Bianca, 1965; Thatcher, 1974 ; Thatcher and Collier,
1982, 1986; Gwazdauskas, 1985; Beede and Collier, 1986), an
indication of the tremendous impact of hyperthermia upon
productivity in cattle. Maintenance of homeothermy is given such
a high priority that other physiological systems are challenged
(Thatcher and Collier, 1982), and this homeokinesis results in
depression of many productive traits such as growth, lactation
and reproduction. The prioritization of physiological systems,
though detrimental in terms of agricultural production, is a
characteristic with evolutionary advantages for mammals.

14
Homeokinesis to regulate Tb in response to increasing Ta can
be divided into two main categories; physical and chemical.
Physical adjustments are attempts to promote conductive,
convective and evaporative heat flux. There is dilation of
cutaneous arterial-venous anastomoses to reduce tissue and body-
cover insulation and carry heat from the body core to the
periphery, thus promoting a thermal gradient. Additionally,
increased respiration rate and thermal sweating promote evapora¬
tive heat flux. Chemical adjustments are attempts to reduce
generation of metabolic heat and to support the physical
adjustments. Nutritional changes as well as alterations in water
turnover and intake, electrolyte balance, and osmoregulation
occur when Tb increases; many of these changes are integrated by
alterations in endocrine activity.
Cardiovascular changes
At rest, cutaneous blood flow accounts for a very small
fraction of cardiac output (Hertzman, 1959) . Increased skin
temperature results in increased cardiac output (Bianca and
Findlay, 1962; Ingram and Whittow, 1963; Whittow, 1965; Marple
et al. , 1974) , primarily a result of increased heart rate because
stroke volume decreases (Whittow, 1965). Elevated temperature
also results in dilation of cutaneous arterial-venous anas¬
tomoses and capillaries (Whittow, 1965), and increased cutaneous
blood flow (Hales, 1983 ; Hales et al. , 1984). Peripheral
vascular tone is under sympathetic neuronal control, and regula¬
tion occurs chiefly in the periphery (Brown and Harrison, 1981) .

15
Movement of blood from the body core to the skin results in
increased skin temperature, creating a sensible heat-loss
gradient that allows thermal energy to move toward the environ¬
ment and away from the animal. In particular, blood flow to the
extremities, i.e., the legs, tail, ears, snout and dewlap in
cattle, increases during periods of elevated temperature
(Whittow, 1962) , while abdominal and thoracic blood flow remain
relatively constant. Alteration of blood flow patterns affects
organ perfusion, such is the case in heat-stress-induced
decrease in uterine blood flow (Gwazdauskas et al., 1973;
Alexander and Williams, 1971; Brown and Harrison, 1981).
Seasonal changes in blood volume occur in humans (Yoshimura,
1958), and warmer seasons are characterized by increased blood
volume in animals (Dale et al., 1956; Bianca, 1957; Whittow,
1965).
Changes in respiration rate
An important response to elevated temperature is increased
respiration rate. Respiration rate may increase from 30 to 300%
in pigs, sheep, goats and cattle (Riek and Lee, 1948; Appleman
and Delouch, 1958; Judge et al., 1973; Baldwin and Parrott,
1984) . Initially, increased respiration rate is characterized
by rapid, shallow breathing resulting in increased respiratory
volume (liters per min) and lower tidal volume (liters per
breath), while not affecting alveolar gas exchange (Bianca and
Findlay, 1962; McDowell et al., 1969). The purpose of this
panting is increased evaporative heat flux from the respiratory

16
tract (Whittow and Findlay, 1968; Ames et al., 1971). Findlay
(1956) determined that a rectal temperature of 40.5°C was the
threshold for increased respiration rate in calves. At severe
hyperthermia, the respiratory pattern may change to a lower-
frequency, higher-tidal-volume pattern called second-stage
breathing (Bianca and Findlay, 1962). During second-stage
breathing, the potential for blood alkalosis occurs (Brown and
Harrison, 1981) as the increased alveolar ventilation rate
results in decreased blood pC02 and increased blood pH (Dale
and Brody, 1954; Bianca and Findlay, 1962; Judge et al., 1973;
Brown and Harrison, 1981) .
One consequence of increased respiratory rate is increased
thermal energy production due to utilization of metabolic
energy. Oxygen consumption of hyperthermic cattle (Tb = 41.5°C)
increased 67% (Whittow and Findlay, 1968) . The increase in 02
consumption is not linear with increasing rectal temperature,
however (Whittow and Findlay, 1968) . By observing this and other
aspects of increased 02 consumption, Whittow and Findlay (1968)
and Ames et al. (1971) determined that panting in the cow and
sheep accounted for only a small portion (about 10%) of the
increase in metabolic energy utilization. Ames et al. (1971)
concluded that most of the increase in 02 consumption above the
upper critical temperature was due to the Q10 effect.
Changes in physiological fluids
Water intake of animals exposed to elevated temperature
increases as both volume and frequency of drinking increase

17
(Appleman and Delouch, 1958; Johnson and Yeck, 1964; Kelley et
al., 1967 ; McDowell et al. , 1969; Gengler et al., 1970; Wettemann
et al., 1988). Influx of cool water provides immediate relief
from hyperthermia as heat can be exchanged down a thermal
gradient from the body to the contents of the gut, whereas intake
of warm water elicits no such immediate effect (Bianca, 1964).
Total body water content increases in water buffaloes and
Friesian cattle during the summer (Kamal and Seif, 1969) . Blood
volume increases in cattle exposed to elevated temperature (Dale
et al. , 1956; Bianca, 1957 ; Whittow, 1965); however, when water
intake is restricted during hyperthermia, osmolality of the
blood serum increases (El-Nouty et al. , 1980). Indirect evidence
of increased blood volume, in the absence of a pathological
state, is a reduction of the volume of packed red blood cells per
unit volume of whole blood, i.e., the hematocrit. Dale et al.
(1956) measured both blood volume, using dye-dilution methods,
and hematocrit; blood volume increased and hematocrit decreased
in heat-stressed heifers. The hematocrit is lower during
hyperthermia in pigs (Forsling et al., 1976), goats (Appleman and
Delouch, 1958), heifers (Dale et al., 1956) and lactating cows
(McDowell et al. , 1969).
Skin evaporation increases during exposure to elevated
temperature (Riek and Lee, 1948; Joshi et al., 1968; McDowell et
al., 1969; Ames et al., 1971) and there is increased salivation
and licking (Riek and Lee, 1948) . Animals such as sheep, goats,
horses and cattle have sweat glands associated with the hair

18
follicles called epitrichial sweat glands (Allen and Bligh,
1969). These glands are surrounded by a layer of myoepithelium
which is stimulated to contract by epinephrine and norepi¬
nephrine; the glands actively secrete fluid in response to
increasing skin temperature as well as in response to adrenergic
stimulation, though no direct innervation has been demonstrated
(Joshi et al., 1968; Allen and Bligh, 1969) . Sweating can occur
due to increased glandular secretion rate of fluid to the skin
surface (bulk flow) or due to contractions of myoepithelial cells
to expel fluid (Allen and Bligh, 1969) . Allen and Bligh (1969)
examined patterns of sweating in several species of domestic
animals. Sheep and goats do not sweat continuously, but rather
in bursts, apparently in response to stimulation of the myoepi¬
thelium. This stimulation was mimicked with injections of
adrenaline (Allen and Bligh, 1969). Cattle exhibit continuous
sweating with periodic increases in discharge, also a result of
stimulation of myoepithelial contractions (Joshi et al. , 1968;
Allen and Bligh, 1969). The skin secretions of cattle contain
sodium, potassium, magnesium, chloride, phosphate, lactate and
nitrogen (Joshi et al., 1968; Jenkinson and Mabon, 1973; Singh
and Newton, 1978) in concentrations greater than in blood plasma.
Electrolytes are actively transported into the duct lumen
creating an osmotic gradient which causes water to move into the
lumen; as the fluid moves through the duct on its way to the skin
surface, the electrolytes may be recovered from the fluid and
conserved (Guyton, 1981). In contrast to humans which secrete

19
sweat high in sodium (Guyton, 1981), the secretions of cattle
contain 3 to 5 times as much potassium as sodium (Johnson, 1970) .
Water is conserved by increased reabsorption in the kidney
as a result of increased antidiuretic hormone (ADH) concentra¬
tion (Forsling et al., 1976; El-Nouty et al., 1980). Cattle
retain water during periods of heat stress, but in contrast to
nonruminants, urinary sodium concentration and urine osmolality
do not change significantly, this is a result of low aldosterone
concentrations (El-Nouty et al., 1980). During dehydration,
aldosterone concentrations do rise in cattle (El-Nouty et al.,
1980) . During heat stress in nonruminants, the serum aldosterone
concentration is high and stimulates reabsorption of sodium from
the urine (Guyton, 1981). This further concentrates the urine,
which already has a low volume due to increased ADH activity.
The nonruminant utilizes sodium in the skin secretions and is
predisposed to conserve the body store for this purpose; the
extracellular concentration of sodium increases during warm
seasons (Yoshimura, 1958). The ruminant, on the other hand,
utilizes greater amount of potassium in the skin secretions
(Johnson, 1970) and sodium conservation is less critical.
Seasonal changes in water content and electrolyte concentra¬
tions in body fluids occur in humans. Blood concentrations of
serum protein, sodium, chloride and potassium are lower in summer
(June, July and August) than in winter (December, January,
February) in humans (Yoshimura, 1958). The sodium to potassium
ratio is higher in summer as is total blood volume, serum volume

20
and total sodium in extracellular fluid (Yoshimura, 1958), the
result of a sodium-conserving mechanism.
Changes in nutritional patterns
Feed intake is reduced in response to elevated temperature
in cattle, leading to reduced heat generation during ruminal
fermentation and to lower body metabolism in cattle (Johnson and
Yeck, 1964; McDowell et al., 1969; Gengler et al., 1970; Baile
and Forbes, 1974) , goats (Appleman and Delouch, 1958) and sheep
(Cartwright and Thwaites, 1976). Dry matter intake begins to
decline in lactating cattle at ambient temperature of about 25°
to 27 °C (Beede and Collier, 1986), possibly as a result of
depression of the hypothalamic appetite center (Baile and
Forbes, 1974). Alternatively, reduced gut motility and rate of
feed passage might lead to depressed appetite (Beede and Collier,
1986) and alteration of blood flow patterns away from the
alimentary tract may reduce nutrient absorption. When feed
intake of cattle was controlled by intraruminal infusion, the
ratios of volatile fatty acids were altered by elevated tempera¬
ture as acetic acid content of the rumen increased and propionate
concentration decreased (Kelley et al. , 1967) . When feed intake
declines, so do fatty acid concentrations (Gengler et al. , 1970).
In lactating cattle, the decrease in digestible energy intake was
exceeded by the decrease in milk energy output, indicating that
maintenance requirements had increased during heat stress
(McDowell et al., 1969). Increased maintenance requirements
coupled with reduced intake of digestible energy and altered

21
volatile fatty acid concentrations leads to lowered productivity
of lactating or growing cattle.
Changes in endocrine function
Short-term exposure to elevated temperature results in
increased concentrations of catecholamines in the blood of
cattle (Robertshaw and Whittow, 1966; Alvarez and Johnson,
197 3) . Removing the neural link between the hypothalamus and the
adrenal medulla abolishes this increase in catecholamines and
induction of heat-loss responses during hypothalamic warming
(Robertshaw and Whittow, 1966). Increased concentrations of
catecholamines result in increased 02 consumption in cattle
(Whittow and Findlay, 1968).
Many endocrine changes during hyperthermia are consistent
with reduction of activities which might generate excess
metabolic heat. Thyroid activity is reduced during hyperthermia
(Brooks et al., 1962; Bianca, 1965; Thompson, 1973; El-Nouty et
al. , 1976; Collier et al. , 1982) as a result of depressed release
of thyroid-stimulating hormone from the anterior pituitary
(Krulich et al., 1976). Ruminal heating in heifers results in
lower thyroid activity and metabolic heat production, as
measured by indirect calorimetry (Yousef et al. , 1968) . Reduced
thyroid activity results in lowered 02 consumption as metabolic
rate declines (Guyton, 1981).
Short-term heat exposure causes increased secretion of growth
hormone (GH) in cattle (Mitra and Johnson, 1972) possibly through
neural stimulation of the hypothalamus as a result of increased

22
skin temperature. The rise in GH concentration in the blood
preceded the rise in rectal temperature; additionally, the
decline of GH at the end of the 4 h heat exposure preceded the
decline in rectal temperature (Mitra and Johnson, 1972) . Long¬
term exposure to elevated temperature causes a depression in GH
secretion (Mitra et al., 1972) . Heat stress increases turnover
rate of GH in pigs following an initial, transient rise in plasma
concentrations of GH (Marple et al. , 1972) . Depressed GH during
thermal stress may represent a mechanism to prevent hyperthermia
because injection of GH during hyperthermia in cattle resulted
in increased thyroid activity, increased 02 consumption, and
increased heart and respiration rate (Yousef and Johnson, 1966) .
In addition, blood concentrations of insulin are depressed by
heat stress (Kamal et al., 1970; Thompson, 1973).
Episodic and estradiol-induced surges of luteinizing hormone
(LH) secretion in ovariectomized ewes were not affected by
hyperthermia (Schillo et al., 1978) and there was a slight
depression in basal serum-concentrations of LH. Elevated
temperature depressed basal and peak LH concentrations in cyclic
heifers (Madan and Johnson, 1973; Miller and Alliston, 1973).
Frequency of LH peaks also was reported to decline in the summer
in cyclic cows (McNatty et al. , 1984) . Vaught et al. (1977) and
Gwazdauskas et al. (1981) found no effect of summer heat stress
on LH secretion in lactating cows or cyclic heifers, respec¬
tively, however. Hyperthermia depresses LH secretion in hens

23
through depression of hypothalamic LH-releasing hormone
(Donoghue et al. , 1989) .
Hyperthermia results in increased prolactin secretion in
heifers (Wettemann and Tucker, 1974) and ovariectomized ewes
(Schillo et al., 1978). Serum prolactin concentrations are
elevated during the summer (Koprowski and Tucker, 1973) and the
prolactin release following injections of thyroid-stimulating
hormone is greater in heat-stressed cattle (Wettemann and
Tucker, 1974; Roman-Ponce et al., 1981). Schillo et al. (1978)
found that the hyperthermia-induced rise in prolactin secretion
preceded the rise in rectal temperature, indicating that neural
stimulation, perhaps as a result of increased skin temperature,
is responsible for the increased secretion of prolactin.
Short-term hyperthermia results in increased concentrations
of circulating glucocorticoids secreted from the zona fasiculata
and zona reticularis of the adrenal cortex in response to
adrenocorticotropic hormone (ACTH) in lactating and nonlactating
cyclic cows (Christison and Johnson, 1972; Alvarez and Johnson,
1973) , dogs (Chowers et al. , 1966) and pigs (Marple et al. , 1972;
1974) . Local heating of the preoptic area, exposure to elevated
temperature and injection of pyrogens (lipopolysaccharide) all
caused increased cortisol release from the adrenal gland in dogs
(Chowers et al., 1966). Cortisol release in response to exposure
to elevated temperature preceded the rise in rectal temperature
(Chowers et al. , 1966). Chronic hyperthermia causes depression
of glucocorticoid secretion. Glucocorticoid concentrations

24
after 24 h of heat stress were below pretreatment values in
nonlactating cows (Christison and Johnson, 1972; Alvarez and
Johnson, 1973? Abilay and Johnson, 1975), lactating cows (Stott
and Robinson, 1970) and pigs (Marple et al., 1972). Gluco¬
corticoid release in response to ACTH injection is lower in heat-
stressed cattle (Gwazdauskas et al., 1981; Roman-Ponce et al.,
1981). In pigs, the clearance rate of ACTH is decreased during
hyperthermia and although ACTH levels may rise, glucocorticoid
secretion is depressed (Marple et al., 1974). Chronic stress
elicits specific responses to heat stress such as reduction of
calorigenic hormones, i.e., glucocorticoids, thyroxine, GH and
insulin.
The excretion of water by the kidney is controlled primarily
by antidiuretic hormone (ADH) (Houpt, 1977). Antidiuretic
hormone is released from the hypothalamus in response to
increases in plasma osmolarity and exerts its effect on the cells
lining the collecting tubules of the kidney to stimulate
reabsorption of water from the urine (Houpt, 1977) . The result
of increased water reabsorption is decreased plasma osmolarity.
Secretion of ADH is elevated in cattle during periods of exposure
to elevated temperature (El-Nouty et al. , 1980).
Sodium excretion is regulated by aldosterone (Houpt, 1977).
Aldosterone is secreted by the cells of the zona glomerulosa of
the adrenal cortex in response to stimulation by products of
renin metabolism, i.e., angiotensin, which is activated by low
sodium concentrations in serum (Houpt, 1977) . Aldosterone acts

25
in the kidney at the level of the distal tubule to stimulate
reabsorption of sodium from the urine. If ADH is present, the
osmotic gradient across the tubule (a result of the [Na+]
difference) causes increased water reabsorption out of the lumen
of the collecting duct (Houpt, 1977) . During hyperthermia,
aldosterone is depressed in ruminants if water availability is
adequate (El-Nouty et al., 1980). If water intake is limited,
there is a rise in aldosterone secretion (El-Nouty et al. , 1980) .
In humans there is sodium loss during periods of elevated
temperature, in skin secretions as well as urinary excretion,
resulting in lower serum concentrations of sodium; this results
in increased ADH and aldosterone secretion and ultimately in
lower urine output (Guyton, 1981) . In ruminants there is greater
loss of potassium in skin secretions than of sodium (Johnson,
1970) ; sodium concentrations in the serum do not decrease and
aldosterone secretion remains low. As a result, reabsorption of
urinary sodium remains low. During dehydration associated with
heat stress in cattle, ADH remains elevated and aldosterone
concentration increases slightly to cause decreased urinary
output (El-Nouty et al. , 1980) .
Concentrations of ovarian steroids have been reported to be
affected by elevated temperature. Gwazdauskas et al. (1981)
reported that estradiol concentrations were depressed following
induced luteolysis in heat-stressed cows. Vaught et al. (1977)
found progesterone concentrations of lactating cows were

26
elevated during the summer, but there was no increase in non-
lactating cows. McNatty et al. (1984) found, however, that
progesterone concentrations were higher in cows during the
winter. Miller and Alliston (1973) also reported effects of heat
stress on progesterone concentrations in cyclic heifers.
Maximal serum concentrations were reached two days earlier in the
estrous cycle of heat stressed animals and had begun to decline
before peak concentrations were established in the control
heifers. A potential source of increased progesterone con¬
centration is increased adrenal synthesis (Stott and Robinson,
1970). Injection of ACTH during heat stress in cattle causes
increased serum progesterone concentrations to appear in
addition to increased concentrations of glucocorticoids (Abilay
et al., 1975). Depressed ACTH turnover during heat stress
coupled with a reduction in adrenal 17a-hydroxylase activity,
adaptations designed to depress glucocorticoid secretion, might
result in increased progesterone secretion from the adrenal
cortex. Increased concentrations of progesterone caused by heat
stress in ewes are not sufficient to suppress pituitary release
of LH, however (Sheikheldin et al., 1988).
Changes in behavior patterns
Animals can relieve some effects of hyperthermia through
alteration of behavior patterns. In a manner similar to that
adopted by ectothermic animals, homeotherms may seek shade or
decrease daytime activity in favor of nighttime activity (Seath
and Miller, 1946; Robertshaw, 1986). Holstein cattle on the

27
island of Fiji did 67% of their grazing at night (Payne et al. ,
1951) , contrasted with about 16% in more temperate climates
(Seath and Miller, 1946). Animals may also utilize changes in
posture to increase convective surface area (Curtis, 1981) and
increase evaporative heat flux by licking and salivating (Riek
and Lee, 1948) . Sexual behavior is altered during periods of
elevated ambient temperature, shortening the period of estrus
in cattle (Hall et al., 1959; Madan and Johnson, 1973; Monty et
al., 1974; Gwazdauskas et al., 1981). Alteration of estrous
behavior may be due to lower concentrations of plasma estradiol
(Gwazdauskas et al., 1981) which, along with progesterone, is
responsible for alterations of neuronal activity in areas of the
central nervous system resulting in disinhibition of mating
behavior at the appropriate time (Parsons and Pfaff, 1985).
Bos Taurus Versus Bos Indicus Cattle
Bos indicus, or zebu, cattle evolved in tropical and subtrop¬
ical regions of the earth and exhibit phenotypic characteristics
which provide advantages in the maintenance of homeothermy
during periods of elevated ambient temperature. Among these are
a high-density hair coat, with hair of short length, generally
of a light color and arranged to produce a sleek appearance
(Robertshaw, 1986). All of these characteristics contribute to
a reduced absorption of radiant energy (Finch, 1986). Zebu
cattle also have lower amounts of body-cover insulation because
fat distribution is localized to the inguinal region and the area
above the shoulders rather than covering the thoracic and

28
abdominal regions as in Bos taurus (Ledger, 1959) . Storage of
fat in anticipation of periods of lowered food availability is
a characteristic of both Bos taurus and Bos indicus cattle. Zebu
cattle, however, evolved in regions where reduced food avail¬
ability is generally correlated with reduced water availability
(Ledger, 1959) . Taurean cattle, on the other hand, evolved in
temperate regions where food availability might decline, but
water supplies remain relatively constant. Thus an insulative
body fat cover, an advantage in temperate regions where seasonal
temperatures become low, is not a disadvantage for taurean cattle
during periods of elevated temperature because capacity for
evaporative heat flux without dehydration is not limiting
(Ledger, 1959). Zebu cattle have a higher density of epitricheal
sweat glands than taurean cattle (Robertshaw, 1986), although
rates of secretory output are similar (Joshi et al. , 1968) . Zebu
cattle also have a higher blood volume than Bos taurus cattle
(Howes et al., 1957), indicating increased water retention
capability. Bos indicus cattle also generate less metabolic heat
than Bos taurus (Kibler, 1957; Finch, 1986).
As compared to Bos taurus, Zebu cattle exhibit shorter
estrous periods, less intense estrous behavior, including almost
no homosexual behavior, more "quiet ovulations" with no detected
estrus and a shorter interval from estrus to ovulation (Plasse
et al. , 1970; Randel, 1980; Randel, 1984). Randel (1980) found
lower levels of luteinizing hormone during the preovulatory
surge in Zebu heifers. The preovulatory surge is also of lower

29
amplitude and shorter duration than in taurean cattle (Randel,
1984) . Corpora lútea of Zebu cattle are smaller and they
synthesize and release less progesterone than corpora lútea of
Bos taurus (Adeyemo and Heath, 1980; Rhodes et al., 1982;
Segerson et al., 1984). Synthesis of estradiol by ovarian
follicles is also lower in Zebu than taurean cattle (Randel,
1980; Segerson et al., 1984). In the study of Segerson et al.
(1984) comparing morphological aspects of the reproductive
tracts of Angus and Brahman (Zebu) cows, ovaries of Brahman cows
were less asymmetric than ovaries of Angus cows in terms of
differences between the ovary on which ovulation had occurred and
the ovary contralateral to it. Ovaries of Brahman cows had
greater overall weight, greater follicular fluid weight and, on
the ovary contralateral to the corpus luteum, larger numbers of
follicles. Angus cows had greater amounts of uterine luminal
protein at day 17 of pregnancy, a result of greater numbers of
endometrial glands and greater cell height of luminal epithelium
(Segerson et al. , 1984).
Reproductive performance of Bos indicus cattle in tropical
and subtropical climates can exceed that of Bos taurus (Wilkins,
1986). There is seasonal variation in fertility of Bos indicus
in more temperate climates (Randel, 1984) . This may account for
observations that reproductive performance of Zebu cattle is
poor in temperate climates (Jochle et al. , 1973) . In Zebu cattle
in winter, frequency of LH surges declines, responsiveness of the
CL to exogenous LH challenge is depressed and conception rates

30
are lower than in summer (Randel, 1984) . Conception rates of Bos
taurus cattle, on the other hand, show seasonal variation with
lowest conception rates in summer (Salisbury et al., 1978),
even at latitudes as far from the equator as Minnesota where
conception rates in dairy cows decline by 11% in August (Udom-
prasert and Williamson, 1987).
One reason for increased resistance to heat stress-induced
infertility in Zebu cattle is the fact that they are better able
to maintain homeothermy in the face of higher ambient tempera¬
tures. It is unclear whether a given increase in Tb causes a less
severe reduction in fertility in Zebu. Turner (1982) analyzed
the relationship between conception rates of crossbred cattle
of either Bos indicus or Bos taurus breeding under sub-tropical
conditions in northern Australia. It was concluded that the
depression in conception rate per unit increase in Tb did not
differ among breed types. However, the Y-intercepts of the
regression curves were greater for Zebu-crossbred cattle than
for Bos taurus indicating greater fertility under subtropical
conditions. While not statistically significant, the percentage
decrease in fertility per unit elevation of body temperature was
less for Zebu than Bos taurus cows.
Peacock et al. (1971) found pregnancy rates for Brahman
cattle (Zebu) to be 10% greater than for Shorthorn cattle in the
subtropical environment of Florida. A similar finding was
reported in a study of crossbreeding using Brahman, Angus and
Charoláis sires (Peacock et al., 1977) during a March to July

31
breeding season. In both of these studies, a significant breed
of sire effect was seen for pregnancy rate as cows mated to
Brahman and Brahman crossbred sires had the highest conception
rates, perhaps indicating improved genetic potential for
survival of Brahman or Brahman-crossbred embryos during periods
of elevated temperature. Among all possible crossbreeding
combinations of Brahman, Angus and Charoláis cows and bulls over
an 11 year period, the highest conception rates were attained in
Brahman x Brahman matings (84%) (Peacock et al., 1977) . Preg¬
nancy rates for cows mated to Brahman sires (82%), Angus sires
(74%) and Charoláis sires (79%) were significantly different
(Peacock et al. , 1977) . There was no difference in breed of dam
as all three breeds had a conception rate of 78%. Taken together
with the data of Turner (1982) , these results suggest the possi¬
bility exists that Zebu cattle exhibit advantageous traits at the
level of the conceptus and/or the female reproductive tract that
provide benefit during periods of elevated temperature.
Breed affects seasonal variation in fertility within Bos
taurus cattle. Stott (1961) reported that Jersey cows in Arizona
showed no seasonal depression in fertility whereas Guernseys and
Holsteins exhibited steep depressions. Individuals within a
breed may also vary in their thermal adaptiveness. El-Nouty et
al. (1976) found that thermal adaptability varied among Hereford
heifers and that animals exhibiting the greatest adaptability
also had the lowest levels of thyroxine secretion in response to
elevated temperature.

32
Effects of Hyperthermia on Reproduction: Effects at the
Reproductive Tract Level
While much attention has rightly been given to heat stress-
induced infertility as a problem in tropical, subtropical and
arid climates it is important to consider that heat stress is a
problem not unique to these areas. In Minnesota, an 11% decline
in fertility in dairy cows during the month of August has been
reported (Udomprasert and Williamson, 1987) and Erb (1940)
reported that conception rates of dairy cows in the Purdue
University herd were lowest in August. If, as pointed out by
Beede and Collier (1986), trends toward global warming continue
and higher environmental temperatures are realized, then heat
stress-induced infertility of domestic animals will become an
increasingly significant problem at latitudes further from the
equator.
Failure to maintain homeothermy results in depression of
fertility. Negative effects of hyperthermia upon establishment
and maintenance of pregnancy can be manifested at a number of key
stages, from mating to parturition. Hyperthermia during
gestation may also affect subsequent lactation (Collier et al. ,
1982) , thereby impacting upon the health and performance of the
neonate. Avenues for heat-induced effects include direct
thermal effects on gametes and on the preimplantation embryo
resulting directly in developmental abnormality. There may
direct effects of elevated temperature on oviductal and uterine
secretory function. Additionally, interactions between

33
conceptus and maternal systems may be altered through changes in
uterine blood flow and alterations of uterine-endometrial
secretory and transport activity as the hyperthermic animal is
subject to various metabolic, endocrine, cardiovascular and
nutritional perturbations.
Uterine Blood Perfusion
Uterine blood perfusion is the ultimate source of luminal
nutrients, oxygen and water for the developing conceptus.
Uterine blood flow is also responsible for dissipation of uterine
metabolic heat (Gwazdauskas et al. , 1974). Vasoregulation in the
uterine vascular bed is controlled by catecholamines, par¬
ticularly norepinephrine, which exert effects locally in the
uterus (Roman-Ponce et al., 1978a; Brown and Harrison, 1981) and
whose effect is modulated by concentrations of circulating
steroids (Ford, 1982). Estradiol-induced protein synthesis
blocks synthesis and release of the a-adrenergic-stimulator
norepinephrine, thus blocking vasoconstriction. Progesterone
enhances norepinephrine-induced vasoconstriction through lower
clearance rate of secreted norepinephrine (Ford, 1982).
Additionally, prostaglandins exert vasoregulatory effects in the
uterine vascular bed, also modulated by steroids. Prostaglandin
E exerts vasodilatory effects by blocking norepinephrine release
while F series prostaglandins exert vasoconstrictor effects
under appropriate conditions, i.e., when estradiol is low, by
potentiating norepinephrine release (Ford, 1982) .

34
Heat stress causes reduction of uterine blood flow in cattle
(Roman-Ponce et al., 1978b), sheep (Oakes etal., 1976; Alexander
et al., 1987; Bell et al., 1987) and rabbits (Leduc et al.,
1972), though apparently not in swine (Wettemann et al. , 1988).
The cause remains uncertain, although endocrine alterations
associated with heat stress in cattle, such as elevated catechol¬
amine concentrations (Whittow and Findlay, 1968; Alvarez and
Johnson, 1973) and decreased estradiol: progesterone ratio
(Gwasdauskas et al., 1981; Vaught et al., 1977; Roman-Ponce et
al., 1981), are consistent with increased vasoconstrictor
effects in the uterine vascular bed. Elevated concentrations
of progesterone depress estradiol-induced increases in uterine
blood flow in ovariectomized sheep and cattle (Catón et al.,
1974; Roman-Ponce et al., 1978a,b). In hepatocytes in vitro,
heat shock causes transient inhibition of the estrogen-mediated
increase in trancription of the vitellogenin gene with a
concomitant decrease in the number of nuclear estrogen-receptors
(Wolffe et al., 1984). Such an alteration of receptors in
addition to the inhibitory effects of elevated progesterone
might explain the refractoriness of uterine blood flow to
estradiol injection.
Elevated environmental temperature is associated with
reduced uterine blood flow and increased uterine luminal
temperature (Gwazdauskas et al., 1973; Thatcher, 1974). The
critical nature of uterine blood perfusion for dissipating
uterine heat is demonstrated by the fact that uterine temperature

35
increases more rapidly than arterial blood temperature as the
uterine blood flow diminishes (Gwazdauskas et al., 1973).
Increased uterine luminal temperature around the time of
insemination is associated with depressed conception rates in
cattle (Gwazdauskas et al., 1973; Thatcher, 1974). The role of
uterine blood flow during later stages of pregnancy and its
effect on fetal growth is discussed later.
Gamete Development
In the dairy industry where artificial insemination is
prevalent, the effect of heat stress upon the male gamete has not
been greatly considered with respect to embryonic mortality.
Exposure of spermatozoa to elevated temperature in vitro
(Burfening and Ulberg, 1968), in útero (Howarth et al. , 1965) or
while in the epididymas (Bellvé, 1972) results in no adverse
effect on fertilization but subsequent embryonic survival is
greatly reduced. Chromosomal abnormalities induced by heat
shock and carried over into embryonic development are the most
likely cause (Ulberg and Sheean, 1973; Waldbeiser and Chrisman,
1986) . Lenz et al. (1983) found that bovine spermatozoa exposed
to 40°C in vitro had lower viability and reduced potential for
capacitation. Alterations of uterine and/or oviductal factors
responsible for interactions with spermatozoa (Sutton et al.,
1984b) , as part of the process of sperm transport (Hunter et al.,
1983) and capacitation (Bedford, 1969; Bedford, 1970), as well
as direct thermal effects on the gametes, are potential causes

36
of fertilization failure or subsequent embryonic loss due to
maternal heat stress.
In vivo heat-stress in the 2 or 3 days prior to ovulation is
associated with low conception rates in cattle (Stott and
Williams, 1962; Ingraham et al. , 1974). A number of studies make
this association (Dutt et al., 1959; Stott and Williams, 1962;
Ingraham et al., 1974) but cannot relate the effect temporally
to the complex series of events (Moor and Gandolfi, 1987;
Wassarman, 1988) that occur prior to ovulation of a fully
competent oocyte.
Fertilization rate in vivo is probably not affected in sheep
(Dutt, 1963), cattle (Monty, 1984; Putney et al. , 1989a) or pigs
(Warnick et al., 1965; Edwards et al., 1968) following hyper¬
thermia on the day of ovulation. Putney etal. (1989a) subjected
superovulated heifers to elevated temperature for 10 h, begin¬
ning at the onset of estrus. Heifers were inseminated 15 and 20
h after onset of estrus and reproductive tracts were flushed at
7 days after estrus to recover embryos. While heat-stress caused
more retarded and abnormal embryos to be present, there was no
difference in ovulation rate or numbers of fertilized ovum
recovered, compared to unstressed superovulated controls. In
vitro, the numbers of bovine oocytes that make the transition
from meiotic prophase I to metaphase II is reduced by elevated
temperature (40°C) as is the fertilization rate (Lenz et al.,
1983) .

37
In mice, heat stress of females prior to ovulation does not
affect fertilization rate but subsequent embryonic survival is
significantly reduced (Bellvé, 1972; Baumgartner and Chrisman,
1988) . This is associated with an increased incidence of chromo¬
somal non-dysjunction and resultant polyploidy in the embryos
(Baumgartner and Chrisman, 1988). Non-dysjunction may result
from altered tubulin synthesis, which is depressed as a result
of heat shock (Thomas et al. , 1982) .
Exposure of gilts to elevated temperature for several days
prior to breeding affects neither ovulation rate nor fertiliza¬
tion rate (Warnick et al. , 1965; Edwards et al. , 1968) . As in the
case of the spermatozoa, alterations of oviductal factors
responsible for interactions with the gamete or zygote (Shapiro
et al. , 1974 ; Kapur and Johnson, 1985; Leveille et al., 1987), as
well as direct thermal effects on the gamete or zygote, are
potential causes of failure of fertilization or loss of embryonic
viability.
In the sexually mature adult, the ovaries contain pools of
oocytes arrested since fetal development in prophase of the first
meiotic division. Fully grown oocytes resume meiosis and are
ovulated during each reproductive cycle. After the preovulatory
surge of LH, oocytes progress from meiotic prophase I to meta¬
phase II in a process called oocyte maturation (Wassarman, 1988) .
This event is characterized by germinal vesicle breakdown,
condensation of the chromatin, spindle formation in preparation
for diakinesis, and extrusion of the first polar body (Moor and

38
Gandolfi, 1987; Wassanan, 1988). In preparation for spindle
formation, nearly two percent of the total protein in the oocyte
is tubulin (Wassarman, 1988) . Tubulin synthesis in heat-shocked
cells is depressed (Thomas et al. , 1982) . High levels of cyclic
AMP in the oocyte inhibit maturation (Eppig and Downs, 1988) ,
these levels may be maintained through regulation by calcium-
calmodulin dependent processes, activation of adenylate cyclase
and inhibition of phosphodiesterase in the oocyte (Eppig and
Downs, 1988 ; Wassarman, 1988) , effects which are reversed by LH
resulting in the trigger for maturation immediately prior to
ovulation (Wassarman, 1988). Elements important for meiotic
maturation such as synthesis of cytoskeletal tubulin (Thomas et
al., 1982), calcium-calmodulin activation (Weigant et al. , 1985;
Stevenson et al. , 1986) and patterns of steroid hormone (Roman-
Ponce et al., 1981) and LH secretion (Madan and Johnson, 1973;
Miller and Alliston, 1973) are all potentially altered by
elevated temperature.
Preimplantation Embryonic Development as Affected by
Hyperthermia
Heat shock during the zygote to morula stages
After sperm-egg fusion and fusion of the male and female
pronuclei, the initial cell cycle of embryonic development
begins and the first cell division occurs about 12 h later
(Pederson, 1988). Embryonic development is dependent upon
temporal coordination of a number of processes, many of which,
such as cell-cycle regulation, may be disrupted by hyperthermia.

39
Subtle disruptions may result in carry-over effects of hyper¬
thermia that cause embryonic death several days or weeks after
the stress has passed (Elliott et al. , 1968; Wildt et al. , 1975) .
Mouse embryos exposed in vivo to elevated temperature during the
first cell division suffer an increased incidence of post¬
implantation death (Elliott et al., 1968). Likewise, many
porcine embryos exposed to high temperatures during placento-
genesis (days 14 to 25) appear normal at day 25, but have
degenerated by day 42 of pregnancy (Wildt et al., 1975). Through
the initial cell divisions (actual number varies among species)
following fertilization, the embryo is dependent upon mater¬
nally-derived mRNA which was synthesized during the period of
oocyte growth preceding meiotic maturation and ovulation
(Wassarman, 1988) . Activation of the embryonic genome occurs at
the 2-cell stage in the mouse (Bolton et al., 1984; Bensaude et
al. , 1984) and 8 to 16-cell stage in the cow (King et al. , 1985?
Camous et al., 1986; Frei et al., 1989). Protein synthesis by
the sheep embryo is very active in the first two cycles of cell
division followed by a 95% reduction in the third cycle (Crosby
et al. , 1988) . In the fourth and fifth rounds of cell division,
16 and 32-cell stages, protein synthesis increases once again
as a result of activation of the embryonic genome.
The early embryo is highly sensitive to hyperthermia and a
large reduction in embryonic survival, as much as 80% loss,
follows heat stress in the first days after fertilization in
cattle (Dunlap and Vincent, 1972; Stott and Wiersma, 1976; Putney

40
et al., 1989), sheep (Dutt et al., 1959; Alliston and Ulberg,
1961? Dutt, 1963), mice (Elliott and Ulberg, 1971; Ulberg and
Sheean, 1973) , rabbits (Ulberg and Sheean, 1973) and pigs
(Warnick et al., 1965; Tompkins et al., 1967; Edwards et al.,
1968; Wildt et al., 1975). Gwazdauskas et al. (1973) found the
greatest correlation between environmental temperature and
conception rate on the day of insemination and on the day after
insemination.
Effects of hyperthermia in all the species listed above
occurred during the period of the first one to three cleavage
divisions when the embryos are present in the oviduct. The
embryo remains in the oviduct for 90 h in cattle, 72 h in sheep,
50 h in pigs (Hafez, 1974), and 68 to 77 h in mice (Biggers et
al. , 1971) . Adverse effects of hyperthermia have generally been
attributed to direct thermal effects upon the embryo rather than
alteration of oviductal-embryonic interactions. There is good
evidence for direct effects of elevated temperature on embryonic
development in sheep (Alliston and Ulberg, 1961), rabbits
(Alliston and Ulberg, 1965), mice (Elliott et al. , 1968; Wittig
et al. , 1983) and cattle (Putney et al., 1988a) .
Nonetheless, it is possible that a portion of the effect of
elevated temperature could be due to alterations of oviductal
function. The volume of oviductal secretions are diminished by
heat stress in rabbits (Thorne et al., 1980). The importance of
the oviductal environment for early embryonic development has
been illustrated in experiments demonstrating superior in vitro

41
development of embryos cultured in the presence of oviductal
cells (Gandolfi and Moore, 1987; Eyestone et al., 1987? Rexroad
and Powell, 1988) or oviduct-conditioned culture medium (Kalt-
wasser et al. , 1987) . It may reasonably be assumed that some of
the effects of the oviduct are mediated by secretory proteins.
Proteins secreted by the oviduct have been implicated in a number
of functional roles, including binding to zona pellucida
(Shapiro et al. , 1974; Kapur and Johnson, 1985; Leveille et al.,
1987) and spermatozoa (Sutton et al. , 1984b), sperm capacitation
(Bedford, 1970) and inhibition of antibody-complement-mediated
immune function (Oliphant et al., 1984b). Stage-specific
changes in total protein secretion rate by bovine oviducts during
the estrous cycle (Geisert et al., 1987; Killian et al., 1987)
and appearance of individual stage-specific proteins from
oviducts of the rabbit (Oliphant et al. , 1984a) , pig (Buhi, 1985;
Buhi et al. , 1989) , cow (Geisert et al. , 1987) , sheep (Sutton et
al., 1984a), mouse (Kapur and Johnson, 1985), rat (Wang and
Brooks 1986), baboon (Fazleabas and Verhage, 1986), and human
(Verhage et al., 1988) all point to the potentially critical
nature of oviductal function around the time of fertilization and
during early pregnancy.
The capacity, or lack of capacity, of maternal mRNA-directed
protein synthesis to respond to stressors such as elevated
temperature may be a key to the susceptibility of the early
embryo to hyperthermia. It is unclear whether heat sensitivity
is temporally related to genomic activation; although in the

42
mouse embryo, RNA production is more sensitive to heat shock at
the time of genomic activation (2-cell stage) than at any other
time (Bellvé, 1976) . Probably as a consequence of maternal mRNA-
directed protein synthesis, embryos are apparently not able to
synthesize heat-shock proteins until the early blastocyst stage
(Wittig et al., 1983 ; Morange et al., 1984; Heikkila and Schultz,
1984) although mouse embryos are capable of constitutive (i.e. ,
non-temperature regulated) synthesis of members of the 70-kDa
heat-shock protein family at the two-cell stage (Bensaude et al. ,
1986; Howlett, 1986).
The Blastocyst and Heat Shock
The embryo remains susceptible to adverse effects of elevated
temperature throughout the preimplantation period as a number of
events critical to embryonic development occur. Following the
early rounds of cell division, the blastomeres form junctional
complexes (Ducibella et al., 1975) and become very tightly
associated. Compaction of the blastomeres occurs at 8-cells in
the mouse (Johnson and Pratt, 1983) , 16-cells in the sheep
(Crosby et al., 1988) and 32-cells in the cow (Barnes et al.,
1987) . The cell-cell adhesion required for compaction is
calcium-dependent (Ogou et al., 1982). Following compaction,
the embryo is referred to as a morula. The next major event of
embryonic development is blastocoele formation. Fluid transport
in the trophectoderm driven by newly synthesized Na+-K+ ATPase
(Beños and Biggers, 1981; Overstrom et al., 1989) pumps water
into the center of the tightly associated cells and a hollow

43
cavity, the blastocoele, is formed and expanded. Early differen¬
tiation events result in asynchronous rates of cell division and
positioning of cells whose developmental fates begin to be
established as restriction of potency begins to occur, generally
around the 32-cell stage (Pederson, 1988) . Inner and outer cell
compartments form and the trophectoderm and inner cell mass
become distinguishable. The blastocyst at this stage contains
16 cells in hamsters and pigs, 32 cells in mice, 64 cells in sheep
and 128 cells in the rabbit (Pederson, 1988) . In addition, free
amino acid content of embryos increases steadily from the 2-cell
stage through blastocyst development (Sellens et al., 1981).
Both Na+-dependent and independent amino acid transport systems
are present in the early embryo, Na+-dependent transport develops
at the morula stage in mouse embryos (Kaye et al. , 1982) . These
transport systems have K„ of 10'5 to 10'6 M (Kaye et al. , 1982) .
As the blastocyst grows, it sheds the zona pellucida and
expands within the uterine lumen to form contacts with the
uterine vasculature (species having invasive implantation) or
the endometrial epithelium (noninvasive) and establish interac¬
tions with the maternal system. Successful establishment and
maintenance of pregnancy involves a close communication between
the mother and the developing conceptus. The critical importance
of synchrony in the development of these two biological systems
as pregnancy progresses has been demonstrated by embryo transfer
studies in domestic animals (Rowson et al., 1972; Wilmut and
Sales, 1981; Wilmut et al., 1985). Steroid-induced alterations

44
in the reproductive tract during the early period after estrus
are critical to embryonic survival (Miller and Moore, 1976; Moore
et al., 1983; Wilmut et al., 1985) and the rate of embryonic
development in the sheep and cow can be altered by the uterine
environment (Wilmut and Sales, 1981; Wilmut etal., 1985; Garrett
et al., 1987) . Presumably, many of the steroid-driven functions
of the uterus are mediated by secretory proteins. Stage-speci¬
fic secretion of proteins by the bovine uterine endometrium has
been reported (Roberts and Parker, 1974, 1976; Bartol et al.,
1981a,b). Endometrial proteins play roles as transport mole¬
cules (Buhi et al., 1982; Pentacost and Teng, 1987), protease
inhibitors (Fazleabas et al., 1982), lysosomal enzymes (Hansen
et al., 1985) and immunoregulatory molecules (Murray et al.,
1978; Hansen et al., 1989) .
Heat stress 8 to 16 days after fertilization results in sig¬
nificant embryonic loss in sheep (Dutt, 1963; Dutt and Jabora,
1976) and cattle (Stott and Williams, 1962; Wise etal., 1988).
In several cases, prolonged lifespan of the corpus luteum in
bred-but-not-pregnant cows has been reported. This may reflect
embryonic death during the period 8 to 16 days after fertiliza¬
tion (Stott and Williams, 1962; Madan and Johnson, 1973; Wise et
al. , 1988) as the embryo survived for a sufficient length of time
to signal its presence to the maternal system and block luteoly-
sis for a short time.
Rescue of the corpus luteum from luteolysis is a critical
physiological event that may be sensitive to heat stress in

45
cattle. Heat stress from 8 to 16 days after insemination caused
alteration of the uterine environment, including elevated
protein and electrolyte concentrations (Geisert et al. , 1988),
reduced weight of corpora lútea and impaired conceptus growth
(Geisert et al. , 1988; Biggers et al., 1987). During this
period, the conceptus secretes the antiluteolysin, bovine
trophoblast protein-1 (bTP-1) (Helmer et al., 1987), an
interferon-like (Imakawa et al., 1989) protein that causes
suppression of uterine prostaglandin (PG) F secretion resulting
in luteal maintenance (Helmer et al., 1989; Thatcher et al.,
1989). Depression of PGF secretion by bTP-1 may be mediated by
an inhibitor of PG synthesis that appears in uterine endometrial
cells during pregnancy (Gross et al., 1988) and after exposure
to bTP-1 (Helmer et al., 1989) . Conceptus weight was signifi¬
cantly reduced when cows were exposed to elevated temperature
from Day 8 to 16 of gestation, although no depression was
observed for subsequent secretion of bTP-1 during in vitro
culture at 37 °C (Geisert et al. , 1988) . Hyperthermia could also
directly alter PGF secretion. Basal and oxytocin-induced PGF
secretion from cultured uterine endometrium from cyclic and
pregnant cows at Day 17 after estrus was increased by exposure
to elevated temperature (Putney et al., 1988; Putney et al.,
1989). Further, nonpregnant cows or cows with retarded embryos
at Day 17 after estrus displayed increased PGF secretion in
response to oxytocin during heat stress in vivo (Putney et al. ,
1989) .

46
Heat Stress During Mid- and Late-Gestation
After hatching from the zona pellucida, the blastocyst
continues to expand through accumulation of water within the
extraembryonic membranes that arise from the embryonic gut.
These are the yolk sac, a transient structure, and the allantoic
sac which continues to expand and, in animals with epithelio¬
chorial placentation, supports expansion of the chorioallantoic
membranes and their apposition with the maternal endometrium
(Bazer et al., 1981). Water and electrolyte transport by the
chorioallantois are critical to successful establishment of
maximally efficient placental exchange between the maternal and
conceptus systems. The basis of water and electrolyte transport
is the establishment by Na*-K+ ATPase in the chorioallantois of
a [Na*] gradient between the allantoic fluid and maternal blood.
Both the active pumping of the ATPase to establish the gradient
and the relative leakiness of the membrane to permit passive
diffusion of sodium-coupled ions and water are affected by the
estrogen: progesterone ratio and by prolactin and placental
lactogen (Bazer et al. , 1981). Alterations in steroid production
and perturbation of prolactin secretion in heat-stressed animals
could result in alterations of establishment and maintenance of
conditions necessary for these processes to occur.
Although the functional roles of many of the proteins that
appear in the uterine lumen during pregnancy remain undefined,
it may reasonably be assumed that uterine and placental secretory
proteins play important roles during pregnancy. These proteins

47
have been implicated as transport molecules (Buhi et al. , 1982) ,
immunomodulators (Hansen et al., 1989), lysosomal enzymes
(Hansen et al., 1985), protease inhibitors (Fazleabas et al.,
1982) and hormones (Talamantes et al., 1980).
Heat stress during mid- to late- gestation causes reduced
fetal and placental weight in cattle and sheep (Alexander and
Williams, 1971; Collier et al., 1982) and reduced cotyledonary
release of estrone sulfate in cattle (Collier et al., 1982).
Retardation of fetal growth caused by heat stress likely results
from perturbation of diverse physiological events. Central to
this phenomenon is a reduction in uterine and placental blood
flow (Oakes et al., 1976; Bell et al., 1987; Alexander et al.,
1987), especially maternal placentomal blood flow in ruminants
(Alexander et al., 1987) . Placental size is depressed by heat
stress (Head et al., 1981; Bell et al. , 1987) and 02 and glucose
transport is reduced (Bell et al. , 1987), possibly an indication
of alteration of the water and electrolyte transport described
by Bazer et al. (1981). Reduced fetal weight is a result of
reduced placental size and transport activity (Alexander and
Williams, 1971; Bell et al., 1987). The depression in maternal
feed intake associated with heat stress is not sufficient to
account for malnutreated condition of fetuses (Cartwright and
Thwaites, 1976; Brown et al., 1977).
The decrease in blood flow to the pregnant uterus and placenta
is actively regulated at the uterine vascular bed since

48
elevated body temperature also reduced response to pharmaco¬
logical stimulation of uterine blood flow in ewes (Roman-Ponce
et al., 1978a) and cows (Roman-Ponce et al., 1978b). Elevated
catecholamine concentrations reduce uterine blood flow (Bell et
al. , 1987) . Increasing pH of the blood, a result of respiratory
alkalosis induced by second-stage breathing (Dale and Brody,
1954; Brown and Harrison, 1981), results in a 30% depression of
umbilical blood flow (Oakes et al., 1976). There is a possib¬
ility that local vasoconstriction during heat stress is caused
in part by changes in PG secretion. Elevated temperature in
vitro can alter the patterns of PG secretion by tissues of the
reproductive tract of the cow (Putney et al., 1988b) and pig
(Wettemann et al., 1988; Gross et al. , 1989). Prostaglandins
affect blood flow (Ford, 1982) through vasoconstrictive and
vasodilatory activities which are modulated by circulating
steroids (Ford, 1982; Vincent et al., 1986). Prostaglandins
produced by the growing blastocyst and placenta may affect per¬
meability of the endometrial vascular bed (Kennedy, 1980; Lewis,
1989) and stimulate fluid and electrolyte transport across the
uterine epithelium (Biggers et al. , 1978).
Effects of Hyperthermia on Cells
Heat stress-induced cell killing involves perturbation of
multiple systems within the cell, which shall be discussed in
turn. Among these are alterations in membranes, oxidation-
reduction balance, second messenger system function,

49
intracellular calcium mobilization, protein synthesis, protein
conformation and stability, enzyme activity, RNA splicing and
DNA repair. Eukaryotic organisms have evolved complex systems
for meeting challenges posed by their environment, these
homeostatic mechanisms include stabilization of membranes and
proteins, the elimination of deleterious products such as oxygen
radicals and reduction of RNA processing and translation as a
precaution against potential errors. Activation of these
various mechanisms through incremental exposure to stress leads
to a state of increased thermotolerance (Henle and Dethlefson,
1979; Li and Werb, 1982; Ashburner, 1982; Nover, 1984), though
the complete response pattern to achieve this tolerant status
remains unclear. Heat-induced cell death is probably the result
of multiple lesions acting at various sites (Jung, 1986).
Heat-shock responses of reproductive tract tissues and
developing conceptuses after activation of the embryonic genome
might be explained at the level of the cellular stress response
of individual cells. Mammalian cells exhibit a characteristic
response pattern to stress, including the production of a set of
proteins, called heat-shock proteins, that are believed to exert
stabilizing effects within the stressed cell (Welch et al.,
1987) . This response is apparently not possible until the early
blastocyst stage in mouse and rabbit embryos (Wittig et al.,
1983 ; Morange et al., 1984; Heikkila and Schultz, 1984) although
embryos are capable of constitutive synthesis of members of the

50
70-kDa heat-shock protein family at the 2-cell stage (Bensaude
etal., 1986; Howlett, 1986).
Heat Shock and Morphological Changes
Heat stressed cells undergo characteristic changes in
morphology, particularly in the areas of the nucleus, nucleolus
and mitochondria. There is general disruption of the nuclear
matrix (Welch and Suhan, 1985) and increased protein content
(Armour et al. ,1988) . Nucleoli of heat-stressed cells appear to
be swollen and decondensed (Pelham, 1984; Welch and Suhan, 1985;
McConnell et al., 1987). Nucleoli and nuclei of heat stressed
cells contain rod-like, fibrous structures shown by Welch and
Suhan (1985) to be actin. Following heat shock, mitochondria of
rat fibroblasts are swollen and exhibit prominent cristae
(Lepock et al., 1982; Welch and Suhan, 1985). Alterations of
mitochondria result in loss of respiratory control and uncou¬
pling of oxidative phosphorylation from electron transport
(Christianson and Kvamme, 1969; Lepock etal., 1982). Addition¬
ally, mitochondria are relocalized in heat-stressed cells to the
area around the nucleus, possibly the result of collapse of
cytoskeletal elements (Shyy et al., 1989) which also results in
aggregation of other cytoplasmic structures such as endosomes
and lysosomes following heat shock (Welch and Suhan, 1985; Shyy
et al. , 1989). Other cytoplasmic changes include fragmentation
or disappearance of the Golgi apparatus and endoplasmic retic¬
ulum (Welch and Suhan, 1985).

51
Heat Shock and Cvtoskeletal Changes
The current concept of subcellular structure is one involving
a three-dimensional skeletal matrix composed of at least three
distinct filamentous components. This network of filaments is
called the microtrabecular lattice. The components of the
lattice include microfilaments, intermediate filaments and
microtubules.
Of the three major networks of cytoskeletal elements in
cells, microfilaments, intermediate filaments and microtubules,
only the microtubules remain relatively unaffected by heat shock
(Welch and Suhan, 1985; Shyy et al., 1989) although tubulin
synthesis stops within a few hours during heat shock (Thomas et
al., 1982). Heat shock caused increased numbers of actin
filaments (stress fibers) to be present in rat fibroblasts (Welch
and Suhan, 1985) but caused disappearance of actin filaments in
mammary epithelial cells (Shyy et al. , 1989) . Within 30 minutes
after heat shock, however, the actin filament network had
reassembled.
The network of intermediate filaments has been shown to
collapse following heat shock resulting in aggregation of
cytoplasmic organelles around the nucleus (Biessman et al.,
1982; Welch and Suhan, 1985; Shyy et al., 1989) . Drummond et al.
(1988) incubated cells in a calcium-free, EGTA-buffered medium
to demonstrate that the heat-induced changes in the intermediate
filament network were not due to increased intracellular
calcium.

52
Heat Shock and Alteration of Membranes
The fluid-mosaic model of membrane structure (Singer and
Nicholson, 1972) based on the phospholipid bilayer with asso¬
ciated peripheral and transmembrane proteins remains the
accepted basis for understanding membrane function. The
capability of biological membranes to compartmentalize and
sequester proteins and nucleic acids is critical to life of all
organisms from unicellular prokaryotes to complex multicellular
organisms such as mammals.
The characteristics of membranes depend upon interactions
between lipid constituents and between lipids and proteins in the
membrane (Quinn, 1981). The lipid composition and relative
amount and type of proteins present in a membrane affect its
physical characteristics. At biological temperatures, membranes
exhibit a considerable degree of disorder and molecular mobility
(Lee and Chapman, 1987). Flexing of hydrocarbon chains by
rotation about carbon-carbon bonds, wobbling of the entire
phospholipid molecule along its longitudinal axis and lateral
displacement of molecules along the plane of the membrane all
occur (Quinn, 1981). Biological membranes are heterogeneous
systems of diverse composition and intermolecular interactions
can result in formation of microdomains within the plane of the
membrane as well as exhibition of different physical properties
in each monolayer of the membrane (Quinn, 1981).
The physical state of the membrane is affected by association
with molecules of water, called lyotropic mesomorphism, and by

53
temperature, called thermotropic mesomorphism (Quinn, 1981).
The transition of membranes from a rigid, gel-crystalline state
to a more disordered, liquid-crystalline arrangement occurs over
a narrow temperature range in an artificial homogeneous lipid
bilayer, but in heterogeneous biological membranes this transi¬
tion occurs over a broader range of temperatures (Quinn, 1981;
Cossins and Raynard, 1987). In addition to the temperature and
hydration state, ions, sterols and proteins all affect membrane
structure and behavior (Cossins and Raynard, 1987). The major
sterol in animal cell membranes, cholesterol, reduces the
enthalpy at transition from gel-crystalline to liquid-crystal¬
line and at high concentrations nearly eliminates the change in
phase as temperature changes (Lee and Chapman, 1987).
Alterations in cell membranes of poikilotherms and bacteria
in response to temperature has led to the hypothesis of homeo-
viscous adaptation (Cossins and Raynard, 1987) . As temperature
changes, the composition of the membrane is altered through
shifts in the degree of saturation of the fatty acyl chains of
the membrane phospholipids (thereby reducing the flexing of the
hydrocarbon chains) and removal or insertion of sterols to
maintain consistent viscosity (Anderson et al., 1981; Cossins
and Raynard, 1987). Anderson et al. (1981) found that mammalian
cells grown at elevated temperature (41°C) altered the choles¬
terol : phospholipid molar ratio in their membranes . Such changes
in membranes of animal cells may not occur at very high tem¬
peratures (42 to 44°C) as no alterations in cholesterol:

54
phospholipid ratio occurred in membranes of cells incubated for
24 h at these temperatures (Anderson and Parker, 1982).
Elevated temperature increases basal membrane permeability
as determined by passive diffusion of K*, increases permeability
to water, alters facilitated transport, i.e., glucose transport,
and decreased Na+-K+ ATPase activity (Burdon and Cutmore, 1982;
Ellory and Hall, 1987) . There is net loss of ion gradients as
leakage increases (Bowler, 1987) . In the case of the glucose
transporter, it was determined that the rate of return of the
unloaded transporter to the outer membrane surface was increased
(Ellory and Hall, 1987). Elevated temperature also results in
aggregation of integral membrane proteins, possibly due to their
denaturation (Lepock et al., 1983 ; Arancia et al. , 1986; Lee and
Chapman, 1987). Alterations of permeability lead to loss of ion
gradients, influx of Na* and Ca*, loss of membrane bioelectric
properties, failure of ion transport and impairment of facili¬
tated transport mechanisms (Bowler, 1987). Additionally,
activity of receptors is impaired (Magun, 1981; Calderwood and
Hahn, 1983). Radiolabeled epidermal growth factor (EGF) bound
to cell-surface EGF receptors of Rat-1 cells with lower affinity
after heat shock (Magun, 1981) . The slope of the Scatchard plot
as well as the abcissa intercept were different between heat-
shocked and control cells indicating both reduced affinity and
receptor number after heat shock (Magun, 1981) . Additionally,
degradation of internalized 125I-EGF in the lysosomes was slower
in heat-shocked cells (Magun, 1981). Calderwood and Hahn (1983)

55
• 125 . . . .
examined I-insulin binding to HA-1 cells and found that
receptor numbers were depressed by heat shock, but affinity was
not affected.
As previously mentioned, heat shock caused mitochondria of
rat fibroblasts to become swollen and exhibit prominent cristae
(Lepock et al., 1982; Welch and Suhan, 1985). Heat-induced
alterations of mitochondrial membranes result in loss of
respiratory control and uncoupling of oxidative phosphorylation
from electron transport (Christianson and Kvamme, 1969; Lepock
et al., 1982) .
Heat Shock Alters DNA Transcription and RNA Processing
Heat shock and DNA
The thermodynamic stability of the DNA double-helix at
elevated temperature is simply based upon the relative adenine-
thymine: guanosine-cytosine ratio of the nucleic acids (Lewin,
1987). Higher concentrations of guanosine-cytosine base-pairs
with their three hydrogen bonds between each nucleotide pair are
more stable than adenine-thymine base-pairs which interact with
two hydrogen bonds (Lewin, 1987) .
Heat shock terminates DNA synthesis (Ashburner, 1982 ; Warters
and Henle, 1982) and results in failure of DNA repair mechanisms
in the cell (Henle and Dethlefson, 1978; Warters and Roti Roti,
1982), possibly due to accumulation of excessive protein in the
nucleus (Armour et al., 1988). Warters et al. (1980) tested the
accessability of DNA to nuclease attack following heat shock.
There were fewer sensitive sites in DNA fractions from heated

56
cells compared to controls, possibly due to accumulation of
proteins associated with the DNA (Warters et al. , 1980) . Failure
of unstressed cells to complete replication of DNA during the S
phase of the cell cycle results in increased strand breakage
during the next replication event (Laskey et al. , 1989) an effect
seen in heat-shocked cells (Warters and Roti Roti, 1982).
Transcription of DNA is altered by heat shock as there is
preferential transcription of genes coding for heat-shock
proteins (Ashburner, 1982; Nover, 1984) under the influence of
specific heat-shock transcription factors (Chousterman et al.,
1987; Wu et al., 1987; Sorger and Pelham, 1988). Temperature-
dependent phosphorylation of a heat-shock transcription factor
has been reported (Sorger and Pelham, 1988) and Rougvie et al.
(1988) found that RNA polymerase was bound to the 5' end of an
uninduced heat-shock protein gene in Drosophila apparently
waiting for a signal to transcribe the gene. Exons of the c-myc
oncogene product turn on heat-shock gene promotors (Kingston et
al., 1984). Ananthan et al. (1985) were able to show that
denatured proteins introduced into the cell (Xenopus oocytes),
either by microinjection of denatured peptides or by insertion
of gene constructs coding fragments of peptides incapable of
normal folding, would induce the promotor region of the
Drosophila 70-kDa heat-shock protein (70-kDa promotor plus a
/3-galactosidase reporter gene) to activate gene transcription.
The same region of DNA in the promotor is responsible for both

57
the response to denatured proteins and for response to heat shock
(Ananthan et al. , 1985).
Heat shock and RNA
Posttranscriptional processing of heterogeneous nuclear RNA
is terminated by heat shock, apparently due to a direct effect of
heat-shock proteins (Yost and Lindquist, 1986). If heat-shock
protein synthesis was blocked with cycloheximide, RNA splicing
was not stopped during heat shock. Ribonucleoprotein particles,
called snRNPs, are the effectors of RNA splicing and are composed
of multiple subunits (Cech, 1986) ; snRNPs are disrupted by heat
shock in stress-susceptible cells but not in thermotolerant
cells (Bond, 1988) .
Heat shock reduces the half-life of mRNA in the cytoplasm by
induction, through synthesis of 2', 5 ' oligoadenylates, of latent
endoribonucleases in a manner similar to the actions of inter¬
ferons in virus-infected cells (Chousterman et al., 1987). Heat-
shocked MDBK cells produced a soluble heat-shock induction
factor that was able to induce 2',5' oligoadenylate synthetase
activity in fresh, unstressed MDBK cells (Chousterman et al.,
1987) . This soluble factor was not a member of any known class
of interferons however.
Heat Shock and the Cell Cycle
The cell cycle is the coordinated series of events by which
the cell accomplishes growth and replication. The rate of
passage through various stages varies with cell type, physio¬
logical state, nutrient availability and through control by

58
other exogenous cues. The G0 phase is considered a subphase of
G1 and is a quiescent state during which the cell awaits an
external signal, such as a particular growth factor(s), to
initiate active replication (Pardee, 1989) . The G1 phase of the
cell cycle is the period during which the cell prepares for the
S phase. There is protein and histone synthesis and some DNA
synthesis (Pardee, 1989) . The cell is induced to enter the S
phase by coordination of cytoplasmic signals (Laskey et al.,
1989) . During the S phase, the entire DNA complement of the cell
must be replicated. This is accomplished through bidirectional
replication at multiple sites (Laskey et al. , 1989) .
Sensitivity of CHO cells to heat shock varies with stage of
the cell cycle. Maximal thermotolerance is manifested at G1,
while the progression through S and G2-M are particularly heat
sensitive (Read et al. , 1983; Rice et al. , 1984a,b). Stabiliz¬
ation of nucleolar components during heat shock is a function
proposed for members of the 70-kDa family of heat-shock proteins
(Pelham, 1984). Association of 70-kDa heat-shock protein with
proteins in the nucleolus varies in a cell-cycle dependent
fashion (Milarski et al., 1989). In G2, three different mono¬
clonal antibodies to 70-kDa heat-shock protein failed to bind to
nucleolar protein in heat-shocked cells, although cells in G2
responded to heat shock with increased heat-shock protein mRNA
and protein synthesis (Milarski et al., 1989). As the cells
proceeded from S through G2 to the next M phase, there was
appearance of antibody binding in the nucleolus followed by

59
disappearance of binding and reappearance again. Three dif¬
ferent antibodies, each recognizing a different epitope of the
70-kDa heat-shock protein, gave three different patterns of
appearance and disappearance of nucleolar binding during G2
(Milarski et al. , 1989) . This variation is believed to be due to
association of the heat-shock protein with nucleolar components,
which block the epitope to antibody binding, and is not observed
at other stages of the cell cycle (Welch and Feramisco, 1984;
Milarski et al., 1989). Mitogen-induced lymphocytes produce
heat-shock proteins through translation of preexisting mRNA
(Colbert et al. , 1987) , and this occurs particularly during the
G0 through G1 transition preceding DNA synthesis (Kazmarek et
al. , 1987; Haire et al. , 1988).
Heat Shock and Metabolic Alterations
Heat shock induces alterations of mitochondrial membranes
resulting in loss of respiratory control and uncoupling of
oxidative phosphorylation from electron transport (Christianson
and Kvamme, 1969; Lepock et al., 1982). Adenosine, adenylate
activity and concentrations of ATP in the cell are not affected
by heat shock until temperatures become relatively high, i.e.,
45 to 48 °C (Calderwood et al. , 1985) .
Heat Shock and Second Messenger Systems
Heat shock causes increased turnover rates of phosphatidyl-
inositol trisphosphate in cells (Calderwood et al., 1987) . The
rate of turnover induced in the cell is rapid enough to quickly
deplete the membrane store of polyphosphoinositides, indicating

60
high activity of phospholipases. The hydrolysis of polyphospho¬
inositides is normally the result of specific activation of a
membrane-bound receptor. There is subsequent activation,
mediated by a G-protein, of specific phospholipases (Berridge,
1984) . Release of diacylglycerol in the membrane and of inositol
trisphosphate into the cytosol results in activation of protein
kinases, including protein kinase C, and release of intracel¬
lular Ca+ stores, probably from the endoplasmic reticulum
(Berridge, 1984). Heat shock may, therefore, perturb regulation
of intracellular Ca+ concentrations and Ca+-dependent events in
the cell as well as the pattern of intracellular protein phospho¬
rylation. The intracellular pattern of tyrosine phosphorylation
is altered by heat shock (Maher and Pasquale, 1989) as is the
phosphorylation state of several protein-synthesis initiation
factors (Duncan and Hershey, 1989) .
During heat shock, and following an increase in inositol
trisphosphate (Calderwood et al., 1987), there is a large
increase in intracellular Ca* concentration (Stevenson et al.,
1986? Calderwood et al., 1988). The complete impact of this
increase in Ca* concentration in the heat-shocked cell is
unclear, although it is of critical importance. Inhibition of
calmodulin activity in the cell increases its sensitivity to heat
(Weigant et al., 1985) and incubating cells in media containing
either low or high Ca* concentrations increases the heat sensit¬
ivity of the cells (Malhotra et al. , 1986) . There is also influx
of Ca* from extracellular sources as a result of membrane

61
leakage. Intracellular Ca+ overload results in activation of
protein kinases, phospholipases and proteinases to perturb
cellular homeostasis (Bowler, 1987).
Heat-Shock Proteins
Among the responses to heat shock is the alteration of protein
synthesis in the cell in favor of the increased transcription
and translation of small number of evolutionarilyconserved
proteins called heat-shock proteins. Enhanced production of
heat-shock proteins in response to cellular stress has been
observed in virtually every species of plant or animal examined
(Ashburner, 1982; Nover, 1984). Since increased production of
these proteins has been observed during treatment with a variety
of other cellular stressors such as hydrogen peroxide, ethanol,
and heavy metals, they have come to also be called stress
proteins (Welch et al., 1982). Heat-shock proteins were first
observed in the early 1960s as products of gene transcription
associated with the heat-induced alteration of chromosomal
puffing in the giant polytene chromosomes of the Drosophila
salivary gland (Ashburner, 1982). Chromosomal puffing was
recognized as a characteristic of areas of active gene transcrip¬
tion, and heat shock became one of the first model systems for
study of gene transcription. Until quite recently, most of the
work on heat shock was related to mechanisms of gene expression
and little attention was given to functional aspects of the heat-
shock proteins.

62
Appearance of heat-shock proteins is correlated with the
acquisition of thermotolerance (LiandWerb, 1982; Landry et al.,
1982 ? Heikkila et al. , 1985) . Activation of various homeostatic
mechanisms of the cell through incremental exposure to stress
leads to a state of increased thermotolerance (Henle and
Dethlefson, 1978; Li and Werb, 1982; Ashburner, 1982; Nover,
1984), the complete response pattern to achieve this tolerant
status remains unclear, however. Temperature sensitivity and
resistance can be created by altering the ability of organisms
to generate heat-shock proteins (Craig and Jacobsen, 1984;
Riabowel et al., 1988; Landry et al., 1989). Riabowel et al.
(1988) inserted antibodies against the 70-kDa heat-shock protein
into fibroblasts by microinjection. Cells containing antibody
to heat-shock protein were more heat-sensitive than cells
injected with control antibody (goat-anti-chicken IgG). The
ability of another heat-shock protein, the human 27-kDa protein,
to confer thermotolerance was demonstrated by placing a gene
construct containing the gene for 27-kDa plus a constitutive
(always on) promotor into mouse cells (Landry et al., 1989).
Transfected cells became thermotolerant, cells transfected with
the promotor elements only did not (Landry et al. , 1989) .
Heat-shock proteins can confer thermotolerance, however it
remains to be determined whether heat-shock proteins are the
primary effector in thermotolerance or a secondary response
since the correlation between appearance of heat-shock proteins
and thermotolerance does not always hold true. In the studies of

63
Hall (1983), Hallberg (1986), Widelitz et al. (1986) and Easton
et al. (1987) a thermotolerant state was achieved in yeast,
tetrahymena, rat fibroblasts and adult salamanders, respec¬
tively, in the absence of heat-shock protein synthesis.
Several heat-shock proteins have been identified in mammalian
cells including a low molecular weight group appearing at about
27 to 28-kDa, 32-kDa and 58-kDa which have yet to be studied
extensively. Little is known about these low molecular-weight
heat-shock proteins and a 110-kDa heat-shock protein other than
their existence in stressed cells and a few physical charac¬
teristics (Welch et al. , 1989) . It is known, in the case of the
27-kDa protein, that thermotolerance is associated with expres¬
sion (Landry et al. , 1989) . By far the most studied are the 70-
kDa group of heat-shock proteins and a related group of glucose-
regulated proteins. Another mammalian heat-shock protein that
has been well studied is the 90-kDa species.
The 70-kDa heat-shock proteins
The 70-kDa family of heat-shock proteins include at least
eight related proteins in Drosophila and yeast, and at least
five, and as many as ten, related proteins in mammals (Zakeri et
al. , 1988) . Some of these are constitutively produced and hence
called heat-shock cognate, while others are induced by heat
shock. Other members of this family of proteins include the dnaK
and groEL proteins of E. coli and the YG 100 and YG 102 proteins
of S. cerevisiae, mutants of which confer temperature sensi¬
tivity (Craig and Jacobsen, 1984). Since it is difficult to

64
distinguish individual members of this family of proteins except
through nucleic acid hybridization experiments, they have been
generally called simply the 70-kDa heat-shock protein.
Several characteristics of 70-kDa proteins provided clues
to potential function in the cell. During stress, these proteins
accumulate in the nucleus and nucleolus of the cell (Welch and
Feramisco, 1984; Pelham, 1984; Lewis and Pelham, 1985).
Nucleolar morphology is altered during heat shock (Pelham, 1984 ;
Welch and Suhan, 1985), presumably due to denaturation and
aggregation of proteins and ribonucleoproteins caused by heat
shock (Pelham, 1984). Transfection of cells with a plasmid which
constitutively expressed 70-kDa increased the rate of nucleolar
recovery after heat shock (Pelham, 1984) . The 70-kDa protein is
also an ATP-binding protein and Lewis and Pelham (1985) found
that the ability of deletion mutants of 70-kDa to migrate to the
nucleolus and aid in recovery was dependent on ATP-binding. They
proposed that ATP-driven cycles of binding and release of 70-kDa
cause solubilization aggregates of proteins and ribonucleopro¬
teins that form during heat shock. Another member of the 70-kDa
family was shown to be active in the ATP-driven process of
removal of clathrin from coated vesicles (Chappel et al. , 1986) .
Another ATP-binding protein related to the 70-kDa family is
the glucose-regulated protein 78, also known as immunoglobulin
heavy chain binding protein or BiP. This protein is present in
the endoplasmic reticulum and binds to unfolded protein subunits
or to improperly folded proteins (Munro and Pelham, 1986).

65
Following proper assembly of proteins in the endoplasmic
reticulum, BiP is released by ATP. Another related protein, the
groEL protein of E.coli, binds to subunits of secretory proteins
in an ATP-dependent manner during their assembly in the endoplas¬
mic reticulum and is essential for correct folding and assembly
(Bochkareva et al. , 1988) . Collectively these data have led to
the hypothesis that members of the 70-kDa heat-shock protein
family are engaged in maintenance of protein and ribonucleo-
protein folding and assembly both in unstressed and in heat-
stressed cells (Welch et al., 1989) .
Constitutive expression implies some function during normal
cell function. Recently, evidence has been accumulated that 70-
kDa proteins are integral to protein translocation across
membranes (Chirico et al., 1988; Deshaies et al., 1988; Murakami
et al., 1988; Flynn et al., 1989). In these studies, protein
translocation into mitochondria (Deshaies et al. , 1988 ; Murakami
et al. , 1988) or into synthetic microsomes (Chirico et al. , 1988)
was dependent upon the presence of 7 0-kDa protein as the addition
and depletion of the 70-kDa protein in the system enhanced and
depressed, respectively, the rate of translocation. Import of
proteins across membranes and into organelles such as mitochon¬
dria requires a high degree of control; sequestration of protein
by membranes is essential to life. The hypothesis of Chirico et
al. (1988), Deshaies et al. (1988) and Murakami et al. (1988) is
that translocation of completely formed protein requires the

66
ATP-driven alteration of conformation through binding with 70-
kDa heat-shock protein. Flynn et al. (1989) demonstrated
specific and saturable binding of BiP to peptides. BiP bound to
both hydrophobic and charged peptide sequences perhaps indi¬
cating steric, or topographic, recognition (Flynn et al., 1989).
The 70-kDa proteins have also been implicated in the mechanisms
of peptide recognition during lysosomal degradation of intra¬
cellular protein as part of normal protein turnover in the cell
(Chiang et al. , 1989) , a finding related to the data of Ananthran
et al. (1985) discussed previously.
The 90-kDa heat-shock protein
The 90-kDa protein is abundant in most cells and in amplified
three- to five-fold following heat shock (Welch et al., 1989).
The 90-kDa protein associates with a number of tyrosine kinases
in the cell, particularly the oncogene product pp60src (Brugge et
al., 1981). Association of 90-kDa with the pp60src apparently
inactivates its tyrosine kinase activity until the protein has
been delivered to the cell membrane (Brugge et al. , 1981) .
The 90-kDa protein is associated with the nontransformed or
non-DNA-binding form of the glucocorticoid and progesterone
receptor (Catelli et al. , 1985; Sanchez et al. , 1987; Howard and
Distelhorst, 1988; Baulieu and Catelli, 1989). Hormone-depen-
dent and temperature-dependent transformation of the receptor
to the DNA-binding form are analogous events (Sanchez et al.,
1987) . In both cases, the 90-kDa protein dissociates from the

67
receptor complex. It is believed that dissociation of the 90-
kDa protein from the receptor complex uncovers the DNA-binding
site of the receptor (Baulieu and Catelli, 1989). Ramachandran
et al. (1988) have shown that synthesis of the 90-kDa heat-shock
protein can be regulated by steroid hormones in reproductive
tissues. Uterine 90-kDa protein was depressed following
ovariectomy in mice, and estradiol injections caused a 4-fold
increase in the concentration of the protein; data were normal¬
ized to total protein in cytosolic extract to account for
estradiol-induced increases in uterine wet weight and protein
concentration. This increase in 90-kDa protein was accompanied
by a similar increase in progesterone receptor concentrations
(Ramachandran et al. , 1988).
Ubiouitin
Ubiquitin is a protein involved in the process of protein
turnover in normal cells, proteins are generally short-lived and
turnover and recycling of amino acids is a normal activity in
most cells (Rechsteiner, 1987). Ubiquitin recognizes and binds
to peptides that are destined for degradation in the cell and
presents them to specific proteases (Rechsteiner, 1987).
Ubiquitin is also a heat-shock protein (Bond and Schlesinger,
1985) . Enhancement of protein turnover by ubiquitin-mediated
activity occurs after heat shock (Carlson et al. , 1987) .
Thermostabilizing Agents
Thermostabilizing agents offer the potential to pharma¬
cologically reduce heat stress-induced embryonic death,

68
particularly during early development when embryos may have no
inherent capability to respond to heat stress.
Amino acids
Vidair and Dewey (1987) found that CHO cells in a nutrient-
deprived state were extremely sensitive to heat shock and that
several hydrophilic, neutral amino acids, including alanine,
would reduce this sensitivity. Henle et al. (1988) found that
alanine exerted this protective effect upon CHO cells in the
absence of any nutrient deprivation-induced hypersensitivity.
In those studies also, concentrations of alanine in the milli-
molar range were required, the effect was time dependent, and
D-alanine was equally effective (Henle et al. , 1988), indicating
that incorporation into protein was not necessary. Sequential
addition and removal of the supplemental alanine around the time
of heat shock demonstrated that alanine was a thermoprotective
agent, i.e., it must be present during the heat shock, rather
than an inducer of thermotolerance whose effect remains for a
time after it is removed (Henle et al. , 1988) . Both Henle et al.
(1988) and Vidair and Dewey (1987) found that alanine thermo¬
protection was evident within 5 min of alanine addition to cell
cultures.
Taurine has been implicated in a variety of protective roles
within cells (Wright et al., 1986) and certain cell types
actively take up taurine. Banks et al. (1989) have shown that
alveolar macrophage, which generate oxidants and are relatively
resistant to oxidant injury, have a specialized transport system

69
for taurine which is, in part, a carrier-mediated transport. In
human lymphocytes, 60% of the intracellular free amino acid pool
is taurine (Fukuda et al., 1982) and taurine will enhance
viability of lymphocytes in vitro in a dose-dependent manner
(Gaull et al., 1983) under homeothermic conditions. Taurine is
abundant in the mammalian reproductive tract (Lorincz et al.,
1968) and is taken up by preimplantation embryos (Kaye et al.,
1986) , making it attractive to speculate that taurine has some
role in the early embryo, possibly as an antioxidant. Like
D-alanine, taurine is not incorporated into cellular protein.
The roles of these amino acids may lie in stabilization of
proteins and organelles or antioxidation in the heat-shocked
cell. Vidair and Dewey (1987) suggested that because amino acids
were required in such large amounts and were not involved in
formation of proteins, stabilization of cellular components such
as proteins by aggregation around the surface of the protein to
cover hydrophobic sites exposed during denaturation, and thereby
impede non-specific aggregation of proteins, was the likely role
of protective amino acids. This type of effect, i.e., non¬
specific aggregation around a protein molecule, is similar to
that suggested for the 70-kDa heat-shock protein (Lewis and
Pelham, 1985) .
Reduced glutathione
The intracellular oxidative-reductive state may be related
to heat shock since oxidative stress also induces heat-shock
proteins (Li and Werb, 1982; Lee et al., 198 3; Drummond et al.,

70
1987) and heat shock induces increases in antioxidant enzymes
such as superoxide dismutase (Omar et al. , 1987) . Additionally,
cellular responses to hydrogen peroxide exposure are similar to
heat-shock responses and development of tolerance to these two
stressors are, to a large degree, interchangeable (Li and Werb,
1982; Spitz et al., 1987) .
Glutathione and glutathione S-transferase play a critical
role antioxidation and detoxification in cells. Glutathione S-
transferase catalyzes the nucleophilic addition of the thiol
group of glutathione to electrophilic acceptors including
peroxides, thiocyanates and alkyl halides (Armstrong, 1987).
Depletion of intracellular glutathione was reported to
increase sensitivity of cells to heat shock (Mitchell et al.,
1983; Russo et al. , 1984) although metabolic inhibitors used to
deplete and prevent new synthesis of glutathione may have created
toxic conditions. Lilly et al. (1986) used a mutant fibroblast
cell line deficient in glutathione production and determined
that these cells were competent to develop thermotolerance even
in the presence of very low glutathione levels. Mitchell et al.
(1983) did find that glutathione concentrations increased in
cells during heat shock and during the development of thermo¬
tolerance .
Vitamin E
Vitamin E is a hydrophobic, peroxyl radical-trapping, chain
breaking antioxidant found in membranes and its principal
function is to protect cells from the effects of spontaneous

71
autooxidation (Burton et al., 1983). Treatment with vitamin E
in vitro protected cells from chemical-induced injury (Pascoe et
al., 1987) .
Cycloheximide
This protein synthesis inhibitor protects cells from heat-
induced cell killing (Lee and Dewey, 1986; Armour et al. , 1988) .
A 95% inhibition of protein synthesis could be attained without
toxicity to cells (Yong and Lee, 1986) and the onset of inhibi¬
tion corresponded to onset of thermal protection. Increased
nuclear and nucleolar protein accumulation was blocked by this
inhibition and failure of DNA repair was also blocked (Armour et
al. , 1988) , suggesting that nuclear and nucleolar functions were
less impaired by heat shock in the absence of intranuclear
protein accumulation.
Others
Other chemical agents have been shown to exert beneficial
effects in reducing death of stressed cells in vitro, including
1 M glycerol (Henle and Warters, 1982) and soybean trypsin
inibitor (Korbelik et al. , 1988) .
Objectives
Elevated environmental temperature results in depressed
fertility in female cattle and other species. This effect is
multifaceted, occurring at various times and at multiple
locations during the establishment and maintenance of pregnancy.
There are both direct effects of hyperthermia on the early embryo

72
and indirect effects on maternal blood flow, hormone concentra¬
tions and metabolism. The objectives of the research described
in this dissertation were to identify and characterize direct
effects of heat shock on tissues of the maternal reproductive
tract and conceptus to identify possible causes of hyperthermia-
induced infertility.
Synthesis and secretion of proteins and prostaglandins by
reproductive tract tissues are critical to early maternal-
conceptus interactions. Disruption of oviductal and endometrial
protein secretion by direct hyperthermia, in addition to changes
caused by endocrine imbalance, may disrupt early maternal
conceptus interactions. Comparisons of tissue responses of Zebu
and Bos taurus cattle were made in order to determine whether
Zebu cattle exhibit greater homeothermic advantages at the
tissue level in response to direct hyperthermia. Cellular
responses to elevated temperature can result in thermotolerance.
Another objective was to determine whether reproductive tract
tissues respond to elevated temperature in a similar manner to
the many cell types that have been examined in vitro, i.e. , with
production of heat-shock proteins.
Alterations in prostaglandin release by the uterine endo¬
metrium at day 17 of pregnancy in the cow as a result of conceptus
signalling allows maintenance of the CL and permits pregnancy to
continue beyond this time. Disruption of these events by direct
hyperthermic effects might result in failure of luteal main¬
tenance. Direct effects of hyperthermia on prostaglandin

73
secretion by endometrium, including effects on regulation of
secretion, were examined in addition to direct effects of
elevated temperature on secretion of bTP-1 by day 17 conceptuses.
Events later in pregnancy are also affected by hyperthermia.
Heat stress during mid- and late-pregnancy reduces fetal and
placental weight and efficiency of placental function. Protein
and prostaglandin synthesis and secretion in response to in vitro
heat shock were examined in ovine uterine and placental tissues
late in pregnancy to determine whether alterations occur that
might impact fetal growth.
Thermostabilizing agents were tested in vitro to determine
the efficacy of exogenous treatments to alter responses to heat
stress. Bovine peripheral lymphocytes and preimplantation mouse
embryos were used to test heat shock-induced killing in the
presence and absence of these agents in vitro. One of these
agents, alanine, was tested in an in vivo experiment with early
pregnant mice to determine whether intraperitoneal injection
could improve embryonic survival during maternal hyperthermia.
Two stages of embryonic development were tested, before and after
development of the ability to produce heat-shock proteins in
response to stress, to determine whether embryonic capacity to
produce heat-shock proteins in response to hyperthermia might
effect embryonic survival.
Some of these experiments, those reported in Chapter 5, were
carried out in collaboration with D. James Putney and these

74
results also appear in his Ph.D. dissertation (University of
Florida, 1988).

CHAPTER 2
DIFFERENCES BETWEEN BRAHMAN AND HOLSTEIN COWS IN
ALTERATIONS OF PROTEIN SECRETION
BY OVIDUCTS AND UTERINE ENDOMETRIUM
INDUCED BY HEAT SHOCK
Introduction
Maintenance of homeothermy is critical to fertility in
cattle (Gwazdauskas et al., 1973; Turner, 1982) and the repro¬
ductive performance of Zebu cows in tropical or sub-tropical
climates is superior to that for European-type cows (Peacock et
al., 1971, 1977; Turner, 1982). Though not statistically
significant, Turner (1982) found a tendency for fertility to
decline less in Zebu cows for each unit increase in body tempera¬
ture than in Bos taurus cows. This suggests that Zebu cattle may
exhibit advantageous characteristics at the level of the
reproductive tract tissues during periods of hyperthermia. If
Zebu cattle are more resistant to heat stress at the cellular and
tissue level, the disruptive effects of heat shock on protein and
prostaglandin secretion might be less severe. The objectives of
the present study were to determine whether tissues of the
oviduct and uterine endometrium of Brahman (Bos indicus) and
Holstein (Bos taurus) cattle differ in the synthesis and
secretion of proteins, prostaglandins and DNA in response to in
vitro heat shock. Cyclic cows were sampled at estrus since this
75

76
represents the time of greatest susceptibility to heat stress in
terms of adverse effects upon conception rates (Dutt et al.,
1959; Dutt, 1963; Dunlap and Vincent, 1971; Gwazdauskas et al.,
1973) .
Materials and Methods
Materials
Eagle's minimum essential medium (MEM) was modified by
supplementation with penicillin (100 units/ml), amphotericin B
(250 ng/ml), streptomycin (100 /ng/ml), insulin (.2 units/ml),
non-essential amino acids (1%, v/v) and glucose (5 mg/ml).
Medium was also supplemented with D-calcium pantothenate (100
Mg/ml), choline chloride (100 /ng/ml) , folic acid (100 Mg/ml),
i-inositol (200 nq/ml) , nicotinamide (100 /ng/ml) , pyridoxal-HCl
(100 /ng/ml) , riboflavin (10 /ng/ml) and thiamine (100 /ng/ml) .
Content of L-leucine was limited to .1 times normal (5.15 mg/1)
to enhance uptake of L- [4,5-3H] leucine added to cultures. Medium
was filter sterilized (.22 /nm) and stored at 4"C. All medium
components were from GIBCO (Grand Island, NY).
Materials used in isoelectric focusing, sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
Western blotting were as follows: tris (hydroxymethyl)amino-
methane (Tris) base, phenylmethylsulfonylfluoride (PMSF),
Nonidet P-4 0, and N, N, N ' , N '-tetramethyl ethylenediamine (TEMED)
were purchased from Sigma Chemical Co. (St. Louis, MO) . Sodium

77
salicylate, 2-mercaptoethanol, glycine, and ammonium peroxydi-
sulfate were purchased from Fisher Scientific (Orlando, FL).
Acrylamide, urea, dithiothreitol, sodium dodecyl sulfate (SDS),
and amido black 10B were purchased from Research Organics
(Cleveland, OH). Bis-acrylamide, gelatin, and Tween-20 were
purchased from Bio-Rad (Richmond, CA) . Carrier ampholytes used
in isoelectric focusing were purchased from Serva (Heidelberg,
FRG) . Lutalyse (dinaprost tromethamine) was obtained from The
Upjohn Co. (Kalamazoo, MI). Synchromate-B was purchased from
Ceva Laboratories (Overland Park, KS) . L-[4,5-3H]leucine
(SA, -150 Ci/mmol) and [ 5,6,8,11,12,14,15-3H] PGF2a (SA,~180
Ci/mmole) were purchased from Amersham Corp. (Arlington
Heights, IL) and [methyl-3H]thymidine (SA,~6.7 Ci/mmol) was from
New England Nuclear (Boston, MA) . Radioinert PGF2a was purchased
from Sigma. Rabbit antiserum to PGF2a was kindly provided by Dr.
T. G. Kennedy at the University of Western Ontario. Enzygnost
serum progesterone kits were a gift from Hoechst-Roussel
Agri-Vet Company (Somerville, NJ). Antibody against a major
mammalian heat-shock protein was generously provided by Dr. W.
J. Welch of the University of California, San Francisco. The
mouse monoclonal antibody (C92) was specific for the inducible
72-kDa heat-shock protein produced by HeLa cells (Welch et al. ,
1982). CHAPS (3-[3-cholamidopropyldimethylammonio]-1-pro¬
pane-sulfonate) was purchased from Calbiochem (La Jolla, CA).
Horseradish peroxidase (HRP) -conjugated second antibody was from

78
Bio-Rad (Richmond, CA) , as was the HRP color development reagent,
4-chloro-l-napthol.
Animals
In experiment 1, six purebred Brahman and five purebred
Holstein cows having active corpora lútea as determined by rectal
palpation received 25 mg of prostaglandin F2a (Lutalyse).
Subsequently cows were given a steroid regimen to ensure that all
cows, irrespective of breed, were exposed to a common hormonal
environment so as to avoid breed-specific variation in steroid
production (Adeyemo and Heath, 1980; Randel, 1980) which might
confound tissue responses to in vitro treatments. Cows received
a norgestomet ear implant (Synchromate-B) 11 d after Lutalyse.
Implants were removed after 14 d and all cows were injected
(i.m.) with 6 mg estradiol cypionate. This pharmacological dose
of a long-acting estrogen was administered to create an estrogen
dominated uterus, as would be the case when embryos are most
susceptible to heat stress. Cows were slaughtered 70 h later,
tissues collected as described below and serum obtained for
determination of progesterone.
Due to the potentially critical nature of events occurring
in the oviduct, the response of oviductal tissue to in vitro heat
shock was examined in a second experiment in order to further
characterize heat-shock induced changes. In this experiment,
oviducts were collected from four cows of various percentages of
Angus and Brahman breeding to examine the effects of in vitro
heat shock on de novo synthesis and secretion of protein by the

79
bovine oviduct. These cows were slaughtered on the day of
naturally occurring estrus and oviducts collected as described
below.
In both experiments, reproductive tracts were collected
following exsanguination and transferred to a sterile laminar
flow hood. The ovaries were examined to determine the site of
preovulatory or recently ovulated follicles. Oviducts were sub¬
sequently identified as ipsilateral or contralateral with
respect to the site of ovulation, hereafter called the active
ovary. Entire oviducts (ampulla and isthmus) were trimmed free
of connective and vascular tissue, sliced longitudinally to
expose inner endosalphinx and cut into 2-3 mm3 cubes. Thus, each
cube contained endosalpinx, myosalpinx and serosal layers.
Explants were weighed and cultured as described below. In each
culture, cubes from both isthmus and ampullary regions were
mixed together so that both types of tissue were present.
Uterine horns were opened just above the external bifurcation.
Intercaruncular endometrial tissue from both uterine horns was
dissected free from myometrium and cut into 2-3 mm3 cubes.
Explant Culture
Tissue explants (500 mg) were placed in sterile plastic 100
mm Petri dishes and cultured in 15 ml of modified MEM under an
atmosphere of 50% N2, 47.5% 02 and 2.5% C02 (v/v/v) . Cultures were
maintained in the dark on rocking platforms. In experiment 1,
explants of oviductal tissue were cultured at homeothermic (10
h, 39 0 C) or heat-shock (6 h, 39°C; 4 h, 43°C) temperature. At 6

80
h, cultures were pulse-chase labeled. For 2 h, endometrium was
cultured in MEM containing 50 /¿Ci L-[ 4,5-3H] leucine. At the end
of this period the medium was replaced with MEM containing 51.5
mg/liter L-leucine. Tissue and medium were harvested 2 h later.
Endometrial explants were cultured similarly except that pulse
labeling was performed for 0 to 30, 30 to 60 and 60 to 90 min
following the onset of heat-shock treatment followed by a 2 h
chase period. Pulse-chase labeling also was done for the first
0 to 15 min after the onset of heat shock and considered separa¬
tely for statistical analysis. Additional endometrial cultures
were incubated for 24 h at homeothermic or heat-shock tempera¬
ture in the presence of 50 [iCi L-[ 4,5-3H] leucine or 25 /iCi
[methyl-3H]thymidine.
In the second experiment, oviduct cultures were incubated
at either homeothermic (24 h, 39°C) or heat-shock (6 h, 39°C? 18
h, 43 °C) temperature in the presence of 50 /xCi L-[4,5-3H] leucine
added after 6 h. For each experiment, cultures were stopped by
the separation of tissue and medium during centrifugation (700
x g; 30 min; 4°C). Tissue was immediately placed in ice-cold
solubilization buffer [50 mM Tris-HCl, pH 7.6, which contained
1 mM phenylmethylsulfonylfluoride, 1 mM EDTA and 2% (w/v) CHAPS]
and homogenized. Samples of tissue and medium were stored at
-20°C until analyzed.
Protein Synthesis and Secretion
Secretion of de novo synthesized macromolecules into
culture medium by oviducts and endometrium was determined by

81
measuring incorporation of [3H]leucine into nondialyzable
macromolecules. Conditioned culture medium was dialyzed
extensively (three changes of 4 liters) against deionized water
using dialysis tubing with a 6,000-8,000 dalton exclusion limit.
Radioactivity in the reténtate was determined by scintillation
spectrometry. Measurement of [3H]leucine incorporated into
macromolecules in solubilized tissue was done by trichloroacetic
acid (TCA) precipitation. Samples (50 nl) of solubilized tissue
were placed onto Whatman 3MM paper (previously saturated with 2 0%
TCA [w/v]) and allowed to dry. Proteins were precipitated onto
filter paper by serial washings with 20% TCA, 5% TCA and 95%
ethanol as described by Mans and Novelli (1961) . Radioactivity
of precipitated protein was determined by scintillation spec¬
trometry.
DNA Synthesis
Incorporation of [3H] thymidine into tissues was determined
by TCA precipitation of nucleic acids onto filter discs (Mani-
atis et al., 1982) . Tissue was solubilized in 10 mM Tris which
contained 1 mM EDTA and centrifuged (2,500 xg, 30 min, 4°C). Two
volumes of ice-cold 95% (v/v) ethanol were added to supernatants
and samples were frozen (-20°C) overnight. Samples were then
centrifuged (12,000 xg, 10 min, 4°C) and the pellet resuspended
in 200 /il of 10 mM Tris containing 1 mM EDTA. An equal volume of
ice-cold 5% (w/v) TCA was then added and samples chilled on ice
for 15 min. An aliquot of each sample was blotted and dried onto
Whatman filters previously treated with 5% (w/v) TCA. Filters

82
were washed five times each with 10% (w/v) TCA and 95% (v/v)
ethanol. Presence of radiolabel was determined by scintillation
spectroscopy.
Measurement of Prostaglandin F
Medium from cultures incubated for 24 h in the presence of
[3H]leucine was assayed for PGF using a RIA procedure (Knicker¬
bocker et al., 1986) modified to use an antibody characterized
by Kennedy (1985) that recognizes F series prostaglandins. The
assay was validated for use with MEM in this culture system as
described in Chapter 5.
Electrophoresis
One-dimensional polyacrylamide gel electrophoresis in the
presence of SDS (1-D SDS-PAGE) was performed using the buffer
system of Laemmli (1970). Tissue homogenates were dialyzed
extensively (three changes of 4 liters) against deionized water
using dialysis tubing with a 6,000-8,000 dalton exclusion limit.
Radioactivity in the reténtate was determined by scintillation
spectrometry. Proteins were prepared in a solubilization buffer
containing 62.5 mM Tris, pH 6.8, 5% (w/v) SDS, sucrose and 5%
(v/v) 2-mercaptoethanol. Solubilized proteins were resolved on
12.5% (w/v) polyacrylamide gels in the presence of .05% (w/v)
SDS. Two-dimensional SDS polyacrylamide gel electrophoresis (2-D
SDS-PAGE) was performed on proteins secreted into the culture
medium using procedures of Roberts et al. (1984). Samples were
dissolved in 5 mM K2C03, pH 10.5, which contained 9.0 M urea, 2%
(v/v) NP-40 and .5% (w/v) dithiothreitol. Proteins were resolved

83
in the first dimension by isoelectric focusing in 4% (w/v)
polyacrylamide disk gels containing 250 mM N, N 1 diallyltartar-
diamide (DATD) , 8.0 M urea, 2% (v/v) NP-40 and 5.1% (v/v)
ampholytes (pH 3 to 10, 5 to 7 and 9 to 11; 50:36:16 by vol.,
respectively) . Disk gels were equilibrated with 60 mM Tris, pH
6.8, 1% SDS (w/v) and 1% (v/v) 2-mercaptoethanol, and subjected
to electrophoresis in the second dimension on 12.5% (w/v)
polyacrylamide gels in the presence of .5% (w/v) SDS. Equal
amounts of radiolabeled protein were loaded onto each gel to
determine qualitative differences due to treatments. Proteins
were fixed in the gels using acetic acid, ethanol and water
(7:40:53, by vol., respectively), and the gels were dried and
fluorographs prepared using sodium salicylate as described by
Roberts et al. (1984) .
Western Blotting
Proteins from control and heat-shocked tissue were resolved
by 1-D SDS-PAGE on 7.5% (w/v) polyacrylamide gels. Gels were
equilibrated for 15 min in 25 mM Tris-HCl buffer (ph 6.8) which
contained 200 mM glycine and 20% (v/v) methanol, overlaid with
nitrocellulose membrane (BA85, .45 ¿im) and subjected to electro¬
phoresis (200 mA, 24 h, 4’C) toward the cathode. Following
electrophoretic transfer the protein binding sites on the
membranes were blocked by incubation with 3% (w/v) gelatin.
Membranes were then incubated with a monoclonal mouse IgG
specific for the inducible 72-kDa heat-shock protein. Duplicate
membranes were incubated with normal mouse serum to determine the

84
extent of nonspecific binding. Membranes were then incubated
with anti-mouse IgG conjugated to horseradish peroxidase.
Presence of the proteins was detected by incubating with .05%
(w/v) 4-chloro-l-napthol in 100 mM Tris containing 20% (v/v)
methanol and .02% (v/v) hydrogen peroxide.
Serum Progesterone Determination
Progesterone concentrations in serum were determined
utilizing the Enzygnost serum progesterone kit of Hoechst-
Roussel Agri-Vet Company. This kit utilizes a competitive
enzyme-linked immunoassay for progesterone and alkaline phospha¬
tase detection system in which color development is read at 490
nm. Standards included in the kit were used to prepare a
standard curve from 0 to 10 ng/nl. Absorbance at 490 nm of
unknown samples were compared to standard curve.
Statistical Analysis
Data were analyzed by least squares analysis of variance
using the General Linear Models procedure of the Statistical
Analysis System (SAS, 1985) . Models used to analyze endometrial
data included effects of breed, cow nested within breed,
incubation temperature of cultures and, for pulse-chase studies,
time of labeling and the interactions of all these effects. To
analyze data from oviductal explants, the model included effects
of breed, cow nested within breed, side of the reproductive tract
relative to the active ovary, incubation temperature, and their
interactions.

85
Results
Animals
At the time of slaughter of cows in experiment 1, 4 of 6
Brahman cows and 3 of 5 Holstein cows had ovulated as evidenced
by the appearance of a recent ovulation point on the ovary and
the lack of large follicles. One Holstein cow had a serum
progesterone concentration of 5.5 ng/ml and was subsequently
dropped from the statistical analyses, leaving four Holstein
cows in the study. Reproductive tracts of remaining animals had
evidence of estrogenic influence, including highly vascularized,
edematous uteri having a high degree of muscle tone. Serum
progesterone concentrations at slaughter were not different
between breeds and averaged less than 1 ng/ml.
At slaughter, none of the crossbred cows in experiment 2 had
yet ovulated. All exhibited large preovulatory follicles. Uteri
were edematous and vascularized and exhibited a high degree of
muscle tone.
Synthesis and Secretion of r3Hl-Labeled Proteins by Oviducts
Secretion of nondialyzable macromolecules into the culture
medium in experiment 1 was affected by a breed x side x tempera¬
ture interaction (P < 0.04). This was the result of several
effects. At 39°C, secretion was similar between breeds and
between sides of the reproductive tract relative to the active
ovary. Incubation at 43°C, however, caused an elevation in the
release of secreted macromolecules by explants from the con¬
tralateral side in both breeds. For tissue from the side

86
ipsilateral to the active ovary, the temperature associated
increase was evident only in the Brahman cattle, while in
Holstein cows, secretion from the ipsilateral side was sup¬
pressed by elevated temperature (Figure 2-1).
Incorporation of [5H] leucine into macromolecules in tissue
homogenates exhibited a side x temperature interaction (P <
0.07). At39°Conly, incorporation was greater for explants from
the ipsilateral side than for the contralateral side. On the
ipsilateral side, incorporation was depressed by incubation at
43°C (Figure 2-2) while on the contralateral side elevated
temperature caused either no effect (Brahmans) or an elevation
in incorporation (Holsteins).
In experiment 2, secretion of nondialyzable macromolecules
by explants of oviductal tissue from the crossbred cows also
exhibited a side x temperature interaction (P < 0.05). There was
increased secretory activity in response to elevated temperature
by tissue obtained from the contralateral side while tissue from
the ipsilateral side was unaffected by elevated temperature
(Figure 2-3) . As in the first experiment, there was also a side
x temperature interaction (P < 0.07) for incorporation of radio¬
label into TCA-precipitable macromolecules in tissue. Tissue
from the ipsilateral side of the reproductive tract exhibited
greater activity at 39 °C than at 43 °C while the opposite was true
for tissue from the contralateral side (Figure 2-3) .

87
DPM/MG TISSUE/2 h
Secreted macromolecules
Figure 2-1. Secretion of radiolabeled, nondialyzable macro¬
molecules into culture medium by oviductal explants. Data are
expressed as least square means. Secretion was affected by a
breed x side x temperature interaction (P < 0.04). At 39°C,
secretion was similar between breeds and between sides of the
reproductive tract relative to the active ovary. Incubation at
43 °C, however, caused an elevation in the release of secreted
macromolecules by explants from the contralateral side in both
breeds. For tissue from the side ipsilateral to the active
ovary, the temperature associated increase was evident only in
the Brahman cattle. In Holstein cows secretion was suppressed
by elevated temperature. The pooled sem =31.

88
DPM/MG TISSUE/2 h
Macromolecules in tissue
Least-squares means, SEM=488
3000 Y
2500-
2000 -
1500
1000-
500-
ntraiaterai
Brahman
i rpsiiaterai
39 43
Holstein
Figure 2-2. Incorporation of [3H]leucine into TCA-precipitable
macromolecules in tissue homogenates. Data are expressed as
least square means. Incorporation was affected by a side x
temperature interaction (P < 0.07). Incorporation by explants
of oviductal tissue from the ipsilateral side was depressed by
heat shock in both Brahman and Holstein cows. In explants from
the contralateral side, however, incubation at 43 'C caused
either no effect (Brahmans) or an elevation of incorporation
(Holsteins) . The pooled sem = 488.

89
Secretion of Protein by Endometrial Explants in Pulse-Chase
Experiments
Secretion was examined in the first 0 to 15 min after heat
shock and analyzed separately from other times because of
different labeling time (0 to 15 min and 0 to 30 min). Incor¬
poration of radiolabel into macromolecules in the first 15 min
after heat shock and subsequently secreted into culture medium
was 28 ± 5 and 35 ± 5 dpm/mg tissue for explants from Brahman cows
incubated at 39 °C and 43°C, respectively, and 29 ± 6 and 43 ± 6
dpm/mg tissue for explants from Holstein cows incubated at 39 "C
and 43°C, respectively. The increase in secretion rate caused
by the higher incubation temperature tended to be significant (P
< 0.07) .
Secretion of nondialyzable [3H]macromolecules by endo¬
metrial explants in the first 0 to 30, 30 to 60 and 60 to 90 min
after onset of heat shock is shown in Figure 2-4. Secretion of
newly synthesized protein was significantly affected by time (P
< 0.02) and incubation temperature (P < 0.01) . Secretion tended
to be lower for 0 to 30 min than for other times and was increased
by elevated incubation temperature in both Brahman and Holstein
cows. There was no significant effect of breed or interactions.
Amounts of radiolabeled macromolecules in supernatants of
24 h cultures did not differ between breeds or treatments.
Radiolabeled macromolecules present at 24 h were 3865 ± 245 and
4122 ± 245 dpm/mg tissue for explants from Brahman cows incubated
at 39°C and 43°C, respectively; and 4108 ± 287 and 4251 ± 287

90
dpm/mg tissue for explants from Holstein cows incubated at 39 'C
and 43 “C, respectively.
Radiolabeled Tissue Proteins in Endometrium
In the first 15 min following heat shock, incorporation of
[3H] leucine into macromolecules present in endometrial explants
did not differ between breeds or temperature treatments.
Incorporation was 118 ± 28 and 119 ± 28 dpm/mg tissue for
explants from Brahman cows incubated at 39°C and 43°C, and 93 ±
31 and 112 ± 31 dpm/mg tissue for explants from Holstein cows
incubated at 39 °C and 43 "C.
When incorporation of [3H]leucine into tissue macromole¬
cules was examined at 0 to 30, 30 to 60 and 60 to 90 min after heat
shock, a breed x time x temperature interaction was apparent (P
< 0.06) . During the first 30 min after heat shock, incorporation
was lower in Holstein tissue under homeothermic conditions than
in tissue from Brahmans. Furthermore, in the first 30 min,
incubation at 43°C caused an elevation in protein synthesis for
the Holsteins while inhibiting secretion for the Brahmans
(Figure 2-5) . At later times, incorporation was elevated by heat
shock in both breeds. Amounts of radiolabeled macromolecules in
tissue homogenates of 24 h cultures did not differ between breeds
or treatments (21,838 ± 3453 versus 21,963 ± 3453 and 25,3743 ±
841 versus 19,126 ± 3841 dpm/mg tissue for explants from Brahmans
incubated at 39°C v 43°C and Holsteins incubated at 39°C v 43°C,
respectively).

91
DPM/MG TISSUE/24 h
39 43
Secreteó macromolecules
39 43
Macromolecules in tissue
Figure 2-3. Incorporation of [3H]leucine into non-dialyzable
macromolecules in culture supernatants and incorporation of
[3H] leucine into TCA-precipitable macromolecules by explants of
oviductal tissue from crossbred cows. Data are expressed as
least square means. Secretion was affected by a side x tempera¬
ture interaction (P < 0.05). There was increased secretory
activity from the contralateral oviduct in response to elevated
temperature while the tissue from the ipsilateral oviduct was
unaffected. The pooled SEM = 264. Incorporation of radiolabel
into tissue proteins was affected by a side x temperature
interaction (P < 0.07) similar to that for experiment 1. The
pooled sem = 1429.

92
70
DPM/MG TISSUE/30 MIN
Least-spuares means, SEM=6
Secreted macromolecules
0-30 min
30-60 min
60-90 min
Figure 2-4. Secretion of nondialyzable [3H]leucine-labeled
macromolecules by endometrial explants in the first 0 to 30, 30
to 60 and 60 to 90 min after onset of heat shock. Data are
expressed as least square means. Secretion was significantly
affected by time (P < 0.02) and incubation temperature (P <
0.01) . There was no significant effect of breed or interactions.
The pooled sem = 6.

93
Endometrial DNA and PGF Synthesis During 24 H Culture
Incorporation of radiolabeled thymidine into DNA by endo¬
metrial explants was affected by breed and temperature (P <
0.05). Overall, explants from Brahman cows incorporated less
thymidine at 39°C than at 43°C (460 ± 11 versus 770 ± 11 dpm/g
tissue) while the opposite was true for explants (420 ± 11 versus
310 ± 11 dpm/g tissue) .
Prostaglandin F concentrations in endometrium-conditioned
culture medium did not differ between breeds or treatments. Mean
concentrations were 20.8 ± 8.0 and 15.2 ± 7.8 pg/ml/mg tissue for
cultures of Brahman explants performed at 39 "C and 43 °C, respec¬
tively, and 25.0 ± 9.8 and 20.1 ± 10.4 pg/ml/mg tissue for
cultures of Holstein explants at 39 "C and 43 °C.
Analysis of Secretory Proteins by 1-D SDS-PAGE
Supernatants from oviductal cultures were resolved on 12.5%
(w/v) polyacrylamide gels and proteins were visualized by
fluorography. Several major bands of de novo synthesized
proteins were resolved, including ones having molecular weights
of > 97,000, 85,000 to 97,000, 55,000 and 30,000 (Figure 2-6).
This qualitative pattern did not differ between breeds, between
sides of the reproductive tract or between treatments.
Endometrial culture supernatants from 24 h cultures also
were resolved by 1-D PAGE. While there was no effect of tempera¬
ture on qualitative patterns, a difference between breeds was
revealed (Figure 2-7) . A major secretory product of endometrium

94
DPM/MG TISSUE/30 MIN
0-30 mm
30-60 mm
60-90 min
Figure 2-5. Incorporation of [3H]leucine into TCA-precipitable
macromolecules in tissue of endometrial explants. Data are
expressed as least square means. Incorporation was affected by
a breed x time x temperature interaction (P < 0.06). During the
first 30 min after heat shock, incorporation was lower in
Holstein tissue under homeothermic conditions than in tissue
from Brahmans. Incubation at 43 0 C caused an elevation in protein
synthesis for the Holsteins while inhibiting secretion for the
Brahmans. At other times, incorporation was elevated by heat
shock in both breeds. The pooled sem =34.

95
Figure 2-6. Electrophoretic analysis of oviductal secretory
proteins. Non-dialyzable macromolecules in supernatants from
oviductal cultures were resolved on 12.5% (w/v) polyacrylamide
gels and proteins were visualized by f luorography. Equal amounts
of radiolabeled protein were loaded onto each lane. Several
major bands of de novo synthesized proteins were resolved,
including those with molecular weights of > 97 000, 85,000 to
97,000, 55,000 and 30,000 (arrows). The appearance of radio-
labeled macromolecules at the top of each lane represents high
molecular weight material that was unresolved by these gels.
Overall, this qualitative pattern did not differ between breeds,
between sides of the reproductive tract or between treatments.

96
had an estimated molecular weight of 57,500 for tissue from
Brahmans and a lower molecular weight of 55,600 for tissue from
Holstein cattle. This difference was seen for all cows. The
secreted proteins of Brahman and Holstein endometrial explants
were resolved by 2-D SDS-PAGE (Figure 2-7). Both the 57,500 Mr
protein in explant cultures from Brahman cows and the 55,600 Mr
protein in explant cultures from Holstein cows each were composed
of multiple isoforms having a range of pi = 4.8 to 5.8.
Analysis of Tissue Proteins
Tissue homogenates of endometrial and oviductal explants
after 24 h of culture were resolved on 12.5% (w/v) polyacrylamide
gels. Equal amounts of radiolabeled protein were loaded to
examine the qualitative array of proteins produced. The pattern
of polypeptides present after 24 h did not appear to differ
between breeds or between sides of the reproductive tract. There
was an effect of incubation temperature, however, as there was
enhancement in appearance of two major proteins having molecular
weights of about 72,000 and 90,000 (Figure 2-8).
Analysis of 72-kDa Heat-Shock Protein
Western blot analysis of the appearance of the 72-kDa heat-
shock protein in cultured tissue 2.5, 3 and 3.5 h after onset of
heat shock is shown in Figure 2-9. While immunoreactive heat-
shock protein appeared in both control and heat-shocked tissues,
amounts were enhanced in heat-shocked tissue. Accumulation of
72-kDa heat-shock protein did not appear to differ between

Mrx 10
97
Figure 2-7. Electrophoretic analysis of endometrial secretory
proteins. On the left, non-dialyzable macromolecules in endo¬
metrial culture supernatants from 24 h cultures were resolved
by 1-D PAGE on 12.5% (w/v) polyacrylamide gels and proteins were
visualized by fluorography. Equal amounts of radiolabeled
protein were loaded onto each lane. While there was no effect of
temperature on qualitative patterns, a difference between breeds
was revealed (arrows) . A major secretory product of endometrium
had an estimated molecular weight of 57,500 for Brahman and a
lower molecular weight of 55,600 for Holstein cattle. This
difference was seen in all cows. The endometrial secretory
proteins were also resolved by 2-D SDS-PAGE (Panel B). The
57,500 protein in Brahman explant cultures and the 55,600 protein
in Holstein explant cultures had similar isoelectric variants
(arrows) , having a range of pi = 4.8 to 5.8 in both breeds. The
55,600 spot, pi = 8.8, that appears on the Holstein gel also
appeared on some gels for Brahmans.

98
39 43 39 43
Brahman Holstein
Figure 2-8. Electrophoretic analysis of endometrial and tissue
proteins. Non-dialyzable macromolecules in tissue homogenates
of after 24 h of culture were resolved on 12.5% (w/v) polyacryl¬
amide gels. Egual amounts of radiolabeled protein were loaded
to examine the qualitative array of proteins produced. The
pattern of polypeptides present after 24 h did not differ between
breeds. There was an effect of incubation temperature, however,
as there was consistent enhancement in appearance of two major
proteins having molecular weights of about 72,000 and 90,000
(arrows).

99
timel time2 time3 timel time2 time3
Brahman
Holstein
Figure 2-9. Western blot analysis of the appearance of inducible
heat shock protein in representative samples obtained 2.5 h after
the onset of heat shock treatment (Lanes 1-4). Tissue proteins
were resolved on 7.5% (w/v) polyacrylamide gels and electropho-
retically transferred to nitrocellulose membranes. Membranes
were incubated with a mouse monoclonal antibody specific for an
inducible, 72,000 molecular weight heat shock protein. Second
antibody conjugated to horseradish peroxidase was used to
visualized labeled protein. Immunoreactive heat shock protein
appeared in both control and heat shock cultures and was enhanced
by heat shock. Accumulation did not differ between breeds.
Duplicate membranes incubated with normal mouse serum to
determine the extent of nonspecific binding revealed no cross
reactivity with normal serum components (data not shown).

100
breeds. Duplicate membranes incubated with normal mouse serum
to determine the extent of nonspecific binding revealed no cross
reactivity with normal serum components (data not shown).
Discussion
In the present experiment, there was some evidence of breed
effects on cellular responses to heat shock. It remains to be
determined whether these differences contribute to the greater
heat tolerance of Zebu-type cattle.
Heat shock caused an increase in overall de novo protein
synthesis and secretion by oviducts and uterine endometrium in
the first few hours following hyperthermia. The finding that
protein synthesis and secretion is temporarily increased during
heat shock, is consistent with findings that heat-stressed cows
have higher amounts of protein in the uterine lumen than control
cows (Biggers et al., 1987? Geisert et al., 1988). When
examined over a 24 h period, effects of heat shock were general¬
ly nonsignificant, suggesting that heat-shocked tissue became
stabilized over time. This is what would be expected for a
tissue adapted to heat shock (Li and Werb, 1982) and agrees with
other findings that heat shock did not alter the 24 h synthesis
and secretion of proteins from endometrium collected at Day 2,
5, 8 and 17 of the estrous cycle (see Chapters 3 and 5). Some
heat-shock induced disruption in protein synthesis and secretion
over a 24 h culture period did occur for some oviductal cultures
however. Also, heat shock caused an increase in accumulation of

101
medium and tissue proteins throughout a 24 h culture period in
endometrium collected at estrus and caused selective alteration
in secretion of some uterine proteins at other stages of the
estrous cycle (see Chapter 3) .
The responses to heat shock observed for endometrial and
oviductal tissue are atypical of the classical heat-shock
response, in which general protein synthesis is clearly depress¬
ed by heat shock (Nover, 1984) . This system may respond diff¬
erently because tissues were partially adapted to heat shock
before treatment. Though less than for tissues at 43 °C, tissues
at 39"C contained the inducible 72-kDa heat-shock protein and
amounts of this protein appeared to increase with time in
culture. Synthesis of heat-shock proteins is associated with
induced tolerance in many cell types (Mizzen and Welch, 1988) .
The cause of appearance of the heat-shock protein in control
tissues is not certain although preparation of explants from
whole tissue has been reported to induce heat-shock protein
synthesis (Hightower and White, 1981). Appearance of the 72-kDa
heat-shock protein has recently been reported in HeLa cells
cultured at 37°C (Welch and Mizzen, 1988) and the possibility
remains that heat-shock proteins function in normal processes of
living cells not under stress (Catelli et al. , 1985; Chappell et
al., 1986; Haire et al., 1988; Howard and Distelhorst, 1988;
Pelham, 1988).
Generally, the heat-shock response in tissue from Brahman
cattle was similar to that for Holstein cattle. There were,

102
however, some differences between the breeds. Brahmans exhibi¬
ted an initial decline in de novo protein synthesis and secretion
by endometrium in the first 30 min following onset of heat shock.
This decline, which may reflect the shut down of protein syn¬
thesis characteristic of the classical heat-shock response
(Nover, 1984) , did not occur for tissue from Holstein cows. The
failure of tissue from Holsteins to experience this initial
decline could contribute to a decreased resistance to heat
stress because this initial decrease may reduce the amount of
denatured protein formation in non-tolerant tissue (Hightower,
1980). Differences between the breeds could also reflect an in
vitro artifact. Protein synthesis and secretion by explants from
Holstein cows was very low in the first 30 min after heat shock,
even in tissue that remained at 39°C. Perhaps the tissue from
the Holstein cattle had a different resistance to the explant
preparation procedure than the Brahmans and this resulted in
lower initial activity for these explants.
Synthesis of new DNA by endometrial explants also differed
significantly between breeds. Increasing incubation temperature
from 39 °C to 43°C caused increased [3H]thymidine incorporation
into DNA for tissue from Brahmans and a decrease for tissue from
Holsteins. Heat shock affects proliferation in a number of cell
types and the response appears to be dependent upon stage of the
cell cycle during the heat-shock treatment (Read et al., 1983;
Rice et al. , 1984) . There was no effect of breed, heat shock, or
the interaction on the release of PGF. Though heat shock can

103
increase in vitro secretion of PGF by endometrial explants at Day
17 (see Chapter 5) , a time when PGF secretion is elevated in the
cycling animal (Shemesh and Hansel, 1975), it is apparent that
heat shock does not increase PGF synthesis at estrus, a time when
PGF synthesis by endometrium is low (Shemesh and Hansel, 197 5) .
There was also some variation between breeds in the pattern
of proteins released by endometrium. In particular, a secretory
protein with a molecular weight of 57,500 in culture supernatants
from explants obtained from Brahmans existed as a lower molecular
weight form of 55,600 in the cultures prepared from Holstein
tissue. These proteins are analogous to an endometrial secre¬
tory protein designated as endometrial-secretory protein number
10 (see Chapter 3) . This protein was the most abundant secretory
protein at estrus and decreased in secretion rate from estrus
through d 8 of the estrous cycle. Segerson et al. (1984)
reported that several proteins resolved by 1-D PAGE and quan¬
titated by densitometric scanning were present in greater
concentration in uterine flushings of Angus cows compared to
Brahmans but they did not find any differences in specific
proteins produced. The discrepancy with the present results may
arise from the fact that they examined cows during diestrus.
There was no effect of breed on the array of de novo synthesized
and secreted proteins by explants of the oviduct. The major
proteins in culture supernatants appear identical to those
reported in Chapter 4 for Brangus cows.

104
Bovine endometrium collected from the side of the reproduc¬
tive tract ipsilateral to the corpus luteum or preovulatory
follicle is more likely to be negatively affected by elevated
incubation temperature (see Chapter 3) . Side differences with
respect to uterine and ovarian blood supply (Lamond and Drost,
1974 ; Ginther and DelCampo, 1974) and distribution of progester¬
one from the corpus luteum (Pope et al., 1982; Weems et al. ,
1988) have been reported and Bennett et al. (1988) observed
greater muscular activity by the oviduct ipsilateral to the
active ovary in dairy cows. It is likely, therefore, that
steroids or other molecules released from the preovulatory
follicle or corpus luteum exert local effects on protein
synthesis and secretion by oviducts and endometrium. In the
present experiment the oviduct ipsilateral to the active ovary
also was more likely to be affected by heat shock. For both
experiments 1 and 2, incorporation of [3H]leucine into tissue
proteins was reduced by heat shock for the ipsilateral oviduct
but not for the contralateral oviduct. In addition, protein
secretory activity by the ipsilateral oviduct of Holstein cows
(though not Brahman or crossbred cows) was reduced by heat shock.
In neither experiment was the secretory activity of the con¬
tralateral oviduct reduced by heat shock. In fact, protein
secretion was increased by elevated incubation temperature, as
might be expected for a heat resistant tissue. Segerson et al.
(1984) found that ovaries of Brahman cows were less asymmetric
than those of Angus cattle and this is consistent with our

105
findings that oviductal secretory response to heat stress was
affected by location relative to the site of ovulation in
Holsteins but not in Brahmans.
Heat shock at 43°C must be considered an extreme elevation
in temperature relative to normal physiological ranges in the cow
and these data must therefore be interpreted with caution. We
have measured rectal temperature as high as 42.8°C in heat
stressed cattle, and Wegner et al. (1973) recorded rectal
temperatures of 43.3°C. Nontheless, hyperthermia-induced
responses in tissue activity may occur soon after loss of
maintenance of homeothermy and it is not certain whether the
tissue-specific changes reported here would also occur at
temperatures lower than 43°C. Interestingly, one major effect
of in vitro heat stress seen here, increased endometrial
secretion of protein, is consistent with findings that heat
stressed cows have greater amounts of protein in the uterine
lumen than control cows (Geisert et al. , 1988) .
Variation exists between Brahman and Holstein cattle in
response of reproductive tract tissues to in vitro heat shock.
In addition, slight breed variation exists in the qualitative
pattern of proteins secreted by endometrial explants in vitro.
Whether such variations are sufficient to cause increased
resistance to heat stress and exert effects upon fertility in
heat-stressed cattle remains to be determined.

CHAPTER 3
EFFECT OF DAY OF THE ESTROUS CYCLE, SIDE OF THE REPRODUCTIVE
TRACT AND HEAT SHOCK ON IN VITRO PROTEIN SECRETION BY BOVINE
ENDOMETRIUM
Introduction
Successful establishment and maintenance of pregnancy
involves a close communication between the mother and the
developing conceptus. The critical importance of synchrony in
the development of these two biological systems as pregnancy
progresses has been demonstrated by embryo transfer studies in
domestic animals (Rowson et al., 1972; Wilmut and Sales, 1981;
Wilmut et al., 1985). Steroid-induced alterations in the
reproductive tract during the early period after estrus are
critical to embryonic survival (Miller and Moore, 1976; Moore et
al., 1983; Wilmut et al., 1985) and the rate of embryonic
development in the sheep and cow can be altered by the uterine
environment (Wilmut and Sales, 1981; Wilmut et al. , 1985; Garrett
et al., 1987) . Presumably, many of the steroid-driven functions
of the uterus are mediated by secretory proteins. Stage-speci¬
fic secretion of proteins by the bovine uterine endometrium has
been reported (Roberts and Parker, 1974, 1976; Bartol et al.,
1981a,b). Endometrial proteins play roles as transport mole¬
cules (Buhi et al., 1982; Pentacost and Teng, 1987), protease
inhibitors (Fazleabas et al., 1982), lysosomal enzymes (Hansen
106

107
et al., 1985) and immunoregulatory molecules (Murray et al.,
1978; Hansen et al. , 1989) .
Because of the potential importance of interactions between
the conceptus and maternal endometrium that are mediated by
endometrial secretory proteins, the objectives of the current
experiment were to characterize the pattern of endometrial
protein secretion during the first 8 days after estrus and to
determine whether factors related to the success of pregnancy
are associated with changes in endometrial protein secretion.
Embryos transferred into the uterine horn ipsilateral to the
corpus luteum in cows have an increased chance of survival
compared to embryos transferred into the contralateral horn
(Newcomb and Rowson, 1976; Christie et al., 1979; Newcomb et al.,
1980). The physiological basis for this phenomenon is unclear
and one goal was to identify differences in endometrial protein
secretion between the uterine horns ipsilateral and contra¬
lateral to the ovary bearing the corpus luteum. Secondly, it
was tested whether elevated temperature, which is associated
with a marked increase in embryonic mortality during early
pregnancy in the cow (Thatcher and Collier, 1986), also alters
endometrial protein secretion. Heat shock alters protein
secretion and development in a variety of cell types (Ashburner,
1982;Nover, 1984) and heat induced alteration of the endometrial
protein secretory pattern characteristic of the early period
after estrus may play a role in failure of pregnancy.

108
Materials and Methods
Materials
Eagle's minimum essential medium (MEM) was modified by
supplementation with penicillin (100 units/ml), amphotericin B
(250 ng/ml), streptomycin (100 ^g/ml), insulin (.2 units/ml),
non-essential amino acids (1%, v/v) and glucose (5 mg/ml).
Medium was also supplemented with D-calcium pantothenate (100
nq/ml) , choline chloride (100 nq/ml) , folic acid (100 ¿xg/ml) ,
i-inositol (200 /ig/ml) , nicotinamide (100 /¿g/ml) , pyridoxal-HCl
(100 Mg/ml), riboflavin (10 nq/ml) and thiamine (100 /¿g/ml) .
Content of L-leucine was limited to .1 times normal (5.15 mg/1)
to enhance uptake of L- [ 4,5-3H] leucine added to cultures. Medium
was filter sterilized (.22 ^m) and stored at 4°C. All medium
components were from GIBCO (Grand Island, NY).
Materials for electrophoresis, Western blotting and sample
preparation were as follows: tris (hydroxymethyl)aminomethane
(Tris) base, Nonidet P-40, phenylmethylsulfonylfluoride (PMSF)
and N,N,N',N'-tetramethyl ethylenediamine (TEMED) were
purchased from Sigma Chemical Co. (St. Louis, MO). Sodium
salicylate, 2-mercaptoethanol, glycine, and ammonium peroxy-
disulfate were purchased from Fisher Scientific (Orlando, FL) .
Acrylamide, urea, dithiothreitol, sodium dodecyl sulfate (SDS),
and amido black 10B were purchased from Research Organics (Cleve¬
land, OH). Bis-acrylaraide, gelatin, and Tween-20 were purchased
from Bio-Rad (Richmond, CA) . Carrier ampholytes used in
isoelectric focusing were purchased from Serva (Heidelberg,

109
FRG) . Lutalyse (dinaprost tromethamine) was obtained from
Upjohn Co. (Kalamazoo, MI). L-[4,5-3H]leucine (specific
activity -150 Ci/mmol) was purchased from Amersham Corp.
(Arlington Heights, IL). Antibody against a major mammalian
heat-shock protein (hsp) of 72-kDa was the generous gift of Dr.
W. J. Welch (University of California, San Francisco, CA) . The
mouse monoclonal antibody (C92) was specific for the 72,000 Mr
inducible hsp-72. Horseradish peroxidase (HRP)-conjugated
second antibody was from Bio-Rad, as was the HRP color develop¬
ment reagent, 4-chloro-l-naphthol.
Collection of Uterine Endometrium
Sixteen crossbred (Brahman x Angus) beef cows received 25
mg PGF2a (Lutalyse) during the luteal phase of the estrous cycle
and were observed for onset of estrus. Cows were assigned
randomly to be killed on Day 0 (estrus) , 2, 5 or 8 of the estrous
cycle. After exsanguination, reproductive tracts were collected
aseptically and transferred to a sterile laminar flow hood. The
ovaries were examined to determine the ovary from which the most
recent ovulation had occurred. Three of 4 cows killed at Day 0
had ovulated, while the 4th cow had a clearly identifiable
preovulatory follicle; all cows killed at other times had
ovulated. Tissues were subsequently identified as being
ipsilateral or contralateral with respect to the active ovary.
Uterine horns were opened just above the external bifurcation
and intercaruncular endometrial tissue dissected free from

110
myometrium, blotted on sterile gauze, cut into 2-3 mm3 cubes and
cultured as described below.
Explant Culture
Endometrial explants (500 mg) were placed in sterile
plastic 100 mm Petri dishes and cultured in 15 ml modified MEM
containing 50 nCi L-[ 4,5-3H] leucine under an atmosphere of 50%
N2, 47.5% 02 and 2.5% C02. Cultures were maintained in the dark on
rocking platforms at 39 °C for 24 h (homeothermic treatment) or
were acclimatized at 39 °C for 3 h followed by 43 °C for 21 h (heat-
shock treatment). Cultures were stopped by the separation of
tissue and medium during centrifugation (700 x g; 30 min; 4°C) .
Tissue was placed immediately in ice-cold solubilization buffer
(described below) and homogenized. Samples of tissue and medium
were stored at -20°C until analyzed.
Protein Synthesis and Secretion
The secretion of de novo synthesized polypeptides into
culture medium by endometrium was determined by measuring the
incorporation of [3H]leucine into nondialyzable macromolecules.
Culture medium was dialysed extensively (3 changes of 4 liters
each) against deionized water using dialysis tubing with a 6,000
to 8,000 dalton exclusion limit. Radioactivity in the reténtate
was determined by scintillation spectrometry (LKB Wallac 1219
Rackbeta Spectral LSC, Wallac Oy, Finland).
Tissue samples were solubilized in 50 mM Tris-HCl, pH 7.6,
which contained 1 mM PMSF, 1 mM EDTA and 2% (v/v) NP-40. Measure¬
ment of [3H] leucine incorporated into tissue macromolecules was

Ill
by TCA precipitation. Samples (50 ill) of solubilized tissue were
placed onto Whatman 3MM paper (previously saturated with 20% TCA
[w/v]) and allowed to dry. Proteins were precipitated onto
filter paper by serial washings with 20% TCA, 5% TCA and 95%
ethanol as described by Mans and Novelli (1961). Radioactivity
of precipitated protein was determined by scintillation spec¬
trometry.
Electrophoresis
One-dimensional polyacrylamide gel electrophoresis (PAGE)
in the presence of SDS was performed on tissue proteins using
the buffer system of Laemmli (1970). Proteins were resolved on
12.5% (w/v) polyacrylamide gels in the presence of 5% (v/v) 2-
mercaptoethanol.
Two-dimensional SDS-PAGE was performed on proteins secreted
into the culture medium using procedures described by Roberts et
al. (1984). Equal amounts of radiolabeled protein were loaded
onto each gel to determine qualitative differences due to
treatments. Samples were dissolved in 5 mM K2C03, pH 10.5, which
contained 9.0 M urea, 2% (v/v) NP-40 and .5% (w/v) dithio-
threitol. Proteins were resolved in the first dimension by
isoelectric focusing in 4% (w/v) polyacrylamide disk gels
containing 250 mM DATD, 8.0 M urea, 2% (v/v) NP-40 and 5.1% (v/v)
ampholytes (pH 3-10, 5-7 and 9-11; 50:36:16 byvol., respective¬
ly). Disk gels were equilibrated in 50 mM Tris-HCl, pH 6.8,
containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol and
subjected to electrophoresis in the second dimension on 12.5%

112
(w/v) polyacrylamide gels in the presence of .5% (w/v) SDS. Slab
gels were equilibrated in acetic acid, ethanol and water (7:40:53
by volume, respectively), stained with .125% (w/v) Coomassie
Blue R-250 in the same solvent, destained in acetic acid, ethanol
and water (7:10:83, by volume, respectively), soaked in deion¬
ized water (30 min) , equilibrated with 1 M sodium salicylate (30
min) and dried. Fluorographs were prepared with Kodak XAR x-ray
film.
Analysis of Proteins Resolved by Two-Dimensional SDS-PAGE
Fluorographs were examined for the presence of radiolabeled
protein spots. Prominent or frequently occurring spots were
categorized according to apparent molecular weight and iso¬
electric point. Using the fluorographs as templates, these
protein spots were excised from each gel. A stainless-steel cork
borer was used to remove an equivalent area from each gel. The
borings of polyacrylamide were solubilized by incubating in 30%
(v/v) hydrogen peroxide (500 /il) at 70°C for 48 h; ascorbic acid
(500 mM; 100 ill) was then added to neutralize the hydrogen
peroxide. Afterwards, the samples were stored in the dark for
24-48 h to reduce chemiluminescence and radioactivity was
quantified by scintillation spectrometry. Since equal amounts
of radiolabeled protein (500,000 dpm) were loaded onto each gel,
the amounts of individual radiolabeled proteins recovered from
each gel were corrected mathematically to reflect the amount of

113
radiolabeled protein associated with each spot in the total 15
ml culture supernatant.
dpm/culture = (dpm/qel spot) x (vol/culture) x (dpm/unit vol)
500,000 dpm/gel
Western Blotting
Proteins from control and heat-shocked endometrial tissue
were resolved by one-dimensional SDS-PAGE on 7.5% polyacrylamide
gels as described above and electrophoretically transferred to
nitrocellulose membranes (see Chapter 2) . Other protein binding
sites on the membranes were blocked by incubation with 3% gelatin
and membranes were then incubated with a monoclonal mouse IgG
specific for the inducible 72-kDa hsp. Duplicate membranes were
incubated with normal mouse serum to determine non-specific
binding. Membranes were then incubated with anti-mouse IgG
conjugated to horseradish peroxidase. Presence of the proteins
was detected by adding peroxidase substrate, 4-chloro-l-naphthol
in methanol in the presence of hydrogen peroxide.
Statistical Analysis
Effects of day of the estrous cycle, side of tract and
incubation temperature upon the incorporation of [3H]leucine
into macromolecules by the endometrium were analysed by least-
squares analysis of variance using the General Linear Models
procedure of the Statistical Analysis System (SAS, 1985).

114
Results
Overall Synthesis and Secretion of r5HlLabeled-Proteins
Secretion of nondialyzable, [3H]leucine-labeled macro-
molecules by endometrial explants was greatest at Day 0 (estrus) ,
declined through Day 5 and increased again at Day 8 (Table 3-1) .
There was a day x temperature interaction (P < 0.0002) because
secretion was most greatly affected by temperature at Day 0. At
estrus, tissue incubated at 43 °C secreted more protein than did
tissue at 39°C while at other days examined temperature did not
affect protein secretion. Results for tissue proteins were
similar to those for secretory proteins? incorporation of
[3H] leucine into TCA-precipitable proteins in endometrial tissue
was greatest at Day 0 (Table 3-1) , declined during Days 2 and 5
and was again elevated at Day 8. Effects of heat shock were not
significant. There was no significant effect of side of the
uterus with respect to the active ovary on amounts of secretory
or tissue proteins.
Analysis of Individual Endometrial Secretory Proteins
Since total secretion of nondialyzable protein provides no
information on distribution of protein subunits or on secretion
rates of individual proteins, patterns of proteins secreted by
endometrial explants were analysed by two-dimensional SDS-PAGE
(Figure 3-1). The most complex array of proteins appeared at
estrus. All fluorographs were exposed for similar lengths of
time, thus relative changes in intensity of development reflect

115
quantitative variation in the relative proportions of radio-
labeled polypeptides. The lower incorporation of radiolabel
into the major secretory proteins at Days 2-8 is reflected in
lack of appearance of the numerous polypeptides which appear at
Day 0. Of the most prominent proteins resolved by two-dimen¬
sional SDS-PAGE, 25 were excised from the gels and radioactivity
associated with the proteins was determined. Of these 25
proteins, 14 were affected by day of the cycle (P < 0.05) (Table
3-2) , with amounts in all cases being greatest at estrus. Bovine
endometrial secretory proteins (bESPs) 10, 15, 21 and 2 3 were the
major estrus-associated proteins. Together, these 4 proteins
accounted for 37% of the radioactivity at estrus associated with
the 25 proteins examined.
Elevated incubation temperature reduced secretion rates of
bESPs 9 (P < 0.05) and 15 (P < 0.06) (Table 3-3) . A day x temp¬
erature interaction was significant for Protein 10 because
secretion was not affected by temperature at estrus, Day 5 or 8
while being reduced at Day 2. While the day x temperature
interaction for Protein 9 and 15 was not significant, the effect
of incubation temperature varied between days of the estrous
cycle, with negative effects of high incubation temperature
being greatest at estrus. A side x temperature interaction (P
< 0.05) was observed in bESPs 7, 9, 10, 11 and 19 (Table 3-4);
this interaction tended to be significant (P<0.08) for proteins
16 and 20. These interactions resulted from two causes. First,
endometrium from the ipsilateral side tended to secrete these

116
Table 3-1. In vitro synthesis and secretion of radiolabeled,
macromolecular [3H]leucine by endometrial explants obtained
during early stages of the estrous cycle.
Incubation Temperature
39 °C 43 °C
Day of
Estrous
Cycle Secreted3 Tissueb Secreted3 Tissue5
0
7,599
22,472
10,174
28,849
+
858
+
3,516
+
742
+
4,222
2
4,786
11,033
5,166
15,005
±
659
+
3,636
+
766
±
3,876
5
2,589
9,524
2,616
8,687
±
147
+
1,620
+
185
±
1,151
8
3,718
16,197
4,257
19,581
±
626
+
5,103
±
1,075
±
6,531
aResults are nondialyzable [3H]leucine in culture supernatants
expressed as dpm/mg explant tissue (mean ± sem). Effects of day
(P < 0.002), temperature (P < 0.0006) and day x temperature (P
< 0.0002) were observed. Effects of side of uterus (ipsilateral
vs. contralateral to active ovary) and of interactions with side
were not significant; data are therefore presented as pooled
across classification.
hTCA-precipitable [3H]leucine in tissue homogenates (dpm/mg
explant tissue; mean ± sem). Incorporation of [3H]leucine was
affected by day of cycle (P < 0.08) ; no significant effects of
temperature, side or their interactions were observed.

117
Table 3-2. Amounts of individual radiolabeled proteins secreted
by cultured endometrial explants from early stages of the estrous
cycle and isolated from electrophoretic gels.
Day of the Estrous Cycle
Protein Mr x 10'3 pi 0 2 5 8
1
> 97
03
•
03
3,647
± 839
2,258
± 752
562
± 109
1,068
± 238
2
> 97
8.8
1,843
± 419
1,114
± 277
905
± 498
857
± 128
3a
> 97
8.5
2,243
± 429
1,556
± 359
495
± 55
971
± 125
4
> 97
8.2
2,829
± 731
2,478
± 558
1,388
± 589
1,077
± 304
5
58
8.8
4,132
± 1,371
2,056
± 607
1,308
± 667
1,797
± 875
6a
> 97
5.9
4,966
± 1,067
3,511
± 968
1,182
± 217
1,380
± 317
-j a,d
> 97
5.9
8,112
± 1,234
3,474
± 675
987
± 190
1,320
± 329
86
95
6.0
3,368
± 715
1,631
± 367
679
± 165
858
± 141
9a«b 95
5.9
6,816
± 1,239
3,497
± 720
1,245
± 265
1,667
± 427
10a,c.d.e
55
5.9
21,807
± 4,337
7,607
±1,496
1,714
± 337
1,858
± 260
lld
42
5.9
2,533
± 1,069
1,116
± 379
729
± 145
1,477
± 256
12
22
5.7
7,297
± 1,406
8,419
±3,063
5,081
±2,131
3,783
±1,530
13
76
5.0
3,762
± 1,208
2,176
± 472
2,341
± 856
1,185
± 383
14a
46
5.3
3,893
± 859
1,611
± 275
686
± 105
736
± 97
15a,b,e
35
5.1
12,593
± 3,235
4,715
±1,117
1,642
± 285
2,455
± 595

118
Table 3-2--continued.
Protein
Mr X 10'
3 Pi
Day of the
Estrous Cvcle
0
2
5
8
16a,d
34
4.9
7,358
1,649
541
645
± 1,977
± 310
+
52
± 108
17a
36
4.9
3,808
1,741
465
780
± 1,036
± 487
+
91
± 206
18
35
4.8
2,174
1,231
391
438
± 398
± 310
+
39
± 97
19d
> 97
4.0
7,561
2,615
850
1,229
±2,071
± 588
+
308
± 427
2 0a,d
> 97
4.0
4,856
1,737
554
963
± 1,066
± 315
+
68
± 393
2 Ia
58
4.7
9,034
2,705
1
, 340
1,664
± 1,620
± 478
+
236
± 521
22a
29
8.0
2,746
1,495
667
1,027
± 447
± 242
+
119
± 250
2 3a
> 97
5.5
9,534
3,220
1
,387
1,525
± 2,377
± 800
+
171
± 347
24a
38
4.0
5,854
2,472
780
1,447
± 1,626
± 712
+
140
± 493
25
20
8.5
2,009
1,693
795
855
± 491
± 294
+
125
± 215
Note: Results are expressed as dpm/total culture (mean ± sem) .
Data are pooled across temperature and side of ovulation.
Significant effect of day of the estrous cycle (P < 0.05).
“significant effect of temperature (see Table 3-3).
“Significant day x temperature interaction (see Table 3-3) .
“significant side x temperature interaction (see Table 3-4) .
“Significant day x side interaction (see Table 3-5).

119
proteins in greater amounts than endometrium from the contra¬
lateral side. This difference in secretion rate between
endometrium from the ipsilateral and contralateral sides tended
to be greater when tissue was cultured at 39 "C than when cultured
at 43 °C for Proteins 7, 9, 10, 19 and 20. This effect, in turn,
was caused in part by the ipsilateral side being more likely to
be inhibited by 43 °C incubation temperature. This was true for
bESPs 7, 9, 10, 11, 19 and 20.
Overall, side of the uterus relative to the active ovary did
not significantly affect secretion of any proteins, although the
means of 20 of 25 proteins were greater for the ipsilateral side.
There was, however, a day x side interaction for Proteins 8 (P <
0.05), 10 (P < 0.05) and 15 (P < 0.07) (Table 3-5). These
interactions occurred because differences in secretion rates
between the ipsilateral and contralateral sides were greatest at
Days 0 and 2. In addition, the side x temperature interactions
for bESPs 7, 9, 10, 11 and 19 (Table 3-4) indicate that at an
incubation temperature of 39°C, secretion rates for these
proteins were greater for the side of the uterus ipsilateral to
the active ovary. This result was masked at a 43°C incubation
temperature by the greater inhibitory effect of heat shock on the
ipsilateral side.
Production of Heat-Shock Proteins of 70,000 and 90,000 Mr
To determine qualitative changes in tissue proteins synth¬
esized de novo, equal amounts of radiolabeled protein were

120
Table 3-3. Effect of incubation temperature upon secretion of
individual radiolabeled proteins by cultured endometrial
explants obtained during early stages of the estrous cycle.
Day of
Estrous
Incubation Temperature
Protein
Cycle
39 ° C 43 ° C
9a
0
8,670
± 1,916
4,591
+
821
2
4,384
± 1,239
2,709
+
775
5
1,004
± 174
1,426
+
448
8
2,021
± 612
1,047
+
393
10b
0
21,483
± 5,500
22,195
+
7,618
2
9,491
± 2,563
5,934
+
1,612
5
1,355
± 435
1,984
+
494
8
1,956
± 352
1,687
+
415
15a
0
14,539
± 4,866
10,258
±
4,421
2
4,813
± 611
4,627
+
2,103
5
2,000
± 443
1,373
+
368
8
1,952
± 701
3,335
+
1,056
Note: Results shown are expressed as dpm/culture (mean ± sem)
pooled across side of the uterus.
Significant effect of temperature (P < 0.05).
Significant day x temperature interaction (P < 0.05).

121
Table 3-4. Side x temperature interactions affecting secretion
of individual proteins from cultured endometrial explants
obtained during early stages of the estrous cycle.
Protein
Incubation
Temperature
(°C)
Side of tract relative to
active ovarv
Ipsilateral
Contralateral
7a
39
4,814 ± 1,292
2,477 ± 742
43
3,300 ± 930
2,685 ± 827
9a
39
5,253 ± 1,131
2,589 ± 964
43
2,254 ± 540
2,614 ± 648
10a
39
11,376 ± 3,513
5,183 ± 1,616
43
8,628 ± 3,267
5,517 ± 2,512
IIa
39
1,854 ± 474
800 ± 155
43
1,781 ± 855
1,000± 253
19a
39
4,153 ± 1,371
1,533 ± 612
43
3,586 ± 1,472
2,064 ± 762
16b
39
3,129 ± 1,139
2,566 ± 1,374
43
2,451 ± 1,173
1,014 ± 245
20b
39
2,445 ± 809
1,363 ± 510
43
2,137 ± 738
1,611± 500
Note: Results are shown as dpm/culture (mean ± sem) pooled
across day of the estrous cycle. For each protein listed there
was a side x temperature interaction (aP < 0.05; bP < 0.08) .

122
Table 3-5. Day of cycle x side interactions affecting secretion
of individual proteins from cultured endometrial explants.
Day of
Estrous
Side of tract relative
to active ovarv
Protein
Cycle
Ipsilateral
Contralateral
8
0
4,033 ± 994
2,205 ±
719
2
2,072 ± 564
1,239 ±
471
5
836 ± 249
522 ±
217
8
803 ± 160
923 ±
261
10
0
24,707 ± 5,826
16,730 ±
6,338
2
10,633 ± 2,660
4,918 ±
1,014
5
1,634 ± 350
1,795 ±
607
8
1,768 ± 337
1,966 ±
443
15
0
15,229 ± 4,727
7,981 ±
2,564
2
5,731 ± 2,184
3,812 ±
888
5
1,866 ± 460
1,417 ±
351
8
2,509 ± 761
2,389 ±
1,036
Note: Results are shown as dpm/culture (mean ± sem) pooled
across incubation temperature. For each protein listed there
was a (P < 0.05 ) day x side interaction.

123
resolved by one-dimensional SDS-PAGE. Representative fluoro-
graphs of one-dimensional SDS-polyacrylamide gels of tissue
homogenates are shown in Figure 3-2. Heat shock altered the
array of radiolabeled proteins in the tissue as indicated by the
enhancement at 43 °C of proteins migrating at 70,000 and 90,000
Mr compared to tissue incubated at 39 °C.
Western Blotting of the Heat-Shock Protein of 70,000 M|
To determine whether the enhancement of the 70,000 Mr bands
of the tissue incubated at 43 °C was due to the presence of
proteins homologous with the major heat shock protein of 72,000
Mr described by Welch et al. (1982) , the tissue homogenates were
screened with antibody (C92). Results of Western blotting are
shown in Figure 3-3. The protein was present in the 39 °C lane to
a slight degree and, to a greater degree, in the 43 °C lane.
Discussion
This model involved determination of de novo synthesis and
secretion of proteins by cultured endometrium that was exposed
to endogenous fluctuations in steroid concentrations normal for
the bovine estrous cycle. The elevated secretion by endometrium
at estrus is probably due to estrogens, which are elevated at
estrus (Peterson et al. , 1975) and enhance protein secretion by
endometrium in cows (Bartol et al., 1981b) and other species
(Geisert et al. , 1982 ; Miller and Moore, 1983 ; Komm et al. , 1985?
Kuivanen and DeSombre, 1985? Lejeune et al., 1985? Salamonsen et

124
al., 1985). Bartol et al. (1981a) found total luminal protein
of cyclic and pregnant cows to be greater at Day 4 of the estrous
cycle than at later times through Day 14. The elevated secretion
of proteins at estrus could also be caused in part by the low
concentrations of progesterone present at that time, since
progesterone can inhibit the secretion of certain endometrial
proteins (Salamonsen et al. , 1985) . There was a notable increase
in total protein synthesis and secretory activity of endometrial
explants between Days 5 and 8. This may signal the onset of
progesterone-mediated uterine secretory activity since proges¬
terone is elevated after about Day 6 in the cow (Kindahl et al. ,
1976).
Of the individual endometrial proteins that varied with day
of the estrous cycle, 14 of 25 appeared to be estrogen-enhanced
because their levels in culture medium declined after the
post-estrus fall of estrogen concentrations. Secretion of two
major proteins, bESPs 10 and 15, were reduced by about 90 and
80%, respectively, from estrus to Day 8 of the cycle. The array
of proteins secreted by endometrial explants from cows during
the late luteal phase of the estrous cycle was not examined
because an experiment on that subject was reported by Bartol et
al. (1985b). Comparison of fluorographs of secretory proteins
of uterine explants from Day 19 cyclic cows resolved by two-
dimensional SDS-PAGE from their experiment with those from cows
at estrus in the current study reveal a number of proges¬
terone-induced proteins in the luteal phase. In particular,

M, x 10“
125
Figure 3-1. Representative fluorographs of two-dimensional
SDS-PAGE of secretory proteins from bovine endometrial explants
obtained at Days (a) 0 (estrus), (b) 2, (c) 5 and (d) 8 of the
estrous cycle. Proteins were separated in the first dimension
by isoelectric focusing and in the second dimension by SDS-PAGE
using 12.5% (w/v) polyacrylamide gels. Equal amounts of
radioactivity were loaded for each gel. The greatest number of
spots observed on fluorographs were from culture supernatants at
Day 0. The 2 5 most frequently occurring spots were characterized
according to pi and molecular weight. Individual regions at
these coordinates were excised and radioactivity determined even
in cases in which a spot could not be identified by fluorography
(see Table 3-2 for results) . Individual spots that were excised
and counted are identified by number in (a) .

126
M, x 1(T3
Figure 3-2. Representative fluorographs of one-dimensional
SDS-PAGE of tissue proteins solubilized from bovine endometrial
explants incubated at (a) 39°C or (b) 43 °C. Equal amounts of
radioactivity were loaded for each lane and proteins were
separated by SDS-PAGE using 12.5% (w/v) polyacrylamide gels. Two
proteins, at 70,000 Mr and 90,000 Mr, were enhanced by incubation
at 4 3 *C (arrows) . Note that other bands in the 43 °C lane appear
diminished compared to those in the 39 °C lane.

127
C92
97 -
75 -
69 -
57 -
39°C 43"C
Figure 3-3. Western blotting of bovine heat-shock proteins.
Proteins from control and heat-shocked endometrial tissue were
resolved by one-dimensional SDS-PAGE on 7.5% polyacrylamide
gels, electrophoretically transferred to nitrocellulose
membranes and incubated with a monoclonal mouse IgG (C92)
specific for only the inducible hsp-72. Membranes were then
incubated with anti-mouse IgG conjugated to HRP and localized
by addition of enzyme substrate.

128
Bartoletal. (1985b) described a group of high molecular weight,
acidic proteins (Mr > 150,000 , pi < 5.1) and several low molecular
weight species in the neutral isoelectric range (30,000 - 50,000
Mr, pi 6-8) that appeared during the late luteal phase- These
proteins were not observed in the present experiment.
One must interpret statistical analyses of secretion rates
of individual proteins with caution. Given that secretion rates
of 25 individual proteins were analyzed statistically, it is
possible that some statistically significant effects occurred
due to chance. In particular, effects of incubation temperature
and interactions between day of the estrous cycle and temperature
and between day of cycle and side of the reproductive tract
occurred for only 1 to 3 of 25 proteins (Tables 3-3 and 3-5) .
Nonetheless, secretion rates of several proteins were altered by
side and by temperature in a generally consistent manner (Table
3-4) and it is likely that effects of side and temperature were
not simply artifacts of the statistical analysis. While side of
the reproductive tract relative to the active ovary had no sig¬
nificant effect upon total protein secretory activity, the
secretion by endometrial explants of a number of proteins was
greater for tissue from the ipsilateral side, particularly when
cultured at 39°C (Table 3-4). In addition, secretion rates of
proteins 8, 10 and 15 were greater for endometrium from the
ipsilateral horn at Days 0 and 2 but not at Days 5 and 8 (Table 3-
5) . This suggests a transient local effect of ovulation. Side
differences with respect to the uterine and ovarian blood supply

129
(Lamond and Drost, 1974; Ginther and DelCampo, 1974) and
distribution of progesterone from the corpus luteum (Pope et al. ,
1982) have been reported and it is likely that steroids or other
molecules released from the preovulatory follicle or corpus
luteum induce local effects to alter oviductal and endometrial
protein secretory activity.
Elevated temperature, which is associated with embryonic
mortality in cattle (Thatcher and Collier, 1986) , also altered
protein secretion by the endometrium, though in a manner atypical
of the classic heat-shock response. Total protein synthesis has
been reported to decrease drastically in heat-shocked cells of
many species of plants and animals (Ashburner, 1982; Nover,
1984) . This effect was not seen in bovine reproductive tract
tissues. Instead, heat shock induced an overall elevation in
protein synthesis and secretion at Day 0 but not at other times
examined. A possible explanation is that the ability of endo¬
metrium to regulate rates of metabolic activity in the face of
heat shock was enhanced as estrogen declined so that rates of
chemical reactions that had been accelerated at 43 "C by the
effect were brought under control at days of the estrous cycle
other than estrus.
De novo synthesis and secretion of certain specific
proteins were decreased by heat shock even at Day 0, especially
in tissue collected ipsilateral to the active ovary. Taken
together, the results in Tables 3-4 and 3-5 suggest that the two
uterine horns are different with respect to the secretion of

130
certain proteins, especially immediately after estrus, and that
heat shock alters that response of side by reducing protein
secretion in the ipsilateral but not the contralateral horn.
Therefore, heat shock of endometrium may be associated with
suppression of specific proteins rather than a more general
inhibition. Differences in messenger RNA turnover, transcrip¬
tion or intracellular protease concentration may make protein
secretion by endometrium ipsilateral to the active ovary more
sensitive to inhibition by heat stress. It is not certain
whether the subtle effects of heat stress on endometrial function
observed in the present experiment would be sufficient to cause
embryonic mortality. If ovarian processes alter the local
uterine horn as a necessary step to prepare the uterine environ¬
ment, alteration of that priming by heat shock could induce
asynchronous development of the endometrium and conceptus
resulting in increased embryonic mortality.
Heat shock results in specific changes in the patterns of
protein synthesis by mammalian cells, characterized by the
synthesis of a small number of intracellular proteins referred
to as heat-shock or stress proteins that may provide a degree of
tolerance to stress (Ashburner, 1982; Nover, 1984). The
endometrium of cows produces heat-shock proteins of 70,000 Mr and
90,000 Mr. In the present study, proteins in bovine endometrium
cross-react with antibody generated to a heat-shock protein
purified from HeLa cells (Welch et al., 1982). The antibody,
which recognizes a heat-shock protein of 72,000 Mr that is

131
produced in response to heat shock, reacted with an endometrial
protein of 72,000 Mr that also was greatly enhanced after heat
shock.
In summary, secretion of proteins by the uterine endome¬
trium during the first 8 days of the estrous cycle in cows is
significantly affected by day of the estrous cycle, suggesting
that some of the requirements for synchrony between mother and
embryo (Rowson et al., 1972; Wilmut and Sales, 1981; Wilmut et
al., 1985) are a result of the requirements of the embryo for a
specific milieu of endometrial proteins for proper development.
That the function of the endometrium is related to embryonic
survival is suggested by findings that two factors that alter
embryonic survival, side of the uterus relative to the corpus
luteum (Newcomb and Rowson, 1976; Christie et al., 1979; Newcombe
et al. , 1980) and heat shock (Alliston and Ulberg, 1961; Alliston
et al., 1965; Elliott et al., 1968; Ulberg and Sheenan, 1973),
also are associated with changes in endometrial protein secr¬
etion.

CHAPTER 4
SECRETION OF PROTEINS BY CULTURED BOVINE OVIDUCTS
COLLECTED FROM ESTRUS THROUGH EARLY DIESTRUS
Introduction
The importance of the oviductal environment for early
embryonic development has been illustrated in experiments
demonstrating superior in vitro development of embryos cultured
in the presence of oviductal cells (Gandolfi and Moor, 1987;
Eyestone et al., 1987; Rexroad and Powell, 1988) or oviduct-con¬
ditioned culture medium (Kaltwasser et al., 1987). It may
reasonably be assumed that some of the effects of the oviduct are
mediated by secretory proteins. Proteins secreted by the oviduct
have been implicated in a number of functional roles, including
binding to zona pellucida (Shapiro et al., 1974; Kapur and
Johnson, 1985; Leveille et al., 1987) and spermatozoa (Sutton et
al., 1984b), sperm capacitation (Bedford, 1970) and inhibition
of antibody-complement-mediated immune function (Oliphant et
al. , 1984a). Stage-specific changes in total protein secretion
rate by bovine oviducts during the estrous cycle (Geisert et al. ,
1987; Killian et al., 1987) and appearance of individual
stage-specific proteins from oviducts of the rabbit (Oliphant et
al., 1984b), pig (Buhi, 1985), cow (Geisert et al. , 1987), sheep
(Sutton et al., 1984a), mouse (Kapur and Johnson, 1985), rat
(Wang and Brooks 1986), baboon (Fazleabas and Verhage, 1986),
132

133
and human (Verhage et al., 1988) all point to the potentially
critical nature of oviductal function around the time of
fertilization and during early pregnancy.
The initial objective of the current study was to charac¬
terize the pattern of oviductal protein secretion during the
first 8 days of the estrous cycle of the cow in order to identify
proteins associated with the period of fertilization through
conceptus migration to the uterus (Day 3.5 to 4; Noden and de
Lahunta, 1985). Second, the local effect of the ovary upon
oviductal protein secretory activity was examined because of the
potential effect of steroids and proteins released from the
preovulatory follicle or corpus luteum upon oviductal activity.
Such a local effect of the ovary has been proposed in the pig by
Hunter et al. (1983), who described a possible mechanism for
products of the preovulatory follicle to be transferred by
vascular and lymphatic pathways and influence oviductal activity
related to sperm transport.
Materials and Methods
Materials
Eagle's minimum essential medium (MEM) was modified by
supplementation with penicillin (100 units/ml), amphotericin B
(250 ng/ml) , streptomycin (100 ¿ng/ml) , insulin (.2 units/ml),
nonessential amino acids (1%, v/v) and glucose (5 mg/ml). Medium
was also supplemented with D-Ca pantothenate (100 ¿ng/ml) ,
choline chloride (100/ng/ml) , folic acid (100/ng/ml), i-inositol
(200 /ng/ml), nicotinamide (100 /ng/ml), pyridoxal-HCl (100

134
Mg/ml) , riboflavin (10/xg/ml) and thiamine (100/xg/ml). Content
of L-leucine was limited to .1 times normal (5.15 mg/1) to
enhance uptake of L-[4,5-3H]leucine added to cultures. Medium
was filter sterilized (.22 /im) and stored at 4°C. All medium
components were from GIBCO (Grand Island, NY).
Acrylamide, urea, dithiothreitol and sodiumdodecyl sulfate
(SDS) were purchased from Research Organics (Cleveland, OH).
Coomassie Blue R-250 was from Polysciences, Inc. (Warrington,
PA). Bis-acrylamide and N,N1-diallyltartardiamide (DATD) were
purchased from Bio-Rad (Richmond, CA) . Ampholytes used in
isoelectric focusing were from Serva (Heidelberg, FRG).
Lutalyse (dinaprost tromethamine) was from Upjohn Co. (Kalama¬
zoo, MI) . L-[4,5-3H]leucine (SA,~150 Ci/mmol) was obtained from
Amersham Corp. (Arlington Heights, IL). Nonidet P-40 (NP-40),
Phenylmethylsulfonylflouride (PMSF), Tris and N,N,N',N'-tetra-
methylethylenediamine (TEMED) and ascorbic acid were purchased
from Sigma Chemical Co. (St. Louis, MO) . Spectrapor dialysis
tubing was from Spectrum Medical (Los Angeles, CA). Sodium
salicylate, 2-mercaptoethanol, glycine, trichloroacetic acid
(TCA), and ammonium peroxydisulfate were purchased from Fisher
Scientific (Orlando, FL) . Whatman 3MM paper (Clifton, NJ) was
utilized for TCA precipitation. X-omat XAR film was from Kodak
(Rochester, NY).
Animals
Sixteen crossbred cows of varying proportions of Angus and
Brahman breeding received 25 mg PGF2a (Lutalyse) during the
luteal phase of the estrous cycle and were observed for onset of

135
estrus. Cows were randomly assigned to be killed on either Day
0 (estrus) , 2, 5, or 8 of the estrous cycle. Three of four cows
killed at estrus had ovulated, and the fourth had a clearly
identifiable preovulatory follicle. Oviductal tissue from these
cows was cultured as described below and incubated at 39 °C
(homeothermic temperature) to characterize patterns of in vitro
synthesis and secretion.
Collection of Oviducts
Following exsanguination, reproductive tracts were
collected aseptically and transferred to a sterile laminar flow
hood. The ovaries were examined to determine the ovary on which
the most recent ovulation had occurred. Tissues were sub¬
sequently identified as ipsilateral or contralateral with
respect to the active ovary. Entire oviducts (ampulla and
isthmus) were trimmed free of connective and vascular tissue,
sliced longitudinally to expose inner epithelium, and cut into
2-3 mm3 cubes. Thus, each cube contained epithelial, muscular,
and stromal layers. Explants were weighed and cultured as
described below. In each culture, cubes from both isthmus and
ampullary regions were mixed together so that both types of
tissue were present.
Explant Culture
Oviductal explants were placed in sterile plastic 100 mm
Petri dishes and cultured in 15 ml of modified MEM containing 50
/iCi L-[4,5-3H] leucine per 500 mg tissue under an atmosphere of
50% N2, 47.5% 02, and 2.5% C02. A constant ratio of tissue,
medium, and radiolabeled precursor was held. Cultures were

136
maintained in the dark on rocking platforms at 39°C for 24 h.
Cultures were stopped by the separation of tissue and medium
during centrifugation (700 x g; 30 min; 4°C). Tissue was
immediately placed in ice-cold solubilization buffer (described
below) and homogenized. Samples of tissue and medium were stored
at -20°C until analyzed.
Protein Synthesis and Secretion
The secretion of de novo synthesized macromolecules into
culture medium by oviducts was determined by measuring the
incorporation of [3H]leucine into nondialyzable macromolecules.
Conditioned culture medium was dialyzed extensively (three
changes of 4 liters) against deionized water using dialysis
tubing with a 6,000 to 8,000 dalton exclusion limit. Radio¬
activity in the reténtate was determined by scintillation
spectrometry (LKB Wallac 1219 Rackbeta Spectral LSC, Wallac Oy,
Finland).
Tissue samples were solubilized in 50 mM Tris-HCl, pH 7.6,
which contained 1 mM PMSF, 1 mM EDTA, and 2% (v/v) NP-40.
Measurement of [3H]leucine incorporated into tissue macro¬
molecules was done by TCA precipitation. Aliquots (50 /¿l) were
blotted and dried onto Whatman 3MM paper that had been previously
saturated with 20% (w/v) TCA. The protein was precipitated onto
the paper by 20% (w/v) TCA and nonproteinaceous material removed
by serial washing with 20% (w/v) TCA, 5% (w/v) TCA, and 95%
ethanol (Mans and Novelli, 1961). Radioactivity on the paper was
determined by scintillation spectrometry.

137
Electrophoresis
One-dimensional polyacrylamide gel electrophoresis in the
presence of SDS (1-D SDS-PAGE) was performed on tissue proteins
using the buffer system of Laemmli (1970). Proteins were
resolved on 12.5% (w/v) polyacrylamide gels in the presence of
5% (v/v) 2-mercaptoethanol. Two-dimensional SDS polyacrylamide
gel electrophoresis (2-D SDS-PAGE) was performed on proteins
secreted into the culture medium using procedures described by
Roberts et al. (1984). Equal amounts of radiolabelled protein
were loaded onto each gel to determine qualitative differences
due to treatments. Samples were dissolved in 5 mM K2C03, pH 10.5,
which contained 9.0 M urea, 2% (v/v) NP-40, and .5% (w/v)
dithiothreitol. Proteins were resolved in the first dimension
by isoelectric focusing in 4% (w/v) polyacrylamide disk gels
containing 250 mM DATD, 8.0 M urea, 2% (v/v) NP-40 and 5.1% (v/v)
ampholytes (pH 3-10, 5-7, and 9-11; 50:36:16 by volume, respec¬
tively) . Disk gels were equilibrated in 50 mM Tris-HCl, pH 6.8,
containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol and
subjected to electrophoresis in the second dimension on 12.5%
(w/v) polyacrylamide gels in the presence of .5% (w/v) SDS. Slab
gels were equilibrated in acetic acid, ethanol, and water
(7:40:53 by volume, respectively), stained with .125% (w/v)
Coomassie Blue R-250 in the same solvent, destained in acetic
acid, ethanol, and water (7 :10 : 83 by volume) , soaked in deionized
water (30 min) , and equilibrated with 1 M sodium salicylate (30
min) , and dried. Fluorographs were prepared with Kodak XAR x-ray
film.

138
Quantitation of Secretion Rates of Individual Proteins
The amounts of radiolabel associated with individual
polypeptides resolved by fluorography were quantified as
follows: Prominent or frequently occurring spots were categor¬
ized according to apparent molecular weight and isoelectric
point (pi). Using the fluorographs as templates, these protein
spots were excised from each gel. A stainless-steel cork borer
was used to remove an equivalent area from each gel. The borings
of polyacrylamide were solubilized by incubating in 30% (v/v)
hydrogen peroxide (500 /¿l) at 70°C for 48 h; ascorbic acid (500
mM; 100 m 1) was then added to neutralize the hydrogen peroxide.
Afterwards, the samples were stored in the dark for 24-48 h to
reduce chemiluminescence and radioactivity was then quantified
by scintillation spectrometry. Since equal amounts of radio-
labeled protein (500,000 dpm) were loaded onto each gel, the
amount of each individual radiolabeled protein recovered from
gels was corrected mathematically to reflect the amount of
radiolabeled protein associated with each spot in the total 15
ml of culture supernatant (see Chapter 3) .
Statistical Analysis
Data were analyzed by least-squares analysis of variance
using the General Linear Models procedure of the Statistical
Analysis System (SAS, 1985).
Results
Total Synthesis and Secretion of f3H1 labeled-proteins
There was an effect (P < 0.04) of day of the estrous cycle
on amounts of nondialyzable, [3H] leucine-labeled macromolecules

139
secreted by oviducts into the culture medium. Total secretion
was greatest at Day 0 and declined through Day 5-8 (Table 4-1) .
Amounts of TCA-precipitable, [3H] leucine-labeled tissue proteins
also were affected by day of the estrous cycle (P < 0.08).
Amounts were greatest at Day 0 and declined through Day 2 and 5
(Table 4-1). There was no significant effect of side of the
reproductive tract relative to the active ovary or side x day of
estrous cycle interaction upon total incorporation of [3H]leu¬
cine into secreted macromolecules or into tissue proteins.
Analysis of Individual Oviductal Secretory Proteins
The pattern of proteins secreted by oviductal explants as
resolved by 2-D SDS-PAGE is shown in Figure 4-1. The array of
radiolabeled polypeptides appeared qualitatively similar on all
days examined and no apparent differences due to side of the
reproductive tract were detected. Two major groups of radio-
labeled proteins predominated; a number of proteins appeared
having apparent pi of approximately 5.0, and their molecular
weights ranged from > 97,000 to < 29,000. A second group all had
apparent pi of around 4.0 and molecular weights ranging from >
97,000 to 30,000. Of these two sets of polypeptides, the
majority had molecular weights of > 97,000, 85,000-97,000 and
50,000-60,000. A third group of basic polypeptides, with pi
estimates of from 7.3 to 8.8 and molecular weight range from >
97,000 to < 29,000, were also present at all days examined.
Prominent or frequently occurring spots were excised from
these gels using the fluorographs as templates. When these spots

140
Table 4-1. In vitro synthesis and secretion of radiolabelled,
macromolecular [3H] leucine by oviductal explants obtained during
early stages of the estrous cycle.
Secreted3 Tissue6
Day of
Estrous
Cycle
Ipsilateral Contralateral
Ipsilateral Contralateral
0
7,932
± 794
6,107
± 552
15,245
± 3,958
11,464
± 2,261
2
6,485
±1,877
5,884
±1,109
7,466
± 3,047
2,262
± 496
5
3,225
± 286
3,797
± 952
5,298
± 1,669
10,782
± 3,902
8
3,607
± 681
3,426
±1,023
10,532
± 2,880
5,231
± 1,589
aResults are nondialyzable [3H]leucine in culture supernatants
(dpm/mg explant tissue; least-squares mean ± sem) . An effect of
day (P < 0.04) was observed but effects of side of tract (ipsi-
lateral vs. contralateral to active ovary) and of interactions
with side were not significant.
bTCA-precipitable [3H]leucine in tissue homogenates (dpm/mg
explant tissue; least-squares mean ± sem). Incorporation of
[3H]leucine was affected by day of cycle (P < 0.08); no sig¬
nificant effect of side or any interactions appeared.

141
Table 4-2. Amounts of individual radiolabeled proteins secreted
by cultured oviductal explants from early stages of the estrous
cycle.
Day of the Estrous Cycle
Protein
Mr
(kDa)
Pi
0
2
5
8
bOSPl
>
97
CO
00
4,096
3,479
3,024
2,376
+
1,045
+
1,150
± 886
± 978
bOSP2
>
97
00
•
CO
4,946
1,787
3,153
2,811
+
1,007
+
639
±1,077
± 889
bOSP3
97
00
CO
5,199
3 , 111
2,927
1,879
+
3,149
+
1,126
±1,139
± 648
bOSP4a
60
00
CO
4,940
2,088
2,575
2,108
+
985
+
501
± 777
± 451
bOSP5
30
CO
•
CO
3,683
1,648
1,837
5,614
+
1,153
±
464
± 467
±2,989
bOSP6
25
CO
CO
4,619
4,466
2,719
5,365
+
2,398
+
1,630
± 681
±1,982
bOSP7
58
7.5
7,589
7,231
4,400
7,757
+
2,137
+
2,488
± 932
±2,638
bOSP8
>
97
7.0
6,775
5,699
3,078
6,468
+
1,338
+
2,377
± 664
±3,817
bOSP9
90
CO
CO
7,154
7,966
2,420
4,068
+
1,411
±
2,700
± 714
±2,421
bOSPIO
29
7.0
2,809
4,404
3,357
3,364
+
800
+
1,755
± 559
±1,042
bOSPll
>
97
o
•
in
4,736
5,915
4,173
6,605
+
1,079
±
1,592
±1,178
±2,621
bOSP12a
>
97
5.0
5,815
4,415
1,104
2,614
+
1,197
+
1,007
± 223
±1,023
bOSP13
>
97
5.0
6,256
5,069
3,840
8,743
+
1,570
+
2,048
±1,454
±3,809
bOSP14a
97
5.0
30,125
17,991
5,810
5,241
±10,114
±
3,103
±1,328
±2,886

142
Table 4-2—continued.
Day of the Estrous Cycle
Mp
Protein
(kDa)
Pi
0
2
5
8
bOSP15
55
5.0
4,576
2,679
1,735
3,986
+
2,694
+
814
± 324
±1,930
bOSP16
45
5.0
2,295
2,514
2,641
4,378
+
495
+
759
± 903
±1,518
bOSP17
29
5.0
4,859
5,628
3,277
3,306
+
700
+
1,499
± 566
±1,299
bOSP18
45
4.5
1,276
2,132
1,152
1,811
+
273
±
737
± 265
± 736
bOSP19
46
4.5
2,628
1,735
1,370
2,200
+
573
+
352
± 339
± 981
bOSP2 0
> 97
4.0
1,798
3,244
1,044
1,219
+
397
+
1,534
± 229
± 377
bOSP2 Ia
97
4.0
5,954
5,262
1,671
2,264
+
1,559
+
1,578
± 382
± 713
bOSP22
90
4.0
5,697
5,754
2,409
3,032
+
1,497
+
1,776
± 418
± 782
bOSP2 3
90
3.9
3,740
9,120
3,953
2,412
+
1,066
+
4,283
± 893
± 548
bOSP24
48
3.9
2,128
1,811
2,675
945
+
498
+
818
± 849
± 175
bOSP25
46
3.9
5,397
5,055
3,814
1,863
+
2,041
+
3,076
±1,339
± 543
bOSP2 6
45
3.9
6,311
2,824
804
1,014
+
3,202
+
1,550
± 64
± 179
bOSP27a
20
5.0
2,783
1,140
763
736
±
706
+
242
± 261
± 173
bOSP28
> 97
7.5
2,393
3,435
1,194
2,172
±
299
+
2,138
± 341
±1,199

143
Table 4-2—continued.
Protein
Mr
(kDa)
Pi
Day of the
Estrous Cycle
0
2
5
8
bOSP29
> 97
7.5
4,931
4,302
1,350
2,943
± 1,451
+
2,355
± 396
±1,795
bOSP30
97
7.5
6,646
2,985
1,748
3,391
± 2,461
+
606
± 359
±1,936
bOSP31
60
ir>
r"
3,262
3,206
2,145
1,521
± 554
+
916
± 400
± 326
bOSP3 2
29
7.5
3,850
2,422
1,550
1,339
± 1,037
+
951
± 356
± 285
Note: Results are shown as dpm/culture (least-squares mean ±
sem) .
Significant effect of day of the estrous cycle (P < 0.05).

144
were solubilized and radioactivity quantified, amounts of radio¬
activity associated with most spots tended to be greatest at
estrus and decline thereafter (Table 4-2). The polypeptide
labeled bOSP14 (97,000 Mr; pi = 5.0) appeared to be the major
secretory product of the oviduct at estrus (18% of total radio¬
activity recovered from gels from estrous cultures) and was
reduced approximately 80% by Day 8 (5% of radioactivity recovered
from gels from Day 8 cultures). This postestrus decline in
synthesis of bOSP14 marks it as the major regulated protein
produced by the bovine oviduct during Day 0-8.
Two other patterns emerged in terms of change in the amount
of radioactivity associated with the spots as the estrous cycle
progressed. In some cases, such as bOSP6, 7, 8, 10 and 13, the
amount of recovered radioactivity remained fairly constant as
the estrous cycle progressed. For other polypeptides examined,
the associated radioactivity increased as the cycle progressed;
in 9 of 32 spots from Day 0 to Day 2, in 2 of 32 spots from Day 2
to Day 5, and in 15 of 32 spots from Day 5 to Day 8. Stage of the
estrous cycle was statistically significant (P < 0.05) for 5 of
32 bovine oviductal secretory proteins (bOSPs) examined (bOSP4,
12, 14, 21, and 27) (Table 4-2). Stage of cycle tended to be
significant (P < 0.09) for two others (bOSP31 and 32). In all
seven cases, radioactivity associated with these spots was
greatest at estrus and declined thereafter.
Effect of side of the reproductive tract relative to the
active ovary was statistically significant (P < 0.05) for 2 of 32
proteins (bOSP2and4) and approached significance (P<0.08) for

145
two others (bOSP3 and 16) . In each of these cases, radioactivity
associated with the spots was less for those samples from
oviducts obtained from the side ipsilateral to the side of
ovulation (2,284 ± 569 versus 4,309 ± 611; 2,225 ± 406 versus
3,855 ± 436? 1,883 ± 1,135 versus 5,109 ± 1,220; 2,135 ± 721
versus 3,887 ± 774 dpm/mg tissue; least-squares means ± sem for
ipsilateral vs. contralateral oviductal explants of bOSP2, 4, 3
and 16, respectively). There were no significant interactions
between side of the reproductive tract and day of the estrous
cycle on radioactivity associated with the excised portions of
the gels.
Analysis of Secretory Proteins by 1-D SDS-PAGE
In order to compare the major peaks of de novo synthesized
and secreted proteins by oviductal explants with reports in other
species where the proteins were resolved by 1-D SDS-PAGE, culture
supernatants from explants collected at estrus were resolved on
12.5% polyacrylamide gels. The pattern of proteins secreted by
oviductal explants as resolved by 1-D SDS-PAGE is shown in Figure
4-2. Regardless of stage of the estrous cycle or side of the
reproductive tract, major secretory products appeared on
fluorographs in four bands having apparent molecular weights of
> 97,000, 85,000-97,000, 55,000 and 30,000. The first of these
bands probably is separated into bOSP3, 9, 14, 21, and 30 by 2-D
SDS-PAGE (see Figure 4-1) . The band at 55,000 is likely resolved
as bOSP4, 7, 15, 24, and 31, and the 30,000 Mp band likely
consists of bOSP5, 10, 17, 26 and 32. Patterns of radiolabeled
proteins in tissue homogenates resolved by 1-D SDS-PAGE did not

146
appear to differ among days of the estrous cycle examined or with
side relative to the active ovary (Figure 4-2). The pattern of
radiolabeled proteins in the tissue homogenate was distinct from
that of radiolabeled molecules secreted into the culture medium,
indicating that proteins in the culture medium represented
secretory proteins and not intracellular proteins that leaked
from cells.
Discussion
As expected, de novo synthesis and secretion of proteins by
cultured oviducts was greatest at estrus and immediately
thereafter. This is the time when many critical events such as
sperm transport, capacitation, fertilization, and early cleavage
events occur in the oviduct. Enhancement of the rate of protein
synthesis and secretion around this time suggests the involve¬
ment of oviductal secretory proteins in these events. Geisert
et al. (1987) found de novo synthesis and secretion of radio-
labeled protein by bovine oviductal explants to be greater at
estrus than at other times examined through Day 19. Killian et
al. (1987) measured in vivo bovine oviductal fluid volume
throughout the estrous cycle and found that this peaked at
estrus. The elevated synthetic and secretory activity by
oviducts at estrus is likely due to estrogens, which are
elevated at estrus in the cow (Peterson et al. , 1975) or due to
the reduction in progesterone levels following the luteolytic
event of the previous estrous cycle (Kindahl et al., 1976).

146
Figure 4-1. Representative fluorographs of 2-D SDS-PAGE of
secretory proteins from cultured bovine oviducts obtained at A)
Day 0 (estrus), B) Day 2, C) Day 5 and D) Day 8 of the estrous
cycle. Proteins were separated in the first dimension by
isoelectric focusing, in the second dimension by SDS-PAGE using
12.5% (w/v) polyacrylamide gels under reducing conditions.
Egual amounts of radioactivity were loaded for each gel.
Individual spots that were excised and counted are identified by
number (panel C).

147
Figure 4-1. Representative fluorographs of 2-D SDS-PAGE of
secretory proteins from cultured bovine oviducts obtained at A)
Day 0 (estrus) , B) Day 2, C) Day 5 and D) Day 8 of the estrous
cycle. Proteins were separated in the first dimension by
isoelectric focusing, in the second dimension by SDS-PAGE using
12.5% (w/v) polyacrylamide gels under reducing conditions.
Equal amounts of radioactivity were loaded for each gel.
Individual spots that were excised and counted are identified by
number (panel C) .

147
Figure 4-2. Representative fluorographs of 1-D SDS-PAGE of de
novo synthesized proteins in culture supernatants (A and B) and
tissue homogenates (C and D) from cultured bovine oviducts. The
pattern secreted protein did not appear different for radiola¬
beled molecules produced at estrus from oviducts A) ipsilateral
and B) contralateral to the side of ovulation. Four major bands
(at 97,000, 85,000 - 97,000, 55,000 and at 30,000 Mp were apparent
(arrows). Similarly, the pattern of tissue labeling was not
different for the C) ipsilateral and D) contralateral oviduct.
Equal amounts of radiolabeled protein were loaded and resolved
on 12.5% polyacrylamide gels under reducing conditions.

148
Figure 4-2. Representative fluorographs of 1-D SDS-PAGE of de
novo synthesized proteins in culture supernatants (A and B) and
tissue homogenates (C and D) from cultured bovine oviducts. The
pattern secreted protein did not appear different for radiola¬
beled molecules produced at estrus from oviducts A) ipsilateral
and B) contralateral to the side of ovulation. Four major bands
(at 97,000, 85,000 - 97,000, 55,000 and at 30,000 Mr were apparent
(arrows). Similarly, the pattern of tissue labeling was not
different for the C) ipsilateral and D) contralateral oviduct.
Egual amounts of radiolabeled protein were loaded and resolved
on 12.5% polyacrylamide gels under reducing conditions.

149
In the present study, amplification of secretion of five
proteins by oviductal explants obtained at estrus was statisti¬
cally significant and several other proteins appeared to be
secreted at enhanced rates at estrus. Increased rates of protein
secretion by the oviduct at around the time of estrus and the
follicular phase of the estrous cycle as well as the appearance
of individual stage-specific proteins produced by the oviduct
have been reported in a number of species, including the rabbit
(Oliphant et al. , 1984b) , pig (Buhi, 1985) , cow (Geisert et al. ,
1987), sheep (Sutton et al., 1984a), mouse (Kapur and Johnson,
1985), rat (Wang and Brooks (1986), baboon (Fazleabas and
Verhage, 1986), and human (Verhage et al. , 1988). In the present
study, there were a number of secretory proteins that were
secreted in the greatest amounts at estrus. However, all
polypeptides examined were present to some degree at all days
examined. In addition, not all individual secretory proteins
examined were secreted in greater amounts at estrus; secretion
rates of some remained constant throughout Day 0-8, while
secretion rates of other polypeptides increased somewhat as the
cycle progressed. These results suggest differential regulation
of protein secretion in the bovine oviduct.
Variation in the regulation of protein secretion between
the ampulla and isthmus of the oviduct has been found in the
mouse (Nieder and Macon, 1987), cow (Geisert et al., 1987) and
pig (Buhi, 1989). In each case, one or two protein spots on
fluorographs of 2-D PAGE separations appear prominent in
secretions from one region but are minor or absent in secretions

150
from the other. One must interpret analyses of individual
secretory proteins with caution. Since a number of proteins
exhibited similar molecular weights and isoelectric points, it
is possible that some overlap of individual proteins occurred
during separation. Excision of individual spots is, however, a
more reliable technique for quantification of relative amounts
of de novo synthesized radiolabeled proteins than comparison of
the relative development of fluorographs.
Based on analysis by 1-D SDS-PAGE, the secretory proteins
of the bovine oviduct appear very similar to those of the sheep.
Sutton et al. (1984b) found that the major classes of sheep
oviductal proteins had molecular weights of 80,000-90,000,
50,000-60,000 and 30,000, values very similar to the groups of
proteins secreted by the bovine oviduct at estrus. The major
protein secreted in vitro by bovine oviducts collected at estrus
appears very similar to a spermatozoa-binding protein secreted
by the sheep oviduct. The protein, bOSP14, of 97,000 Mr and pi =
5.0, represented 18% of the radioactivity recovered from 2-D gels
at estrus. Similarly, the subunits of the estrus-associated,
sperm-binding protein of the sheep oviduct have an apparent
molecular weight of 80,000-90,000 and an pi of 4.7 (Sutton et
al., 1984b; Sutton et al., 1985). It remains to be determined
whether the similarity between the sheep estrus-associated
glycoprotein and bOSP14 extend beyond size and apparent isoelec¬
tric point.
Overall, there was little effect of side of the reproductive
tract on total protein secretion or on secretion of individual

151
polypeptides, although four proteins were secreted in greater
amounts by tissue contralateral to the side of ovulation. The
physiological basis for greater protein secretion by the
contralateral oviduct is unclear. Perhaps these polypeptides
are proteases or other enzymes essential to maintenance of the
oviductal environment during most of the estrous cycle but which
might interfere with sperm activity or events at fertilization.
The possibility of acute local ovarian control over oviductal
function remains. Flickinger et al. (1977) reported differences
in the concentration of estradiol receptors between the fimbrial
ends of the human oviduct and found the concentration of estra¬
diol receptors to be lower on the ipsilateral side. The
combination of local control via blood or lymphatic vasculature
(Hunter et al., 1983) and variation in hormone receptor con¬
centrations might possibly account for asymmetric protein
synthesis and secretion by bovine oviducts.
In this study, estrus-associated changes in the protein
synthetic and secretory activity of the bovine oviduct occurred.
Such stage-specific changes have been seen in all species
examined. If the environment of the oviduct is critical to the
viability of ovum and spermatozoa, the process of fertilization
and early conceptus development, alterations could cause changes
that would hamper fertilization and induce retardation and
reduced viability of the conceptus. Characterization of the
oviductal environment is crucial to understanding its role in the
events of fertilization and early pregnancy and how that role
might be altered in various types of reproductive dysfunction.

CHAPTER 5
HEAT STRESS-INDUCED ALTERATIONS IN SYNTHESIS AND
SECRETION OF PROTEIN AND PROSTAGLANDIN BY CULTURED BOVINE
CONCEPTUSES AND UTERINE ENDOMETRIUM
COLLECTED AT DAY 17 OF PREGNANCY
Introduction
Heat stress-induced hyperthermia can reduce embryonic
survival (Ulberg and Sheean, 1973; Dunlap and Vincent, 1971;
Putney et al., 1988a). Accordingly, pregnancy rates in cattle
are greatly reduced during the summer in regions associated with
elevated ambient temperatures (Scott and Williams, 1962; Posten
et al., 1962; Badinga et al., 1985). One physiological event
that may be sensitive to heat stress is rescue of the corpus
luteum from luteolysis. Heat stress from 8 to 16 days after
insemination caused alteration of the uterine environment
(Geisert et al., 1988), reduced weight of corpora lútea and
impaired conceptus growth (Geisert et al. , 1988; Biggers et al. ,
1987). During this period, the conceptus secretes the anti-
luteolysin, bovine trophoblast protein-1 (bTP-1) (Helmer et al. ,
1987) , a protein that causes suppression of uterine prosta¬
glandin (PG) F secretion resulting in luteal maintenance (Helmer
et al., 1989a,b; Thatcher et al., 1989). Depressed PGF secretion
by bTP-1 may be mediated by an inhibitor of PG synthesis that
152

153
appears in uterine endometrial cells during pregnancy (Gross et
al. , 1988c) and after exposure to bTP-1 (Helmer et al. , 1989a).
The purpose of this study was to determine whether heat
stress in vitro alters uterine synthesis and secretion of protein
and PG by conceptuses and endometrium obtained on Day 17 of
pregnancy to determine whether heat stress-induced alterations
in conceptus and endometrial tissues may play a role in embryonic
mortality.
Materials and Methods
Materials
Radioisotopes L-[4,5-3H] leucine (specific activity [SA] =
-150 Ci/mmol) , [ 125I ] -Na (SA = -16.9 Ci/nq of I) , [5,6,8,11,12,-
14,15-3H]PGF2(J (SA = -160-180 Ci/mmol) and [5,6,8,12,14,15-
3H]PGE2 (SA = -140-170 Ci/mmol) were purchased from Amersham
Corporation (Arlington Heights, IL) . Radioinert PGF2a, PGE2 and
arachidonic acid were purchased from Sigma Chemical Company (St.
Louis, MO). Protein A was obtained from Genzyme (Boston, MA),
and coupled to 125I with IODO-GEN (Pierce Chemical Company,
Rockford, IL) . A PD-10 column was purchased from Pharmacia Inc.
(Piscataway, NJ). Rabbit antiserum to PGF2a was provided by Dr.
T. G. Kennedy, University of Western Ontario, and sheep antiserum
to PGE2 was provided by N. R. Mason from the E. L. Lilly Research
Laboratories (Indianapolis, IN) . Rabbit antiserum to ovine
trophoblast protein-1 (anti-oTP-1), which exhibits binding to
bTP-1 (Helmer et al.,
1987) , was obtained from F. W. Bazer,

154
University of Florida. Preparation of a modified Eagle ' s minimum
essential medium (MEM) and supplies for tissue culture were
described in Chapter 2. Spectrapor membrane dialysis tubing was
purchased from Spectrum Medical (Los Angeles, CA) . Whatman 3MM
paper (Whatman, Clifton, NJ) was utilized for trichloroacetic
acid (TCA) precipitation using TCA from Fisher Scientific
(Orlando, FL) . Nitrocellulose membrane (BA85, .45 /im) was
purchased from Schleicher and Shuell (Keene, NH) .
Supplies for polyacrylamide gel electrophoresis (PAGE) and
Western blotting were as follows: tris (hydroxymethyl)amino-
methane (Tris) base, Nonidet P-40, and N,N,N',N'-tetramethyl
ethylenediamine were purchased from Sigma. Sodium salicylate,
2-mercaptoethanol, glycine, and ammonium peroxydisulfate were
purchased from Fisher. Acrylamide, urea, dithiothreitol, sodium
dodecyl sulfate (SDS), and amido black 10B were purchased from
Research Organics (Cleveland, OH) . Bis-acrylamide, gelatin, and
Tween-20 were purchased from Bio-Rad (Richmond, CA). Carrier
ampholytes used in isoelectric focusing were purchased from
Serva (Heidelberg, FRG) .
Collection of Conceptuses and Uterine Endometrium
Beef cattle (Angus or Brangus) were used for collection of
bovine conceptuses and endometrium. Cattle were observed for
estrus and bred by natural service to Angus bulls and slaughtered
on Day 17 after estrus. Following exsanguination, reproductive
tracts were recovered and transported to a sterile laminar flow
hood. Conceptuses were flushed from uteri with 50 ml MEM, as

155
described by Helmer et al. (1987). Conceptuses were weighed,
placed in fresh MEM and cultured as described below.
After flushing of the conceptus, the uterine horn ipsi-
lateral to the corpus luteum was opened longitudinally along the
antimesometrial border and endometrial slices were excised.
Intercaruncular endometrium was dissected free from myometrial
tissue, blotted on sterile gauze, weighed, cut into 2-3mm3 cubes
and cultured in MEM as described below.
In Vitro Culture
Whole Day 17 conceptuses and endometrial explants (500 mg)
were transferred to sterile plastic 100 mm petri dishes and
cultured in 20 ml of MEM supplemented with 100 ¡iCi L-[4,5-
3H] leucine and 200 ¡xq arachidonic acid under an atmosphere of
2.5% 02, 47.5% C02 (v/v/v) . Cultures were maintained in the dark
on rocking platforms. Control cultures were incubated at 39 °C
for 24 h (homeothermic culture) , conditions representing normal
body temperature of the cow. Heat-stress cultures were accli¬
mated at 39 °C for 6 h then incubated at 43 "C for 18 h. Culture
medium from both treatment groups was sampled (1 ml) at 0, 3, 6,
9, 12, 18 and 24 h after initiation of culture. Samples of medium
were stored at -70 °C until assayed for incorporation of [3H] leu¬
cine into protein and concentrations of prostaglandins.
Preparation of Culture Medium and Tissue for Analysis
At termination of culture, endometrial and conceptus
tissues were separated from culture medium by centrifugation
(3500 x g, 4 ° C, 30 min). Tissues were solubilized in 50 mM

156
Tris-acetate buffer (8 ml, pH 7.5) that contained 1 mM phenyl-
methylsulfonylfluoride, 1 mM ethylenediamine tetraacetic acid
and 2% (v/v) Nonidet P-40. Homogenates were stored at -70°C.
Culture medium recovered at the end of incubation was dialyzed
extensively (three changes of 4 liters) against deionized water
using dialysis tubing with a 6,000 to 8,000 dalton exclusion
limit.
Protein Determination
Incorporation of [3H]leucine into secreted and intra¬
cellular proteins was determined by trichloroacetic acid (TCA)
precipitation. Samples (50 jiil) of solubilized tissue and
conditioned culture medium were placed onto Whatman 3MM paper
(previously saturated with 20% TCA [w/v]) and allowed to dry.
Proteins were precipitated onto filter paper by serial washings
with 20% TCA, 5% TCA and 95% ethanol as described by Mans and
Novelli (1961). Radioactivity of precipitated protein was
determined by scintillation spectrometry.
Electrophoresis
One-dimensional polyacrylamide gel electrophoresis in the
presence of SDS and 2-mercaptoethanol (1D-SDS-PAGE) was done
using the buffer system of Laemmli (1970). Endometrial and
conceptus secretory proteins were resolved on 12.5% (w/v)
polyacrylamide gels.
Two-dimensional (2D) SDS-PAGE was performed according to a
modification of the method described by Roberts et al. (1984).
Endometrial and conceptus tissue proteins were dissolved in .01

157
ml of 5 mM K2C03 containing 9.0 M urea, 2% (v/v) Nonidet P-40 and
0.5% (w/v) dithiothreitol, and resolved in the first dimension
by isoelectric focusing in 4% (w/v) acrylamide tube gels
containing N,N'-diallyltartdiamide, 8.0Murea, 2% (v/v) Nonidet
P-40, and 5.1% (v/v) ampholytes (pi: 3-10, 5-7, and 9-11;
50:36:16 by volume, respectively). First dimension disk gels
were equilibrated in 0.07 M Tris-HCl buffer (pH 6.8), containing
1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol, and subjected to
electrophoresis in the second dimension in 12.5% (w/v) poly¬
acrylamide slab gels. Proteins were localized by Coomassie
Brilliant Blue R-250 staining and fluorographs were prepared
with sodium salicylate as a flúor and Kodak XAR film as described
by Roberts et al. (1984) .
Western Blotting
Conceptus proteins present in culture supernatants were
resolved (ID SDS-PAGE) on 12.5% polyacrylamide gels. After
electrophoretic transfer (see Chapter 2), nitrocellulose
membranes were stained with amido black (Harper et al., 1986),
destained with acetic acid, ethanol and water (7%, 43% and 50%;
v/v/v, respectively) and immunoblotted. Nonspecific binding of
proteins to nitrocellulose was blocked with 10 mM Tris (pH 7.6)
containing 3% (w/v) gelatin, .8% (w/v) NaCl and .05% (v/v) Tween-
20. Membranes were then incubated (2 h at room temperature) with
rabbit antiserum to oTP-1 or with normal rabbit serum at dilu¬
tions of 1:100 in incubation buffer (10 mM Tris-HCl, pH 7.6,

158
containing 1% [w/v] gelatin, .8% [w/v] NaCl and .05% [v/v] Tween-
20) . After incubation, membranes were washed (30 min) in
incubation buffer to remove unbound antiserum and further
incubated (2 h at room temperature) with 125I-labeled Protein A
(106 cpm/ml). Membranes were rinsed with deionized H20, washed
(24 h at 4 ° C) with Tris-HCl (pH 7.6), dried, and Protein-A
antibody-antigen complexes visualized by autoradiography.
Iodination of Protein A
Protein A was iodinated by a modification of the procedure
of Markwell and Fox (1978) . Briefly, 20 nq IODO-GEN was added to
975 jUl of .02 M KP04 (pH 7.0) containing .4 M NaCl. Next, 20 /ng
Protein A and 5 /¿I of carrier-free [125I]-Na (500 /¿Ci) were added
to the reaction tube and incubated for 20 min. Unreacted 125I was
separated from radioiodinated Protein A by chromatography on a
Pharmacia PD-10 column with 1% gelatin in phosphate-buffered
saline as eluent.
Quantification of Proteins Separated by PAGE
Radiolabeled conceptus secretory proteins were resolved
by ID SDS-PAGE on 12.5% gels. Individual lanes, representing
separate conceptus cultures, were isolated and sequentially
sectioned into 2 mm slices to generate a profile of radioactive
proteins. Slices were individually solubilized by incubation in
.4 ml H202 for 2 h at 70'C and mixed with scintillation fluid.
Radioactivity was determined by scintillation spectrometry.

159
Measurement of Prostaglandins
Conceptus- and endometrial-conditioned culture media were
analyzed for PGF2a with a radioimmunoassay (RIA) procedure
(Knickerbocker et al., 1986b) modified to use an antibody
characterized by Kennedy (1985). Standard curves were prepared
in MEM with known amounts of radioinert PGF2a (10-5000 pg) . An
antiserum dilution of 1:5000 with a minimum sensitivity of 25 pg
per tube was used. Cross-reactivity of the PGF2a antiserum with
other PGs were 94% for PGF1a, 2.4% for PGE2, and <0.1% for PGFM,
PGE1 and arachidonic acid. Due to the high cross-reactivity with
PGF1a, measurements are defined as PGF. Unextracted samples (300
111) from tissue incubations contained [3H] leucine (approx.
25,000 cpm) . To correct for this, samples were assayed with and
without the addition of [3H]PGF2a. After charcoal-dextran
isolation of bound material, the bound radioactivity for
duplicate samples without [3H]PGF2a was subtracted from values
for duplicate samples that contained [3H]PGF2a. An inhibition
curve containing PGF2a (5 ng/ml) was assayed serially in 25, 50,
100, 200, and 300 /Ltl volumes (final volume of 300 ¿il with blank
MEM) with [3H]leucine (approx. 25,000 cpm). This inhibition
curve (corrected for [3H] leucine background) was parallel to the
standard curve, and a test for homogeneity of regression
indicated that the curves did not differ. Inter-and intraassay
coefficients of variation were 17.7 and 12.9%, respectively.
Samples were analyzed for PGE2 with an RIA procedure (Lewis
et al., 1978) modified to analyze unextracted MEM. Standard

160
curves were prepared in MEM with known amounts of radioinert PGE2
(10-5000 pg). An antiserum dilution of 1:6000 was used with a
minimum sensitivity of 25 pg per tube. Cross-reactivity of the
PGE2 antiserum with other PGs were 24% for PGE1, 1.7% for PGF2a,
and < .1% for PGFM, PGF1a and arachidonic acid. Correction for
nonspecific binding due to the presence of [3H] leucine in samples
was done as described for the PGF RIA. An inhibition curve
containing PGE2 (5 ng/ml) was assayed serially in 25, 50, 100,
200, and 300 41 volumes (final volume of 300 ¿xl with blank MEM)
with [3H] leucine (approx. 25,000 cpm). This inhibition curve was
parallel to the standard curve and a test of homogeneity of
regression indicated that the curves did not differ. Inter- and
intraassay coefficients of variation were 18.2 and 14.1%,
respectively.
Statistical Analysis
Effect of treatment on conceptus and endometrial secretory
activity was analyzed by least-squares analysis of variance with
the General Linear Models procedure of the Statistical Analysis
System (SAS, 1985). Models used to analyze conceptus data
included effects of temperature and conceptuses nested within
temperature. Endometrial data were modeled with cow, tempera¬
ture and the interaction. Protein and PG secretion rates were
characterized by polynomial regressions for time trends. Tests
for homogeneity of regression were used to detect differences in
secretion rates of the tissues due to temperature.

161
Results
Quantitative Protein Synthesis and Secretion
To examine the effect of heat stress on protein synthetic
rates for conceptus and endometrial tissues, cultures were
maintained at 39° (homeothermic) or 43 °C (hyperthermic) in the
presence of [3H]leucine. Secretion of proteins by tissues was
determined by measuring incorporation of [3H]leucine into TCA-
precipitable proteins released into culture medium. Conceptus
and endometrial tissues remained viable throughout the duration
of culture, as indicated by continued accumulation of newly
synthesized, [3H] leucine-labeled proteins secreted into culture
medium (Figure 5-1). Protein secretion rates varied over the
duration of culture according to tissue type (endometrium <
conceptus) and temperature treatment. Before heat stress,
secretion of proteins by tissues within treatment groups was
similar to thermoneutral control tissues (conceptus: 4888 ± 3363
versus 7654 ± 3684; endometrium: 1161 ± 154 versus 1135 ± 154
dpm/mg tissue/6 h). Elevated incubation temperature reduced
protein synthetic capacity of conceptus tissues, resulting in
a 54.2% decrease (P < 0.01) in incorporation of [3H]leucine into
secretory proteins and a 66.8% decrease (P < 0.01) in incorpora¬
tion into conceptus tissue proteins at the end of culture (Table
5-1) . In contrast, heat stress of endometrium did not affect the
rate of incorporation of [3H]leucine into either secretory or
tissue proteins.

162
Table 5-1. Incorporation of [3H]leucine into TCA-precipitable
protein in tissue and medium after 24 h of culture by conceptuses
and endometrium cultured at homeothermic (39°C) or heat-stress
(43°C) temperatures.
Tissue
i HI-protein (dpm/mg tissue/24 h)
Secreted Tissue
Endometrium
39
43
Conceptus
39
43
8,767 ± 322
8,286 ± 322
64,431 ± 3,656a
29,487 ± 3,656
73,405 ± 4,197
79,914 ± 4,197
167,498 ± 27,247a
55,529 ± 27,247
Note: Data are expressed as least-square means ± sem.
incorporation of radiolabel into de novo synthesized protein
by conceptuses was depressed (P < 0.01) by elevated temperature.
Table 5-2. Concentrations of prostaglandin (PG) F and PGE2 in
medium supernatants collected after 24 h of culture of concep¬
tuses and endometrium at homeothermic or heat-stress tempera¬
tures .
Tissue
Prostaglandin (ng/ml/g tissue/24 h)
PGF PGE2
Endometrium
39
5.6
+
0.7
13.1
+
2.9
43
74.2
+
17.9a
18.6
+
5.4
Conceptus
39
21.5
+
8.8
12.2
+
4.1
43
9.6
+
3.3
43.9
+
33.8
Note: Data are expressed as least-square means ± sem.
aRelease of PGF by endometrium was increased by elevated tempera¬
ture (P < 0.0001).
Release of PGE2 by conceptuses was greater at elevated tempera¬
ture (P < 0.05). Due to heterogeneous variation, data were
transformed to Log10 before analysis.

163
Qualitative Analysis of Proteins
Examination of radiolabeled tissue proteins resolved by 2D
SDS-PAGE revealed a complex array of de novo synthesized, radio-
labeled proteins. Representative fluorographs of protein
patterns are presented in Figure 5-2. Heat stress altered the
array of proteins present in conceptus and endometrial tissues.
In particular, heat stress caused conceptuses to synthesize
proteins with apparent molecular weights of 70,000 (pi 6.2) and
91,000 (pi 5.6) in the range of two of the major mammalian heat-
shock proteins. These proteins were also apparent in low levels
in tissues from endometrial cultures maintained at 39 "C and were
more abundant in endometrial tissues incubated at 43 °C.
Radiolabeled proteins in conditioned medium from conceptus
and endometrial cultures were resolved by ID SDS-PAGE and
radiolabeled proteins visualized by fluorography (Figure 5-3
and 5-4) . The predominant radiolabeled polypeptides detected in
conceptus culture supernatants appeared as three bands of
22,000, 24,500 and 26,000 Mr, the expected range of molecular
weight for bTP-1. The predominant proteins secreted by endo¬
metrium were of 29 , 000 , 38,000 and 50,000 Mr. Heat stress did not
appear to alter the array of radiolabeled proteins secreted into
culture medium by conceptuses or endometrium.
Quantitative Analysis of Proteins
Equal volumes of conditioned medium from each conceptus
were electrophoretically separated (ID SDS-PAGE) , and the radio-
labeled proteins were solubilized from the gel in sequential 2

164
Endometrium Conceptúe
Figure 5-1. Incorporation of [3H]leucine into TCA-precipitable
macromolecules in culture medium by endometrial and conceptus
tissues incubated at homeothermic (solid line) or heat shock
(dashed line) temperature. Shown are least-square means.

165
Endometrium
Homeothermic Heat Shock
co —
14-
-n 1 1 1 1 1 1 '
n k o « l>> O ® ni k o « *■» o
K 4» C ** •» ^ K o' « n (I (i
pi pi
Conceptus
Homeothermic Heat Shock
29 -
20 -
Figure 5-2. Two-dimensional polyacrylamide gel electrophoresis
of proteins present in endometrial and conceptus tissues. Endo¬
metrium and conceptuses were cultured at 39 °C or 4 3 °C. Proteins
were separated in the first dimension by isoelectric focusing and
in the second dimension by SDS-PAGE using 12.5% (w/v) poly¬
acrylamide gels. Radiolabeled proteins were localized by
fluorography. Equal amounts of radioactivity (2 00,000 dpm) were
loaded on each gel. The predominant heat-induced proteins
present in heat-stressed tissues are indicated: (1 = 70,000, pi
6.2; 2 = 92,000, pi 5.6) .

166
39 C 43 C
Figure 5-3 . One-dimensional polyacrylamide gel electrophoresis
of conceptus secretory proteins. Samples of individual concep-
tuses were collected after 24 h of culture. Proteins were
separated by SDS-PAGE using 12.5% (w/v) polyacrylamide gels and
visualized by fluorography. Equal volumes (100 ¡J.1) of medium
were loaded per lane. Fluorographic exposure time differed for
conceptuses (43° > 39°C) in order to examine qualitative
differences in radiolabeled polypeptides in medium between
treatments. The major [3H]leucine-labeled proteins identified
in conceptus secretions were reduced by heat stress (arrows).

167
39 C 43 C
2
20 -
14 -
Figure 5-4. One-dimensional polyacrylamide gel electrophoresis
of endometrial secretory proteins. Proteins were separated by
SDS-PAGE using 12.5% (w/v) polyacrylamide gels and radiolabeled
proteins were visualized by fluorography. Equal volumes (100/xl)
of medium were loaded per lane.

168
mm slices. Profiles of radioactivity were similar between
conceptuses within the same treatment group and were pooled to
examine treatment effects (Figure 5-5). The predominant radio-
labeled polypeptides (representing 27.5% of total radioactivity)
appeared as a single peak with an apparent molecular weight range
of 22,000-26,000, similar to bTP-1 (Helmer et al. 1987).
Overall, heat stress resulted in a 63.2% reduction (P <
0.01) in the total quantity of radiolabeled proteins (94,789 ±
2758 versus 29,064 ± 689 dpm/mg tissue/24 h) . Relative labeling
of secretory protein species with apparent molecular weight
ranges of 50,000 and 85,000 were decreased 76.3 and 76.8%,
respectively. Conceptus secretory proteins migrating coincident
with bTP-1 were reduced 71.7% (p < 0.01) relative to proteins
obtained from homeothermic conceptuses (7391 ± 698 versus 26,099
± 2758 dpm/mg tissue/24 h) .
Immunoblottinq
Antiserum to oTP-1, an immunologically related protein
(Helmer et al. , 1987) , was used to detect bTP-1 in culture medium
by immunoblotting. Autoradiograms of immunoblots are depicted
in Figure 5-6. Antiserum to oTP-1 bound specifically to protein
species with an apparent molecular weight range of 22,GOO-
26, 000. These antibody-reactive proteins were of similar
molecular weight to the predominant protein bands detected by
fluorography (Figure 5-3) as well as those in the major radio¬
active peak solubilized from ID SDS-PAGE gels (Figure 5-5).
Collectively, these different experimental approaches confirm

169
that proteins within this molecular weight range in part
represent the bTP-1 complex. Heat stress resulted in a reduction
in secretory proteins that bound oTP-1 antiserum, as indicated
by a decrease in [ 125I]-Protein A labeling of antibody-bound
proteins. Minor cross-reactivity of antiserum with a polypep¬
tide species of 66,000 Mr was detected. This protein likely
represents bovine serum albumin present in conceptus cultures.
Antibody-protein complexes were not observed on autoradiograms
of nitrocellulose blots incubated with nonimmune rabbit serum.
Prostaglandin Secretion
To examine the effect of heat stress on PG synthesis by con¬
ceptus and endometrial tissues, samples of medium were analyzed
for PGF and PGE2 by RIA. Prostaglandins present in culture medium
of endometrial explants are derived from de novo synthesis of PG
during culture (Thatcher et al., 1984). Endometrium incubated
at 39 °C released PGs primarily during the first 3 h of incubation
(Figure 5-7). Concentrations of PGE2 in culture medium of
homeothermic endometrium increased throughout culture, result¬
ing in higher (P < 0.01) levels of PGE2 than PGF (Table 5-2) at
the end of culture. Prior to heat stress, secretion of PGs by
endometrial tissues was similar to that by homeothermic control
tissues (heat stress vs. homeothermic, PGF: 3.4 ± 0.5 versus 2.8
± 0.5; PGE2: 2.5 ± 0.5 versus 4.1 ± 1.5 ng/ml/g tissue/6 h).
Elevated incubation temperature stimulated endometrial PGF
release, resulting in a 1255% increase (P < 0.01). Release of

170
DPM (x.001) Radiolabeled Protein
Figure 5-5. Electrophoretic profile of [3H]leucine-labeled
polypeptides accumulated in conceptus culture medium after 24
h of culture. Proteins were separated by ID SDS-PAGE using 12.5%
(w/v) polyacrylamide gels and solubilized from sequential 2 mm
slices of the gel matrix with H202. Equal volumes (100 nl) of
conditioned medium from each conceptus were loaded per lane.
Shown are profiles of radiolabeled proteins (dpm/mg tissue) for
homeothermic (solid line) and heat-stressed (broken line)
conceptuses. Peaks in detected radioactivity corresponding to
predominant proteins identified by ID SDS-PAGE and fluorography
were reduced (P < 0.05) by heat stress (arrows).

171
39C 43C 39C
Figure 5-6. Immunoblotting of bovine trophoblast protein-1
(bTP-1) released by conceptuses during 24 h of culture. Pro¬
teins were separated by ID SDS-PAGE on 12.5% (w/v) polyacrylamide
gels, transferred to nitrocellulose membranes, and immunoblotted
with either nonimmune (not shown) or anti-ovine trophoblast
protein-1 (oTP-1) rabbit serum. Equal volumes (100 n 1) of
conceptus-conditioned medium from each conceptus were loaded per
lane. Antibody-protein complexes bound to nitrocellulose were
detected by Ti5I-Protein A labeling of antibody and auto¬
radiography. Specific binding of oTP-1 antibody to bTP-1 was
reduced by heat stress (arrow).

Endometrium
Endometrium
172
Conceptúe
Conceptúe
Figure 5-7. Release of prostaglandins (PGF and PGE2) into
culture medium by endometrium and conceptuses. Samples of
conditioned medium (300 nl) were analyzed for PGs by radio¬
immunoassay. Shown are profiles of PGF and PGE2 for homeothermic
(solid line) and heat-stressed (broken line) endometrium and
conceptuses. Endometrial PGF release (P < 0.001) and conceptus
PGE2 (P < 0.05) were increased in response to heat stress.

173
PGE2 was not affected significantly by heat stress with the
result that heat-stressed endometrium secreted more (P < 0.01)
PGF than PGE2.
Conceptuses released PGs throughout culture, though there
was a trend towards decreased PGF and PGE2 concentrations in
medium as the duration of culture increased at 39 °C (Figure 5-
7) . Imposition of heat stress stimulated conceptus production
of PGE2, resulting in a 360% increase (P < 0.05) . Release of PGF
by conceptuses was not affected by heat stress.
Discussion
Early preimplantation embryos are extremely sensitive to
high environmental temperature (Alliston and Ulberg, 1961; Dutt,
1963; Alliston et al., 1965; Elliott et al., 1968; Elliott and
Ulberg, 1971; Ulberg and Sheean, 1973; Putney et al., 1988a).
The present study of Day 17 bovine conceptuses has shown that
elevated incubation temperature in vitro reduces total protein
synthetic capacity while enhancing the synthesis of 70- and
90-kDa heat-shock proteins. Similar effects of in vivo thermal
stress on subsequent in vitro protein production by conceptuses
have been noted in swine (Wettemann et al., 1984) . These data
suggest that high environmental temperature may alter conceptus
metabolic activity in vivo, leading to reduced growth rates and
failure of conceptuses to produce biochemical signals required
for preventing CL regression. Smaller conceptuses may not
develop the biosynthetic capacity to signal the maternal system

174
to maintain CL function, as evidenced by findings that in vivo
heat stress resulted in reduced CL weight and a trend towards
increased pregnancy failure (Biggers et al., 1987) .
Conceptus-conditioned culture medium was enriched in a
group of low molecular weight proteins (20,000-26,000 Mr) which
cross-reacts with antiserum to oTP-1. This group is referred to
as bTP-1 complex and is believed to be involved in preventing
luteal regression during early pregnancy (Helmer et al. , 1987) .
Heat stress altered total protein synthetic capacity of concep-
tuses and induced a marked reduction in secretory proteins,
particularly proteins within the bTP-1 complex.
Concentrations of PGs released by tissues incubated at 39 °C
were similar to those released in vitro by bovine endometrium
from Day 17 of pregnancy (Thatcher et al., 1984a) . Heat stress
of endometrial tissues resulted in a marked increase in PGF
release. The primary site for the action of heat damage on
tissues is the cell membrane (Bowler et al., 1973; Hahn, 1982),
causing alterations in membrane lipid composition (Anderson and
Parker, 1982) as well as increases in membrane fluidity, phospho¬
lipase activity, and phosphoinositide turnover (Calderwood et
al., 1987; Lee and Chapman, 1988). Heat-induced increases in the
turnover of membrane phospholipids and the release of fatty
acids, such as arachidonic acid, may provide substrates for PG
synthesis (Flint et al., 1986). Increases in endometrial PGF2a
production in vivo in response to heat stress have been reported
for gilts (Wettemann et al. , 1984 ; Hoagland and Wettemann, 1984).

175
Since maintenance of luteal function in cattle is associated with
endometrial PG production (Thatcher et al., 1984a; Gross et al.,
1988a,b), increased endometrial PG release in response to
thermal stress may compromise CL function.
Conceptuses incubated at elevated temperature synthesized
proteins not present in control tissues. The predominant heat-
shock protein was of 70,000 Mp. The mammalian 70,000 Mr heat-
shock protein, is synthesized by mouse (Wittig et al. , 1983), rat
(Mirkes, 1987), and rabbit (Heikkila and Schultz, 1984) embryos
incubated at elevated temperature. It has been assumed that
heat-shock proteins play an essential role in cellular homeo¬
stasis and thermotolerance during periods of environmental
stress (Loomis and Wheeler, 1980; Li and Werb, 1982) .
Incubation of endometrial explants at elevated temperature
did not appear to alter protein synthesis by endometrial tissues.
Endometrial tissues in both treatment groups synthesized
proteins similar in molecular weight (70,000 and 91,000 Mr) to
heat-shock proteins identified in heat-stressed conceptuses.
The intensity of these endometrial proteins was enhanced in
tissues exposed to heat-stress culture conditions. The presence
of a 70,000 Mr heat-stress protein in control tissues was not
unexpected. Two members of the 70-kDa heat-shock family of
proteins have been identified (Welch et al. , 1982) : a constitu-
tively produced 73-kDa heat-shock protein is produced at
homeothermic temperatures, but its production is amplified
during heat stress, and an inducible 72-kDa protein that is

176
produced as a result of tissue shock. Alternatively, trauma of
tissue slicing or incubation conditions of the present experi¬
ment may have resulted in some expression of heat-shock proteins
by endometrium in both treatment groups. Hightower and White
(1981) reported that several high molecular weight, stress-
induced proteins were synthesized by sliced mammalian tissues in
vitro but not synthesized by tissues in vivo.
In summary, elevated incubation temperature induced a large
reduction in conceptus protein synthesis and secretion and
stimulated PGF release by pregnant endometrium. These in vitro
results suggest that exposure of pregnant cows to high environ¬
mental temperature and humidity, as often occurs during summer
months, may disrupt the balance between conceptus and endome¬
trial signals responsible for rescue of the CL and maintenance
of pregnancy.

CHAPTER 6
EFFECT OF IN VITRO HEAT SHOCK UPON SYNTHESIS AND SECRETION OF
PROSTAGLANDINS AND PROTEIN BY UTERINE AND PLACENTAL
TISSUES OF THE SHEEP
Introduction
Heat stress during mid- to late- gestation has been impli¬
cated in reduced fetal and placental weight in cattle and sheep
(Alexander and Williams, 1971; Collier et al. , 1982) and reduced
cotyledonary release of estrone sulfate in cattle (Collier et
al., 1982). Retardation of fetal growth caused by heat stress
likely results from perturbation of diverse physiological
events. Central to this phenomenon is a reduction in uterine and
placental blood flow (Oakes et al., 1976; Bell et al., 1987;
Alexander et al. , 1987) . This decrease in blood flow is actively
regulated at the uterine vascular bed since elevated body
temperature also reduced response to pharmacological stimulation
of uterine blood flow in ewes (Roman-Ponce et al., 1978a) and
cows (Roman-Ponce et al. , 1978b) . One possibility is that local
vasoconstriction during heat stress is caused in part by changes
in PG secretion. Elevated temperature in vitro can alter the
patterns of PG secretion by tissues of the reproductive tract and
early embryo of the cow (Chapters 2, 3, 5; Putney et al., 1988)
and pig (Wettemann et al., 1988; Gross et al., 1989). Prosta¬
glandins affect blood flow (Ford, 1982) through vasoconstrictive
177

178
and vasodilatory activities which are modulated to some extent
by circulating steroids (Ford, 1982; Vincent et al., 1986).
Prostaglandins may also affect permeability of the endometrial
vascular bed (Kennedy, 1980) and stimulate fluid and electrolyte
transport across the uterine epithelium (Biggers et al. , 1978) .
Prostaglandins are also critical to events of parturition
(Thorburn and Challis, 1979) . Accordingly, one objective of the
current experiment was to determine effects of elevated tempera¬
ture upon uterine and placental PG release.
Another objective was to evaluate effects of elevated
temperature on protein secretion by uterine and placental
tissues. Although the functional roles of many of the proteins
that appear in the uterine lumen during pregnancy remain
undefined, it may reasonably be assumed that uterine and
placental secretory proteins play important roles during
pregnancy. These proteins have been implicated as transport
molecules (Buhietal., 1982), immunomodulators (Hansen et al.,
1989), lysosomal enzymes (Hansen et al., 1985), protease
inhibitors (Fazleabas et al., 1982) and hormones (Talamantes et
al. , 1980). In cattle, the secretion rates of certain proteins
were altered by elevated temperature in vitro (Chapter 3) and
heat stress in vivo resulted in increased protein content of the
uterine lumen in cows (Geisert et al., 1988; Biggers et al.,
1987) and pigs (Wettemann et al. , 1988) .

179
Materials and Methods
Materials
A modified Eagle's minimum essential medium (MEM) was
prepared as described previously (Chapter 3) . Materials used in
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) were described previously (Chapter 3). L-[4,5-3H]
leucine (SA,-150 Ci/mmol) , [ 5,6,8,11,12,14,15-3H] PGF2a (SA,~180
Ci/mmole) and [5,6,8,12,14,15-3H] PGE2 (SA,~170 Ci/mmole) were
purchased from Amersham Corp. (Arlington Heights, IL) . Radio¬
inert PGF2a, PGE2, phenylmethylsulfonylfluoride (PMSF) , and
arachidonic acid were purchased from Sigma (St. Louis, MO).
Rabbit antiserum to PGF2a was kindly provided by Dr. T. G. Kennedy
of the University of Western Ontario. Sheep antiserum to PGE2 was
provided by N. R. Mason of the Eli Lilly Research Laboratories,
Indianapolis, IN. CHAPS,3-[3-cholamidopropyldimethylammonio]-
1-propane-sulfonate, was from Calbiochem (La Jolla, CA) .
Animals
Uteri of Suffolk ewes were unilaterally ligated before
mating to isolate the fetal-placental unit to one horn and result
in the occurrence of a gravid and a non-gravid uterine horn
within each animal (Bazer et al., 1979). Ewes were hysterec¬
tomized at either Day 100 (n = 3) or Day 140 (n = 3) of gestation;
tissues were harvested immediately and prepared for explant
culture. Owing to limited amounts of tissue in the non-gravid
horn at Day 100, only tissues from the gravid horn were collected
at this time. Maternal intercaruncular and caruncular uterine

180
endometrium, fetal chorioallantoic membrane and fetal cotyledon
were harvested. Cotyledonary tissue represented that portion of
the placentome which was of fetal origin and the cotyledonary and
caruncular preparations were slightly cross-contaminated owing
to difficulty in complete separation of microvillous attach¬
ments. Other tissues collected from these same animals were
analyzed for protein secretion and results were reported by
Stephenson et al., 1989.
Explant Culture
Tissue explants (500 mg) were placed in sterile plastic 100
mm Petri dishes and cultured in 15 ml of modified MEM under an
atmosphere of 50% N2, 45% 02 and 5% C02 (v/v/v) . Cultures were
maintained in the dark on rocking platforms. Cultures of each
tissue type listed above were prepared in quadruplicate from each
ewe and incubated in the presence of either 200 nq arachidonic
acid (n = 2) , to examine prostaglandin secretion, or 50 /LtCi
[3H] leucine (n = 2) , to measure de novo synthesis and secretion
of protein. Cultures were incubated for 6 h at 39°C followed by
treatment for 18 h at either 39°C or 42°C. After incubation,
cultures were stopped by centrifugation (700 x g, 30 min at 4°C) ,
and tissues were solubilized in 50 raM Tris-HCl, pH 7.6, which
contained 1 mM PMSF, 1 mM EDTA and 2% (w/v) CHAPS. Tissue
homogenates and culture supernatants were stored at -20°C until
analyzed.

181
Measurement of PGs
Culture supernatants were assayed for PGF2a using a radio¬
immunoassay procedure (Gross et al., 1988c) modified for use in
unextracted MEM with an antibody characterized by Kennedy (1985)
that recognized F series PGs. Standard curves were prepared in
unextracted MEM. Inhibition curves prepared from serially dilu¬
ted samples of MEM conditioned by each tissue type were found to
be parallel to the standard curve. Recovery of radioinert PGF
added to unextracted, conditioned MEM averaged 90 percent. All
samples were run in a single assay. Within-assay coefficient of
variation was 8.5 percent.
Supernatants were assayed for PGE2 using the radioimmuno¬
assay procedure of Lewis et al. (1978) modified as described by
Gross et al. (1988c). Standard curves were prepared in unex¬
tracted MEM as described and inhibition curves prepared from
serially diluted samples of conditioned MEM from each tissue type
were found to be parallel to the standard curve. Recovery of
radioinert PGE2 added to unextracted, conditioned MEM averaged
93 percent. All samples were run in a single assay having a
within-assay coefficient of variation of 9.4 percent.
Protein Synthesis and Secretion
The secretion and tissue accumulation of macromolecules
synthesized in culture was determined by TCA precipitation as
described previously (Chapter 3).

182
Electrophoresis
One-dimensional polyacrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate (SDS-PAGE) was performed
using the buffer system of Laemmli (1970) . Tissue proteins were
prepared in a solubilization buffer containing 62.5 mM Tris, pH
6.8, 5% (w/v) SDS, 10% (w/v) sucrose and 5% (v/v) 2-mercapto-
ethanol and resolved on 12.5% (w/v) polyacrylamide gels in the
presence of .05% (w/v) SDS. Measurement of [3H]leucine incor¬
porated into macromolecules in solubilized tissue was done by TCA
precipitation and equal amounts of radiolabeled tissue proteins
were loaded onto polyacrylamide gels to examine qualitative
differences. Proteins were fixed in the gels using acetic acid,
ethanol and water (7:40:53, v/v/v) . The gels were soaked in 1 M
sodium salicylate, dried and fluorographs prepared as described
by Roberts et al. (1984) .
Statistical Analysis
Data were analyzed by least-squares analysis of variance
using the General Linear Models procedure of the Statistical
Analysis System (SAS, 1985) . Models used to analyze endometrial
and placental data included main effects of stage of gestation,
animal nested within stage of gestation, tissue type, incubation
temperature and, for data from Day 140 endometrial tissue, effect
of side of the uterus (i.e. , pregnant versus nonpregnant horn) .
Data were analyzed in three ways; in model 1, all data from the
gravid uterine horns were examined. Model 2 was similar to model
1 except that data from both uterine horns were included. Model

183
3 was used for uterine tissues at Day 140 only and included the
gravid versus nongravid comparison. Probability values are
shown in Table 6-1.
Results
Prostaglandins
All tissues produced greater amounts of PGE2 than PGF at Day
100 and Day 140 (Figures 6-1 and 6-2). Overall, PGE2 release from
tissues was affected by heat shock (P < 0.03 all tissues; P < 0.06
for gravid tissues only) and maternal tissues were more affected
than fetal tissues (temperature x tissue type interaction for
combined data, P < 0.03) . For maternal tissues, PGE2 was reduced
by elevated temperature in all cases except for caruncular tissue
from the gravid horn collected at Day 140. There were no
consistent effects of temperature on PGE2 release by fetal
tissues or any significant effects of elevated temperature or
interactions with temperature upon PGF release.
For PGF and PGE2 secretion by maternal and fetal tissues from
the gravid horn, day of gestation x tissue type interactions (P
< 0.06 for PGE2; P < 0.04 for PGF) were observed. At Day 100,
tissues from the maternal unit (intercaruncular and caruncular
endometrium) secreted more PGs than did tissues of the fetal unit
(the chorioallantois and cotyledon). Between Day 100 and Day
14 0, there was a large increase in fetal PGE2 secretion so that
at Day 140, PG secretion from the fetal tissues was greater than
at Day 100 and fetal PGE2 release at Day 140 slightly exceeded

184
Table 6-1. Probability values for effects of individual sources
of variation upon secretion of PGE2, PGF and [3H] labeled protein
and on accumulation of tissue protein.
Protein
PGE2 PGF Secretion Tissue
1 2
3
1
2
3
1
2
3
1
2
3
Effect
Day3
NS NS
—
NS
NS
—
NS
NS
—
NS
NS
—
Anim (Day)
.01 .01
-
.01
. 01
-
NS
. 02
-
. 05
.01
-
Tissue6
NS .08
.05
.01
. 01
.07
. 01
. 01
. 01
.02
.01
NS
Day x Tissue6
. 06 NS
-
. 04
NS
-
NS
NS
-
NS
NS
-
Tissue x Anim
.01 .01
-
NS
.01
-
NS
NS
-
NS
NS
-
(Day)
Temp
.06 .03
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Day x Tempc
. 07 NS
-
NS
NS
-
NS
NS
-
NS
NS
-
Temp x Anim
NS NS
-
NS
. 07
-
NS
NS
—
NS
NS
—
(Day)
Tissue x Temp
NS .03
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Tissue x Temp
NS NS
-
NS
NS
-
NS
NS
-
NS
NS
-
x Day
Side
NS
- . 07
-
- . 06
-
- . 06
Tissue x Side
- . 09
- . 07
-
NS
-
- . 09
Temp x Side
NS
NS
-
NS
-
NS
Tissue x Temp
NS
NS
-
NS
-
NS
x Side
Note: Model 1. All data from the gravid uterine horns were
examined.
Model 2. All data from both uterine horns were analyzed.
Effects of side were not included because this
effect was represented for only tissues at one
day of pregnancy.
Model 3. Uterine data at day 140 were analyzed with side
as a main effect. Animal was included in the
model.
NS = P > 0.10.
- = effect not included in the model.
aError term in F test = Anim (Day) .
‘’Error term in F test = Tissue x Anim (Day) .
cError term in F test = Temp x Anim (Day) .

185
that of intercaruncular endometrium and greatly exceeded that
of caruncular endometrium (Figure 6-1) .
For maternal tissues at Day 140, secretion of PGF was
affected by a tissue type x side interaction (P < 0.07). For
tissues from the gravid horn, PGF release was slightly greater
for intercaruncular tissue than caruncular tissue. For the
non-gravid horn, secretion was much greater for caruncular
tissue. Similar results were seen for PGE2 secretion (P < 0.09)
(Figure 6-3).
Protein
For tissues of the gravid uterine horn, incorporation of
radiolabeled leucine into newly synthesized macromolecules
present in culture supernatants and in tissue homogenates
differed (P < 0.01 for supernatants; P< 0.02 for tissue) between
tissue types (Figures 6-4 and 6-5), with maternal intercarun¬
cular endometrium producing the greatest amounts of radiolabeled
macromolecules. There was no effect of day of gestation,
temperature, or any interactions on the synthesis and secretion
of radiolabeled macromolecules by tissue explants from the
gravid horn.
For maternal tissues at Day 14 0, elevated temperature did
not affect the synthesis and secretion of [3H]leucine-labeled
macromolecules (Figures 6-4 and 6-5) . Overall, intercaruncular
endometrium secreted more radiolabeled macromolecules (P< 0.01)
than did caruncular endometrium (Figure 6-4) . Incorporation of

Intercaruncular Endometrium
Day 100
Day 140
an
VvvVwvvl
in *
ÜÜ
infera
nn mu
HI
â–  â– 
i
11!
I
39 42 39 42
Car uncu lar Endometrium
39 42 39 42
186
Figure 6-1. Release of PGE2 from ovine tissue explants of
maternal (intercaruncular and caruncular endometrium) and fetal
(cotyledon and chorioallantois) tissues collected at Day 100 or
Day 140 of pregnancy. Data are expressed as means + sem.

Intercaruncular Endometrium
Caruncular Endometrium
187
Chorioallantois Feta! Cotyledon
Figure 6-2. Release of PGF from ovine tissue explants of
maternal (intercaruncular and caruncular endometrium) and fetal
(cotyledon and chorioallantois) tissues collected at Day 100 or
Day 140 of pregnancy. Data are expressed as means + sem.

Intercar un cu lar Endometrium
Caruncular Endometrium
188
Non gravid
1
39 42
40
30
20
10
0
Intercaruncular Endometrium
Caruncular Endometrium
Figure 6-3. Release of PGE2 (upper panels) and PGF (lower panels)
from ovine tissue explants of maternal tissues from the nongravid
and gravid uterine horns collected at Day 140 of pregnancy. Data
are expressed as means + sem. Note different scales of y-axes.

189
[3H] leucine into macromolecules present in culture supernatants
(P < 0.06) and tissue homogenates (P < 0.06) was greater for
tissues from the non-gravid horn than for gravid tissues (Figure
6-6). Effects of side on incorporation of [3H] leucine into
macromolecules in culture medium and tissue were more pronounced
for caruncular tissue than intercaruncular tissue (tissue type
x side interaction, P < 0.09).
Representative fluorographs of tissue proteins as
resolved by SDS-PAGE are shown in Figure 6-7. Several major
differences among the tissue types were evident including
enhanced production of a protein of 28,000 apparent molecular
weight in cotyledonary tissue (arrow 4 in lanes 5 and 6) and of
a group of proteins ranging from 34,000 to 43,000 molecular
weight in chorioallantoic tissue (arrow 3 in lanes 7 and 8).
Heat-shocked tissue contains bands of radiolabeled proteins at
93,000 and 72,000 molecular weight (see arrows 1 and 2) that were
not present in tissues incubated at 39 °C.
Discussion
In the present study, advancing gestation was accompanied
by an increase in PG secretion, primarily of fetal origin. These
results are in agreement with studies in sheep using in vitro

Intercaruncular Endometrium
«000
6000
4000
3000
2000
1000
39 42 39 42
Caruncular Endometrium
Chorioallantois
Fetal Cotyledon
Figure 6-4. Secretion of [3H]leucine-labeled macromolecules
from ovine tissue explants of maternal (intercaruncular and
caruncular endometrium) and fetal (cotyledon and chorioallan¬
tois) tissues collected at Day 100 or Day 140 of pregnancy. Data
are expressed as means + sem.

Intercaruncular Endometrium
Caruncular Endometrium
Chorioallantois Feta! Cotyledon
Figure 6-5. Incorporation of [3H]leucine into macromolecules
present in tissue homogenates of ovine tissue explants of
maternal (intercaruncular and caruncular endometrium) and fetal
(cotyledon and chorioallantois) tissues collected at Day 100 or
Day 140 of pregnancy. Data are expressed as means + sem.

Intercaruncular Endometrium
Caruncular Endometrium
192
Intercaruncular Endometrium Caruncular Endometrium
Figure 6-6. Secretion (upper panels) and tissue incorporation
(lower panels) of [3H] leucine-labeled macromolecules from ovine
tissue explants of maternal tissues from the nongravid and gravid
uterine horns collected at Day 140 of pregnancy. Data are
expressed as means + sem.

193
(Evans et al., 1982; Olsen et al., 1984) and in vivo techniques
(Evans et al. , 1981; Olsen et al. , 1986) . Prostaglandin produc¬
tion during the period before onset of labor is believed to be an
important component in the onset of parturition (Thorburn and
Challis, 1979) . Animals in the present study had not reached the
stage, immediately before parturition, in which fetal adrenal
activity results in increased PGF release. PGE2 also plays
important roles in vasodilation (Ford, 1982) and in maintaining
patency of the ductus arteriosus of the fetal lamb (Challis et
al., 1976). The production of PGs by cultured tissues was
extensive and microgram quantities were released in 24 h. While
this was surprising, Waterman and Lewis (1989) examined the
incorporation of [3H] arachidonate into lipids by ovine chorioal¬
lantoic membranes collected early in pregnancy and found that
precursor was metabolized into PGs in quantities similar to the
present experiment. Adding excess precursor (arachidonic acid)
to the explant preparations likely accounted for this high
production through bypassing regulation of precursor availabili¬
ty by phospholipase A2 activity.
Data of the present experiment support the concept that
changes in PG secretion resulting from elevated temperature
account for a portion of the reduced blood flow to the uterus and
placenta seen in heat-stressed ewes (Oakes et al. , 1976; Bell et
al., 1987 ; Alexander et al. , 1987). For most all cases, maternal
production of PGE2 was reduced by elevated temperature in vitro.

194
Endometrium Caruncle
39 42 39 42
Cotyledon Chorioallantois
39 42 39 42
1 2 3 4 5 6 7 8
Figure 6-7. Representative fluorograph of [3H]leucine-labeled
macromolecules resolved by 1-D SDS PAGE. Equal amounts of
radiolabeled macromolecules were loaded onto 12.5% gels to
determine qualitative differences between tissue types and their
responses to elevated temperature. Several major differences
among the tissue types were evident including enhanced pro¬
duction of a protein of 28,000 Mr in cotyledonary tissue (lanes
5 and 6; see arrow 4) and a group of proteins of 34,000 to 43,000
Mr in chorioallantoic tissue (lanes 7 and 8; see arrow 3).
Appearance in heat-shocked tissue of bands of radiolabeled
protein at 93,000 and 72,000 Mr (see arrows 1-2) that were not
present in tissues incubated at 39°C can be seen by comparing
lanes 2,4,6 and 8 with lanes 1, 3, 5 and 7.

195
This decrease would be especially critical in the placentome
(cotyledon and caruncle) since Makowski et al. (1968a,b)
determined that 84% of uterine blood flow passed through the
caruncular regions of the uterus and that more than 90% of the
fetal umbilical arterial blood flow was through the cotyledonary
chorion. Interestingly, changes in PGE2 due to elevated tempera¬
ture were less in cotyledon and caruncle than in intercaruncular
endometrium. The PGE2-induced tone of the utero-placental
vasculature might be further reduced by heat stress-induced
alterations in estrogen production. The vasoregulatory activi¬
ties of PGs are affected by estrogen (Ford, 1982) and estrone
sulfate of placentomal origin was reduced in heat-stressed cows
(Collier et al. , 1982).
The PG release in response to heat shock in the present
experiment differs from that of bovine and porcine uterine
endometrium collected at the time of expected luteolysis. PGF
release by bovine endometrium in vitro was enhanced by heat shock
while PGE2 was unaffected (Chapter 5). Porcine uterine endo¬
metrium also altered its PG metabolism in response to elevated
temperature (Gross et al., 1989) resulting in elevated PGF
secretion and in perturbation of exocrine versus endocrine
secretion patterns. In particular, the increase in PGF was more
pronounced for secretion towards an endocrine route than for
secretion towards an exocrine route. In tissues collected at
estrus, PGF secretion by bovine endometrium was not affected by

196
elevated temperature (Chapter 2) . Apparently the tempera¬
ture-induced increase in PGF secretion by bovine endometrium
reported in Chapter 5 is not a characteristic response of all
tissues producing PGs, but varies with the physiological state
of the tissue.
Heat shock enhanced the appearance of 72,000 and 93,000
molecular weight proteins in all tissues examined. These
proteins appear to represent members of the groups of 70- and
90-kDa heat-shock proteins that have been well-documented in
other systems and which have been implicated in the development
of thermotolerance in many cell types (Nover, 1984; Mizzen and
Welch, 1988) . This suggests that tissues of the sheep uterus and
placenta, like uterine tissues of the cow (Chapters 2, 3, 5) , can
also respond to stress in this manner. While the response to
heat shock, i.e., production of heat-shock proteins, was as
expected, there was little effect of temperature on de novo
synthesis and secretion of macromolecules by tissue explants.
This finding was similar to previous experiments with bovine
endometrium collected at Days 2, 5, and 8 of the estrous cycle or
Day 17 of pregnancy in which a similar resistance to heat
shock-induced disruption of protein synthesis and secretion was
seen. Nonetheless, heat shock in vitro has been reported to
alter synthesis and secretion of specific uterine proteins
(Chapter 3) and it is possible that subtle changes occurred but
were not seen here due to the methods used. Additionally,

197
reduced uterine and placental blood flow could compromise
protein secretion in vivo.
In the present experiment, local presence of the conceptus
alter the function of caruncular tissue by reducing PGE2, PGF and
protein release and lowering de novo protein synthesis. A
possible explanation for differing responses of gravid and
nongravid horns is that disruption of local microenvironments by
physical separation of the placentomal tissues and resultant
tissue damage at dissection altered function of the tissues in
culture. Additionally, placentomal tissues from the gravid
uterine horn were cross-contaminated due to difficulty of
separation of microvillous and villous attachments. Nonethe¬
less, it is possible that local presence of the conceptus blocked
PG secretion by caruncular tissue. Uterine PG metabolism is
altered by the presence of the preimplantation conceptus around
the time of expected luteolysis in cattle, sheep, pigs and horses
(reviewed by Bazer et al., 1986). Inhibitors of PG synthesis
have been identified in bovine uterine endometrium (Gross et al. ,
1988c; Basu and Kindahl, 1987) , bovine placenta (Shemesh et al. ,
1984), rat placenta (Harrowing and Williams, 1979), in human
amniotic fluid (Saeed et al., 1982) and sheep allantoic fluid
(Rice et al. , 1987) . In at least one case (Gross et al. , 1988a,c;
Helmer et al., 1989a), the conceptus has been shown to enhance
the activity of the inhibitor.
In conclusion, reduction in uterine-placentomal blood
flow by heat stress could be due in part to direct effects of

198
elevated temperature on PG metabolism by maternal tissues.
Changes in PGE2 suggest that the increase in utero-placental PGE2
with increasing gestational age is due to increased fetal
production of PGE2. Fetal tissues exerted effects upon activi¬
ties of uterine endometrial tissue as revealed by differences
between the gravid and non-gravid horns.

CHAPTER 7
REGULATION OF HEAT SHOCK-INDUCED ALTERATIONS IN RELEASE
OF PROSTAGLANDINS BY UTERINE ENDOMETRIUM OF COWS
Introduction
As discussed previously (Chapter 5), one physiological
event that may be sensitive to heat stress is rescue of the
corpus luteum from luteolysis. Heat stress from 8 to 16 days
after insemination caused alteration of the uterine environment
(Geisert et al., 1988), reduced weight of corpora lútea and
impaired conceptus growth (Geisert et al. , 1988? Biggers et al. ,
1987). During this period, the conceptus secretes the anti-
luteolysin, bovine trophoblast protein-1 (bTP-1) (Helmer et al. ,
1987), a protein that causes suppression of uterine prosta¬
glandin (PG) F secretion resulting in luteal maintenance (Helmer
et al., 1989a,b; Thatcher et al., 1989). Depressed PGF secretion
by bTP-1 may be mediated by an inhibitor of PG synthesis that
appears in uterine endometrial cells during pregnancy (Gross et
al. , 1988c) and after exposure to bTP-1 (Helmer et al. , 1989a).
Secretion of bTP-1 was inhibited by in vitro heat stress (Chapter
5). In another study, conceptus weight was significantly reduced
when cows were exposed to elevated temperature from Day 8 to 16
of gestation, although no depression was observed for subsequent
embryonic secretion of bTP-1 during in vitro culture at 37°C
199

200
(Geisert et al. , 1988). Hyperthermia could also directly alter
PGF secretion. Basal and oxytocin-induced PGF secretion from
cultured uterine endometrium from cyclic and pregnant cows at Day
17 after estrus was increased by exposure to elevated temperature
(Chapter 5; Putney et al., 1988b; Putney et al., 1989b).
Further, nonpregnant cows or cows with retarded embryos at Day
17 after estrus displayed increased PGF secretion in response to
oxytocin during heat stress in vivo (Putney et al. , 1989b).
Increased PGF release is not a general response of PG-pro-
ducing tissues to heat shock since explants of bovine uterine
endometrium collected at estrus and explants of ovine uterine and
placental tissues collected during the third trimester of
pregnancy failed to respond in this fashion (Chapters 2 and 6) .
It remains unclear how heat stress acts to increase PGF secretion
of bovine endometrium at Day 17 after estrus in cyclic and
pregnant cows. One possibility is that there is a ther¬
mally-induced increase in reaction rate of arachidonic acid
metabolism to PGs. A second possibility, that increased
precursor (arachidonic acid) mobilization accounts for this
increase, is unlikely since production of PGE2 and PGF were
affected differentially by heat shock and release of PGF from
explants was sensitive to temperature even when excess arachi¬
donic acid was present in culture medium (Chapter 5) . A third
possibility for pregnant tissues is that heat shock results in
loss of functional responsiveness to regulators of uterine PGF

201
synthesis. The first and third possibilities were tested in the
present experiments. One regulator tested was the array of
proteins secreted by Day 17 bovine conceptuses (bovine conceptus
secretory proteins; bCSPs) , which have been shown to suppress PGF
secretion from cultured endometrium (Helmer et al. , 1989a; Gross
et al., 1988c). Another putative inhibitor tested was
platelet-activating factor (l-o-alkyl-2-acetyl-sn-glycer-
yl-3-phosphocholine) (PAF). Platelet-activating factor is a
potent mediator of inflammation which exerts effects through a
receptor-mediated signal transduction that apparently utilizes
cyclooxygenase and lipoxygenase products as second messengers
(Rola-Pleszczynski, 1988) and which has been shown to reduce PGF
secretion from cultured endometrial explants from cows at Day 17
of the estrous cycle (Gross et al., 1989). Finally, it was
tested whether the cytosolic inhibitor of cyclooxygenase found
in endometrium at Day 17 of pregnancy (Gross et al. , 1988c) loses
activity in response to elevated temperature.
Materials and Methods
Materials
A modified Eagle's minimum essential medium (MEM) was
prepared as described previously (Chapter 2) . All medium
components except insulin (Sigma, St. Louis, MO) were from GIBCO
(Grand Island, NY). [5,6,8,11,12,14,15-3H] PGF2 (SA,~180
Ci/mmole) and [5,6,8,12,14,15-3H]PGE2 (SA,~170 Ci/mmole) were

202
purchased from Amersham Corp. (Arlington Heights, IL) . Radio¬
inert PGE2, PGF2a, arachidonic acid and PAF were purchased from
Sigma. Rabbit antiserum to PGF2a was kindly provided by Dr. T.
G. Kennedy (University of Western Ontario) . Cross reactivity of
this antibody with other F series prostaglandins has been
reported (Gross et al., 1988c) and data in the present experi¬
ments are expressed as F series prostaglandins (PGF). Sheep
antiserum to PGE2 was provided by N. R. Mason (Eli Lilly Research
Laboratories, Indianapolis, IN).
Animals
Cyclic cows (Brangus) were slaughtered at Day 17 after
estrus and the reproductive tracts collected. Uterine horns
were opened anterior to the external bifurcation, intercaruncu-
lar endometrium was dissected free from myometrium, blotted on
sterile gauze, cut into 2-3 mm3 cubes and cultured as described
below. Endometrial tissue from cyclic cows rather than pregnant
cows was utilized in experiments with PAF and bCSPs to avoid
effects of previous in vivo exposure to secretory products of the
conceptus.
Additionally, two Brangus cows were slaughtered at Day 70
of pregnancy to examine effects of elevated temperature in vitro
upon PGF and PGE2 secretion by uterine and placental tissues.
Immediately after exsanguination, reproductive tracts were
transferred to the laboratory and samples of intercaruncular
uterine endometrium, caruncular endometrium, cotyledon and
chorioallantois were dissected free and placed in sterile

203
modified MEM. Explants were washed (2X) in sterile medium, cut
into 2-3 mm3 cubes and cultured as described.
Preparation of bCSPs
Bovine conceptus secretory proteins (bCSPs) were obtained
as described by Helmer et al. (1987). Cows were observed for
estrus, bred by natural service, and slaughtered at Day 17 after
estrus. Following exsanguination, reproductive tracts were
removed, transported to a laminar flow hood and trimmed free of
excess tissue. The cervix was clamped and a sterile syringe
fitted with a 16 ga needle used to flush 40 ml of sterile modified
MEM into the uterine lumen through the tubal-uterine junction of
the cornu contralateral to the corpus luteum. Conceptuses were
flushed through an opening at the anterior tip of the ipsilateral
cornu into a sterile 100 mm petri dish. Conceptuses were
transferred to a sterile 100 mm petri dish containing 15 ml
modified MEM and incubated for 24 h under an atmosphere of 50% N2,
47.5% 02 and 2.5% C02 (v/v/v) in the dark on rocking platforms.
Incubation was terminated by removal of conceptuses from
modified MEM. Medium was centrifuged at 12,000 x g for 10 min.
Supernatant fractions from several cultures were pooled and
dialyzed (Mr cutoff = 1000) against modified MEM. Protein
content of conceptus-conditioned, dialyzed culture medium was
determined by the method of Lowry et al. (1951) using BSA as a
standard. Medium was frozen at -20°C.

204
Explant Culture
Explants of intercaruncular uterine endometrium (250 mg)
were placed in sterile plastic 100 mm Petri dishes and cultured
in 8.5 ml of modified MEM under an atmosphere of 50% N2, 45% 02
and 5% C02 (v/v/v). Cultures were maintained in the dark on
rocking platforms. Ten explant cultures were prepared from each
of three cows for examination of the effects of PAF and bCSP. All
cultures contained 100 /¿g arachidonic acid. Stock solution of
PAF (10 jig/ml) was prepared in modified MEM that additionally
contained 10 /¿g/nil BSA. The bCSP stock was prepared at a
concentration of 800 nq protein/ml modified MEM. Duplicate
cultures were prepared with one of five treatments: 1) no
treatment, 2) 2.5 [iq PAF, 3) 5 PAF, 4) 10 nq PAF, or 5) 800 [iq
bCSPs. All cultures were modified to contain the same BSA
concentration (10 vq/culture). Cultures were incubated for 6 h
at 39°C (homeothermic temperature) followed by a 6 h period at
either 39 °C or 42 °C (heat shock) . Cultures were stopped by the
separation of tissue and medium during centrifugation (700 x g;
30 min; 4°C). Culture supernatants were stored at -20°C until
analyzed for PGF and PGE2 by radioimmunoassay.
Explants of intercaruncular and caruncular endometrium,
cotyledon and chorioallantois (500 mg) collected from cows at
Day 70 of pregnancy were placed in sterile plastic 100 mm Petri
dishes and cultured in 15 ml of modified MEM under an atmosphere
of 50% N2, 45% 02 and 5% C02 (v/v/v) . Cultures were maintained in
the dark on rocking platforms. Duplicate cultures of each tissue

205
type were prepared from each cow for examination of the effects
of elevated temperature on PGF and PGE2 secretion. All cultures
contained 200 [iq arachidonic acid. Cultures were incubated for
6 h at 39 0 C followed by an 18 h period at either 39°C or 42°C.
Cultures were stopped and supernatants stored at -20°C as
described earlier.
Cell-Free System
To examine effects of elevated incubation temperature upon
the activity of the cyclooxygenase-endoperoxidase enzyme complex
and the prostaglandin synthesis inhibitor found in the Day 17
pregnant bovine endometrium, a microsomal preparation of term
cotyledon was prepared as described by Gross et al. (1988c).
This preparation, called the cotyledonary PG-generating system,
is rich in cyclooxygenase-endoperoxidase enzyme complex and is
capable of converting arachidonic acid to PGs. Periparturient
bovine cotyledons were collected within 1 h of parturition and
placed in sterile saline. Tissue (20 g/40 ml) was added to .05
M Tris-buffered .25 M sucrose, pH 7.4, and homogenized. Homoge¬
nates were centrifuged (800 x g; 15 min? 4°C) and filtered
through cheesecloth. Filtrates were centrifuged (9000 x g; 15
min; 4°C) to remove nuclei and mitochondria; supernatants were
then centrifuged (100,000 xg; 60 min; 4°C) to yield a microsomal
fraction and a high-speed cytosolic supernatant. Microsomes
were suspended in . 1 M potassium phosphate, pH 7.5, and stored
in aliquots at -70°C.

206
The cytosolic inhibitor of PG synthesis was prepared from
bovine endometrial tissue collected at Day 17 of pregnancy.
Tissue (10 g/20 ml) was homogenized and subjected to centrifuga¬
tion as described for cotyledonary tissue above to obtain a
high-speed cytosolic supernatant. The cytosolic supernatant,
previously demonstrated to contain an inhibitor of endometrial
PG synthesis (Gross et al., 1988c), was stored in aliquots at
-7 O’C.
Cotyledonary microsomes were incubated for 60 min at 1)
39 °C, 2) 39 °C in the presence of the high-speed supernatant of
Day 17 endometrial tissue from pregnant cows, 3) at 42 °C, 4) at
42 °C in the presence of the high-speed supernatant, 5) at 39 °C
with the high-speed supernatant which had been preincubated at
42 °C for 30 min, or 6) at 42 °C with high-speed supernatant
preincubated at 42 "C for 30 min. Each treatment was performed in
triplicate using cotyledonary microsomes (500 mg tissue equiva¬
lent; 0.5 ml in 0.1 M potassium phosphate, pH 7.5) with and
without endometrial high-speed supernatant (250 mg tissue
equivalent; .5 ml) and 100 /ig arachidonic acid (.1 ml) at final
volume of 2 ml (.1 M potassium phosphate) as described by Gross
et al. (1988c). The incubations were terminated by addition of
.25 ml absolute ethanol and the samples centrifuged to remove
ethanol-precipitable material. Supernatants were assayed for
PGF.

207
Measurement of Prostaglandins
Culture supernatants were assayed for PGF and PGE2 using
radioimmunoassay procedures described previously (Gross et al. ,
1988c). The assay for PGF2a used an antibody characterized by
Kennedy (1985) that also recognized PGF1a. Data are therefore
reported as PGF.
Statistical Analysis
Data were analyzed by least-squares analysis of variance
using the General Linear Models procedure of the Statistical
Analysis System (SAS, 1985). The models used for analysis of
PAF and bCSP effects upon PG secretion by Day 17 endometrium
included cow, bCSP treatment or PAF dose, incubation temperature
and interactions. Analysis of endometrial PG synthesis inhibi¬
tor data included effects of treatment, incubation temperature
and the interaction. The model used to analyze effects of
elevated temperature upon tissues of cows collected at Day 7 0 of
pregnancy included cow, tissue type, temperature and interac¬
tions .
Results
Temperature Response of Day 17 Endometrium to bCSP and PAF
Overall, explants produced greater quantities of PGF than
PGE2 (Figure 7-1). Elevated incubation temperature resulted in
a significant increase in PGF secretion (P < 0.02) while not
affecting PGE2 secretion. Addition of bCSPs at 800 /ig/culture
resulted in a significant suppression of PGF release (P < 0.01)

208
at both 39°C (631 + 188 versus 134 + 51 ng PGF/g tissue/ 24 h) and
42 *C (813 ± 88 versus 322 + 170 ng PGF/g tissue/24 h) . There was
no treatment x temperature interaction (P > 0.10) and the
magnitude of the bCSP-induced decrease was similar at 39 °C (-497
ng) and 42 °C (-491 ng) . PGE2 secretion was not measured in bCSP
cultures.
PAF reduced the secretion of PGF (P < 0.02) and had no effect
on PGE2 at 39 °C and 42 °C (Figure 7-1) . There was a significant
treatment x temperature interaction (P < 0.02) for PGF. Elevated
temperature shifted the dose-response to PAF to the left (Figure
7-2) . At 42 °C, inhibition of PGF release was maximal at 2.5 fig
PAF and higher doses of PAF did not increase the inhibition. In
contrast, at 39°C maximal inhibition similar to that seen at 42°C
did not occur until 5 to 10 fig PAF.
Cell-Free System
Production of PGF by the microsomal prostaglandin-gener¬
ating system prepared from periparturient cotyledon was reduced
by 23 percent at elevated temperature (P < 0.01; Table 7-1).
Addition of the high-speed cytosolic supernatant prepared from
Day 17 pregnant endometrium depressed (P < 0.01) the PG gener¬
ating system at both 39°C and 42°C. The cytosolic inhibitor
depressed PGF production to a greater degree when the cotyledo¬
nary generating system was incubated at 42°C. This resulted in
a significant treatment x temperature interaction (P < 0.01).

209
0 2.5 5.0 10 0 2.5 5.0 10 0 2.5 5.0 10 0 2.5 5.0 10
PAF (ug)
Figure 7-1. Effect of PAF and incubation temperature on secre¬
tion of PGF (panel A) and PGE2 (panel B) by endometrial explants.
Data are expressed as means + sem. Elevated temperature in¬
creased PGF release (P < .02) but not PGE2 secretion. PAF reduced
the release of PGF (P < 0.02) at 39°C and 42°C. There was a
significant treatment x temperature interaction (P < 0.02) for
PGF.

210
Table 7-1. Prostaglandin F production from cotyledonary prosta¬
glandin generating system incubated at 39 °C or 42°C in presence
or absence of endometrial prostaglandin synthesis inhibitor.
PGF synthesis
(ng/90 min)
Inhibitory activity
(% reduction in PGF)
Treatment
39 ° C 42 ° C
39 ° C
42 °C
no inhibitor 8
.5 ± 0.1 6.6 ± 0.1
-
-
+ inhibitor 5
.3 ± 0.1 4.3 ± 0.2
38.4 ± 3
34.8 ± 6
+ heat-treated 6
inhibitor
.0 ± 0.1 5.1 ± 0.1
29.7 ± 4
22.7 ± 4
Note: Data are expressed as means ±
affected by temperature (P < 0.01),
treatment x temperature (P < 0.01).
sem. PGF
treatment
synthesis
(P < 0.01)
was
and
Table 7-2. Release of PGF and PGE2 by uterine and placental
tissues collected at Day 70 of pregnancy and incubated at 39 °C or
42 ° C.
PGF
(ng/g tissue)
pge2
(ng/g tissue)
Tissue
39 0 C 42 ° C
39 ° C
42 °C
Intercaruncular
endometrium
745 750
± 108 ± 272
94
± 37
87
± 13
Caruncular
endometrium
1,284 1,112
±1,091 ± 860
266
±229
123
± 67
Cotyledon
1,253 851
± 813 ± 98
159
±102
95
± 50
Chorioallantois
831 722
± 680 ± 95
101
± 33
88
± 49
Note: Data are expressed as means ± sem.

211
Figure 7-2. Dose-response of PGF secretion at 39 °C and 42 °C to
increasing concentrations of PAF. Data are expressed as
inhibition of PGF release to demonstrate the significant
treatment x temperature interaction (P < 0.02) that occurred
because elevated temperature shifted the response to the left.

212
Tissues at Day 70 of Gestation
Release of PGF and PGE2 by uterine and placental tissues at
Day 70 of pregnancy was not affected by elevated temperature
(Table 7-2) . There was a tendency for release of PGF to differ
among tissue type (P < .09) as secretion tended to be greatest
from cotyledon and caruncular tissue explants.
Discussion
Our hypothesis that elevated temperature would cause extra¬
cellular regulatory molecules to lose their capacity to suppress
endometrial PGF release was not borne out. Bovine conceptus
secretory proteins suppressed PGF in a similar fashion at both
39°C and 42°C (i.e., there was no treatment x temperature
interaction) . Additionally, elevated temperature increased the
ability of the bioactive ether-lipid PAF to suppress endometrial
PG secretion since the dose-response relationship was shifted
leftwards at elevated incubation temperature. The results with
PAF indicate that PAF can only partially inhibit PGF (at satura¬
tion by - 25 percent) and that, since PAF is not a protein,
elevated temperature did not cause denaturation but likely
decreased the equilibrium dissociation constant of PAF with its
receptor to reduce the maximum effective dose. Elevated
temperature decreased the activity of the endometrial inhibitor
of PG synthesis present at Day 17 of pregnancy. Putney et al.
(1989b) found similar results when endometrium was subjected to
elevated temperature and the isolated inhibitor tested for

213
activity. Reduced function of this proteinaceous inhibitor of
cyclooxygenase (Gross et al. , 1988c) might account for increased
PGF secretion in pregnant animals but cannot account for
heat-induced PGF release from endometrium of cyclic animals.
Temperature-induced increase of PGF secretion by endometrium was
also likely not due to an increased reaction rate of cyclo¬
oxygenase activity because release of PGF from a cell-free
preparation of cyclooxygenase-endoperoxidase enzyme complex
from periparturient cotyledon was depressed by elevated tempera¬
ture. In fact, results indicate that the temperature-induced
increase in PGF secretion at Day 17 after estrus may occur in the
face of potential partial denaturation of components of the
enzyme complex.
As was found previously (Chapter 5; Putney et al., 1988b,
1989b), elevated temperature did not increase PGE2 secretion,
indicating that elevated temperature affects PG secretion in
some manner specific for PGF. At Day 17, most PGF is released
from the endometrial epithelium while most of the PGE2 originates
in the stroma (Fortier et al. , 1988) . Perhaps high temperature
affects these two cultures differently or preferentially
enhances the activity of endoperoxide F reductase. The increase
in PGF secretion due to elevated temperature did not occur
because of increased 9-keto reductase activity converting PGE2
to PGF (Yamamoto, 1983) because PGE2 secretion did not decrease
upon exposure of explants to 42 °C.

214
As alluded to earlier, temperature-induced secretion of PGF
is not a common response of PG-producing tissues. It remains
unclear how elevated temperature induces an increase in PGF
release from the uterine endometrium at Day 17 of pregnancy, but
not in bovine endometrium at estrus or in uterine and placental
tissues of late pregnant sheep (Chapters 2 and 6) . Additionally,
in the present experiment, PGF secretion by bovine endometrial
and fetal-placental tissues collected from two cows at Day 70 of
pregnancy was not enhanced by elevated temperature.
One possible mechanism for heat-induced PGF release at Day
17 could involve effects on phosphoinositide turnover. Heat
shock alters membrane fluidity (Lee and Chapman, 1987) and
stimulates phosphoinositide turnover directly (Calderwood et
al. , 1987) . Because oxytocin may act to increase endometrial PGF
secretion at Day 17 via increased phosphoinositide turnover
(Flint et al. , 1986) , a heat-induced increase in turnover might
also result in PGF secretion. Data of Putney et al. (1989a)
indicate that heat shock enhances actions of oxytocin on
endometrium of cyclic and pregnant cows at Day 17, perhaps
through alteration of phosphoinositides. Another underlying
cause for the unique response of Day 17 bovine endometrium to
heat shock may be associated with membrane composition. Curl
(1988) found an increase in total phospholipid content and
phosphatidylcholine content in endometrium between Days 17 and
19 after estrus. Some changes in endometrium such as increased
phosphatidylcholine content, indicate a progressive increase in

215
fluidity and permeability as more bulky polar head-groups reduce
close packing in the membrane bilayer (Lee and Chapman, 1987).
Further, phospholipid composition of the membrane may itself
regulate cyclooxygenase activity since fatty acids influence
activity of the enzyme complex (Robak et al. , 1975).
In conclusion, elevated temperature induces secretion of
PGF from uterine endometrium collected at Day 17 after estrus.
Heat shock in vitro does not compromise capacity of extracellular
regulatory molecules involved in suppression of PGF release by
pregnant bovine endometrial tissue at Day 17 . Both proteinaceous
(bTP-1) and lipid (PAF) regulators retained their suppressive
activity at elevated temperature and PAF may have gained
inhibitory activity at 42°C. The regulatory capacity of the
inhibitor of PG synthesis from Day 17 pregnant endometrium was
depressed by heat treatment. The heat-induced increase in PGF
secretion is likely not due to increased cyclooxygenase enzyme
catalysis since heat shock reduced activity of a preparation of
the cyclooxygenase-endoperoxidase enzyme complex. Heat shock
may act to increase PGF secretion through activating phospha-
tidylinositol second messenger pathways.

CHAPTER 8
MODULATION BY ALANINE AND TAURINE OF HEAT SHOCK-INDUCED
KILLING OF BOVINE LYMPHOCYTES
AND MOUSE PREIMPLANTATION EMBRYOS
Introduction
Elevated ambient temperature often results in transient
infertility in mammals (Dutt, 1963; Elliott and Ulberg, 1971;
Ulberg and Sheean, 1973; Badinga et al., 1985). Significant
embryonic losses occur during the early cleavage stages of
preimplantation embryonic development (Dutt, 1963; Elliott and
Ulberg, 1971; Dunlap and Vincent, 1971; Ulberg and Sheean, 1973;
Putney et al., 1988a). Preimplantation embryos of domestic
animals are sensitive to hyperthermia beyond the blastocyst
stage as well: significant embryonic loss due to hyperthermia
have been reported between days 8 and 24 of pregnancy in the ewe
(Dutt, 1963; Dutt and Jabara, 1976), after day 13 in the cow
(Wise et al. , 1988) and between days 8 and 16 in the pig (Omtvedt
et al. , 1971) . Several possible reasons for hyperthermia induced
embryonic loss exist, including alterations of maternal-
conceptus interactions and direct thermal effects on the embryo
itself.
Heat shock-induced disruption of cell function involves
perturbation of multiple systems within the cell including
membrane fluidity (Quinn, 1981; Lee and Chapman, 1987; Lepock et
216

217
al. , 1983) , lipid peroxidation and formation of oxygen radicals
(Lee et al., 1983; Omar et al., 1987), second messenger system
function (Calderwood et al., 1987), including intracellular
calcium release (Calderwood et al., 1987; Landry et al., 1988),
enzymatic activity (Chousterman et al., 1987), protein
conformation and stability (Pain, 1987), RNA splicing (Yost and
Lindquist, 1986) and translation (Lindquist, 1981), and DNA
repair (Henle and Dethlefsen, 1978) .
Several chemical agents have been shown to exert beneficial
effects in reducing death of heat-shocked cells in vitro,
including glycerol (Henle and Warters, 1982), protease
inhibitors (Korbelik et al., 1988), reduced-glutathione
(Mitchell et al., 1983; Russo et al., 1984) and amino acids
(Vidair and Dewey, 1987; Henle et al., 1988). Among amino acids
that were shown to be effective were alanine, which exerted a
transient thermoprotective effective on CHO cells that was
independent of protein synthesis (Vidair and Dewey, 1987; Henle
etal. , 1988), and taurine (2-aminoethane-sulfonic acid) (Wright
et al. , 1986; Banks et al. , 1989) . Taurine is found in a variety
of tissues and is present in millimolar concentrations in certain
tissues, especially those rich in membranes, in exciteable
tissues and in tissues which generate oxidants (for review see
Wright et al. , 1986) and has been implicated in membrane stabili¬
zation, detoxification and antioxidant roles.
Identification of thermostabilizing agents offers the
opportunity to reduce effects of hyperthermia in vivo through

218
provision of these agents during critical periods of heat stress.
The first objective of the current experiments was to examine the
influence of several agents upon heat shock-induced killing of
bovine peripheral lymphocytes to determine the efficacy of
exogenous thermoprotective treatment of cells and determine
optimum concentrations of the agents. A second objective was to
test the efficacy of two of these agents, alanine and taurine,
for protection of preimplantation mouse embryos in vitro.
Materials and Methods
Materials
Materials used in cell culture were from GIBCO (Grand
Island, NY) or Sigma Chemical Co. (St. Louis, MO). Menezo B2
medium was from A.P.I. Systems, S.A. (Vercieu, France). Light
paraffin oil was from Fisher Scientific (Fair Lawn, NJ) and
Siliconol AR200 was purchased from Serva (New York, NY) . Iron-
supplemented calf serum was from Hyclone (Logan, UT). Mice,
C57BL/10J and ICR-random bred, were obtained from the Jackson
Laboratory (Bar Harbor, ME) and from Harlan Sprague-Dawley
(Indianapolis, IN), respectively. Follicle-stimulating hormone
(FSH) and human chorionic gonadotropin (hCG) were purchased from
Sigma or from Calbiochem (LaJolla, CA) . L-alanine, D,L-alanine,
taurine and reduced-glutathione were purchased from Sigma.

219
Bovine Peripheral Lymphocytes
Peripheral blood was collected by venipuncture from mature
cows. Lymphocytes were prepared by density gradient centri¬
fugation as described by Low and Hansen (1988). The final
concentration of lymphocytes was 1 x 106 cells /ml culture medium
(RPMI-1640 supplemented with 10% (v/v) iron-supplemented calf
serum, 100 IU/ml penicillin and 100 nq/ml streptomycin).
RPMI-1640 contains no alanine or taurine.
For each experiment, 1 x 105 cells in 100 /¿I volume were
plated into wells of sterile 96-well Falcon micro test II plates
(Becton-Dickinson, Oxnard CA) and cultured in 5% (v/v) C02.
Plates were prepared and test materials added as described for
each experiment. Test materials were prepared as concentrated
stocks in RPMI+FCS, sterile-filtered (.2 /¿m) and appropriate
volumes (usually 10 to 20 1) added to wells 5 to 10 min prior to
heat shock. All wells were brought to equal final volume of 110
to 120 /Ltl and the plates incubated in 5% (v/v) C02. Immediately
following incubation at appropriate temperatures and times (see
Results) , viability of lymphocytes was determined by trypan blue
exclusion.
Numbers of live and dead cells in each experiment were
analyzed with the CATMOD procedure of the Statistical Analysis
System (SAS, 1985) . Models used to analyze data included effects
of temperature, treatment, and their interaction. For time-
course studies, time and appropriate interactions were included
in the model.

220
Preimplantation Mouse Embryos
Prepuberal female mice (21 to 25 day-old C57BL/10J or
ICR-random bred) on a 14:10 light:dark cycle received FSH (5 IU,
intraperitoneally) , followed 48 h later by hCG (5 IU) . hCG was
given approximately 10 h before the midpoint of the dark cycle
and females were placed overnight with 6 to 10 week old C57BL/10J
males. Females were examined for the presence of a vaginal plug
on the following morning. It was assumed that the endogenous
surge of luteinizing hormone took place at the midpoint of the
dark cycle and that ovulation occurred 3 to 5 h later.
At approximately 63 (4 to 8 cell embryos) or 87 h after hCG
injection (16 cell embryos), females were killed by cervical
dislocation and their reproductive tracts removed. Oviducts and
uterine horns were flushed with sterile Whitten's medium
(Biggers et al., 1971) through a 30 ga needle into a sterile 35
mm petri dish. Embryos were collected with a drawn Pasteur
pipette and washed twice in fresh Whitten's medium which had been
pre-equilibrated to 38 ° C and 5% (v/v) C02. Embryos were cultured
in either Whitten ' s medium or Menezo B2 medium. Whitten' s medium
contains neither alanine nor taurine while Menezo B2 medium
contains 0.7 mM alanine and 0.1 mM taurine. Stock solutions of
media containing appropriate amino acid concentrations were
prepared and sterile-filtered in advance. Microdrops of medium
(5 Ml) were prepared under silicon oil or paraffin oil and
pre-equilibrated to temperature conditions and 5% C02. Embryos
were washed 2 X in appropriate culture medium and moved to

221
microdrops in as small a volume as possible to avoid dilution and
expansion of microdrops. Cultures were incubated under time and
temperature conditions described for each experiment (see
Results).
Embryos were examined immediately after heat shock and 24
h later for morphological state, viability and stage of develop¬
ment. Viability determination was by eosin B exclusion (Dooley
et al., 1987); embryos were placed into Whitten's medium
containing .25 mM eosin B for 15 min, washed and examined
immediately for uptake of the dye. Embryos were categorized as
eosin-negative (no stain in any cell) , eosin-partially positive
(one or more cells stained) or eosin-positive (totally stained;
indicative of a dead embryo). Embryonic morphology was
classified as either good or poor; criteria for classification
as good were equal size of blastomeres, no dark or pyknotic
nuclei, no fragmentation of blastomeres, and no extrusion of
cells from the embryo.
Numbers of embryos in each experiment were categorized by
viability, morphology and development and data analyzed with the
CATMOD procedure of the Statistical Analysis System (SAS, 1985).
Results
Lymphocytes
Experiment 1. In the first experiment, the efficacy of
several agents for enhancing survival of heat-shocked bovine
lymphocytes was tested. Duplicate wells of lymphocytes prepared

222
from 3 cows were supplemented with either 17 mM L-alanine, 17 mM
taurine, 2 % (v/v) glycerol, 10 mM reduced glutathione or an
equal volume of fresh culture medium. Plates were incubated for
1 h at either 38.5°C or 45°C and cell viabilities were deter¬
mined. Incubation at 45°C for 1 h reduced cell viability
(temperature, P < 0.05) ; less than 50% of cells were alive after
heat shock compared to 93% for control cells (Figure 8-1) .
Supplementation with L-alanine and taurine resulted in increased
survival of heat-shocked lymphocytes (temperature x treatment,
P < 0.01) to 89% (alanine) and 71% (taurine) . Addition of 10 mM
reduced-glutathione also increased cell survival (85%) while
addition of 2% (v/v) glycerol killed cells (1% survival).
Experiment 2. The dose-response of lymphocytes to L-ala-
nine and taurine was determined. Lymphocytes from 3 cows in
duplicate culture wells received several concentrations of L-
alanine (.8 to 45 mM) or taurine (1.25 to 17 mM) . To determine
whether response of the cells to alanine and taurine was caused
by changes in osmolarity, duplicate wells were treated with
various concentrations of NaCl (3.5 to 50 mM). Plates were
incubated for 1 h at either 38.5°C or 45°C and cell viabilities
determined.
The effects of alanine and taurine were dose-dependent
(temperature x dose, P < 0.01), with alanine being ineffective
at concentrations below 5 mM and maximally effective at 20 to 50
mM (Figure 8-2). Taurine was effective at concentrations over
2.5 mM (Figure 8-2) . Effects of alanine and taurine were not due

223
Percent Survival
100
37 C 45 C
Control
37 C 45 C
Alanine
37 C 45 C
Taurine
37 C 45 C
Glycerol
37 C 45 C
Reduced
Glutathione
Figure 8-1. Lymphocyte survival after 1 h heat shock at 45°C.
Survival was affected by temperature (P < 0.05). L-alanine,
taurine and reduced-glutathione all increased survival of heat-
shocked lymphocytes, temperature x treatment (P < 0.01).

224
Percent Survival After 1 Hr Heat Stress
Treatment (mM)
Figure 8-2. Dose-response effects of alanine and taurine.
Survival of lymphocytes during a 1 h heat shock was reduced at
L-alanine concentration below 5 mM and at taurine concentration
below 2.5 mM. NaCl concentration over a similar range exerted no
effect (P = .31) upon cell survival in culture.

225
simply to increased osmolarity because increasing NaCl
concentration exerted no effect (P = .31) upon cell survival in
culture (Figure 8-2).
Experiment 3. To examine whether the response of cells to
L-alanine was dependent upon stereospecificity of the amino
acid, triplicate wells of lymphocytes prepared from 1 cow were
supplemented with either 0, 17 or 45 mM D-alanine. Plates were
incubated for 2 h at either 38.5°C or 45°C and cell viabilities
determined. Supplementation of cells with D-alanine resulted
in increased survival (temperature x treatment, P < 0.01) during
incubation at 45°C (Figure 8-3) .
Experiment 4. To determine whether the effect of L-alanine
would diminish over extended heat shock, sixteen wells in each
of two plates were prepared in medium supplemented with 45 mM
L-alanine or in unsupplemented culture medium. Plates were then
incubated at either 38.5°C or 45°C. Cell viabilities were
determined at the beginning of heat shock (time 0) and at 1 h, 3
h, 4 h, 5 h, 6 h, 18 h, and 24 h later. The proportion of live
cells decreased with increasing exposure time to heat (time x
temperature, P < 0.02; Figure 8-4), there was no time x
temperature x treatment interaction however.
Preimplantation Embryos
Experiment 5. Embryos (n = 197) were collected at 63 to 87
h after hCG (4 to 32 cells) and cultured in Whitten's medium
under paraffin oil in 5% C02 at 38°C, 42°C or 45°C for 2 h in the
presence or absence of 50 mM L-alanine. Embryos were examined at

226
Percent Survival After 2 Hr Heat Stress
- D-Alanine
* D-Alanine
Figure 8-3. Effect of D-alanine upon lymphocyte survival.
Supplementation of medium with D-alanine during heat shock
resulted in increased survival (treatment x temperature inter¬
action, P < 0.01).

227
Percent Survival
Duration of Heat Shock
Figure 8-4. Effect of L-alanine upon lymphocyte survival during
prolonged heat shock in vitro. The proportion of live cells
decreased with increasing exposure time to heat (time x tempera¬
ture, P < 0.02) . The beneficial effect of alanine was lost when
cells were cultured for more than 6 to 9 h.

228
24 h after heat shock. Elevated temperature reduced (P < 0.01)
the proportion of embryos that reached the morula stage after 24
h, both at 42“C and 45°C (Figure 8-5) . Addition of 50 mM L-ala-
nine to cultures at 38 °C or 42 °C had no effect on development,
while addition of alanine to embryos at 45°C increased the
percentage of embryos reaching the morula stage (temperature x
treatment, P < 0.01).
Experiment 6. Embryos (n = 47) were collected at 63 h after
hCG (4 to 8-cell stage) and cultured in Whitten's medium under
paraffin oil in 5% C02 at 38°C, 42°C or 45°C for 2 h in the
presence or absence of 50 mM L-alanine. Embryos were examined at
0 and 24 h after heat shock. The number of embryos that reached
the morula stage after 24 h was reduced at 42 °C and 45 °C compared
to embryos cultured at 38°C (P < 0.06) (Figure 8-6). In
addition, heat shock at 42 °C and 45 °C reduced the viability of
embryos as reflected by reduced capacity to exclude eosin B at 24
h after heat shock (P < 0.07) . Heat shock also reduced the number
of embryos classified as having good morphology (P < 0.06) . The
presence of 50 mM L-alanine increased the number of embryos
reaching the morula stage (P < 0.06) at all temperatures.
Alanine supplementation at 45°C, but not at 42°C, increased the
number of embryos classified as eosin negative (temperature x
treatment, P < 0.07). In the presence of alanine, a greater
number of embryos were classified as having good morphology at
all temperatures (P < 0.09) .

229
Percent Embryos to Morula
100
80
60
40
20
38 C
42 C
45 C
Figure 8-5. Effect of heat shock and L-alanine upon mouse embryo
development in vitro. Elevated temperature reduced numbers of
embryos that reached the morula stage both at 42 °C and 4 5 °C (P <
0.01). 50 mM L-alanine at 42°C had no effect, while at 45°C
alanine exerted a beneficial effect improving the percentage of
embryos reaching the morula stage (P < 0.01).

230
De>«iopmant tn Culture
22» no davalopmant
- Aia • Ala
36 C 42 C
VIAbility Staining With EoMn B
nog oooin B atoinlng 233 P04 OOOln B «Ulnlng
12-â„¢"*
0 14 h 0 >4 A
• Aia * Ala • Aia - Ala • Ala
36 C 42 C 46 C
• Ala - Aia • Ala
46 C
Morphological Claaaiflcation
t _ ) good mofptiotogy 5^3 poo* morphology
Tima
0 34 h 0 «4 h 0 14 h 0 34 h 0 74 h
33 C 42 C 46 C
Figure 8-6. Effect of L-alanine upon development and viability
during heat shock in vitro. Heat shock impaired development (P
< 0.06), reduced viability of embryos (P < 0.08) and affected
morphology (P < 0.07) . The presence of 50 mM L-alanine increased
the number of embryos reaching the morula stage (P < 0.06) at
42 ° C and 45°C. Alanine supplementation at 45°C maintained
viability (P < 0.07) and improved embryo morphology (P < 0.09).

231
Experiment 7. Embryos (n = 445) were collected at 63 h
after hCG (4 to 8-cell stage) and cultured in Menezo B2 medium
under paraffin oil in 5% C02 at 38°C, 42°C or 45°C for 2 h in the
presence or absence of 50 mM L-alanine. Embryos were examined at
24 h after heat shock. Exposure to elevated temperature caused
developmental retardation (P < 0.01) and reduced embryonic
viability (P < 0.02) (Table 8-1) . Rate of embryonic development
was affected by alanine treatment (P < 0.01) but not by the
temperature x treatment interaction because D,L-alanine had a
positive effect on development at all temperatures. Viability
was affected by a treatment x temperature interaction (P < 0.03)
as alanine enhanced viability at 42 "C and 45 °C, but not at 38 °C.
Experiment 8. Embryos (n = 151) were collected at 63 h
after hCG (4 to 8-cell stage) . Embryos were cultured in Menezo
B2 medium under silicon oil for 2 h in 5% C02 at 38 °C without
supplementation and at 42 °C in the presence or absence of 50 mM
D,L-alanine, or 50 mM taurine. Embryos were examined at 24 h
after heat shock. Temperature effects were determined by
comparison of results at 38 "C and 42 °C without alanine supplemen¬
tation. Elevated temperature reduced embryonic development in
vitro (P < 0.02) (Table 8-2) ; nearly 50% of embryos at 42 °C were
degenerate and none reached the 16-cell stage. In contrast, only
12% of embryos at 38°C were degenerate and 81% reached the 16-
cell stage. Elevated temperature also altered embryonic
viability (P < 0.01) . All the embryos incubated at 38°C were

232
Table 8-1. Effects of alanine upon development and eosin B
staining of 4 to 8-cell embryos 24 h after heat shock.
Temperature
38 ° C 42 ° C
45 ° C
alanine alanine
- + - +
alanine
+
Development
Degenerate
11
10
28
10
22
11
15%
14%
38%
12%
31%
15%
4 to 8-cell
5
12
11
25
13
24
7%
16%
15%
31%
19%
32%
16-cell
7
0
17
14
10
13
10%
0%
23%
17%
14%
17%
Morula
38
29
18
32
25
27
27%
20%
12%
20%
18%
18%
Blastocyst
10
23
0
0
0
0
14%
31%
0%
0%
0%
0%
Eosin Stainina
Positive
1
4
22
8
24
8
2%
6%
34%
11%
36%
12%
Partial-pos.
19
17
30
40
35
43
31%
25%
46%
56%
53%
64%
Negative
42
47
13
24
7
16
68%
69%
20%
33%
11%
24%
Note: Data are expressed as number of embryos in each category
and as percent of total number of embryos in each treatment.
Exposure of 4 to 8-cell embryos to elevated temperature caused
developmental retardation (P < 0.01) and reduced embryo via¬
bility (P < 0.02).

233
classified as partially positive for eosin B staining (Table 8-
2) . In all cases, one or two cells were stained with the dye and
most developed to 16-cell stage overnight. Effects of D,L-
alanine and taurine were examined by comparing results at 42 °C
with alanine and 42 °C without alanine and by comparing 42 °C with
taurine and 42 °C without taurine. D,L-alanine had no beneficial
effects either on viability (P = .17) or on development (P = .53)
although several more embryos did develop in the presence of
alanine. Taurine in the culture medium caused more embryos to
develop after heat shock at 42“C (P < 0.01) but viability was not
affected (P = .2).
Embryos collected at 63 h after hCG and cultured in Menezo B2
medium under silicon oil in 5% C02 at 38°C overnight (n = 123)
were tested at the 16-cell stage for effects of heat shock and
amino acid supplementation. Embryos were cultured in Menezo B2
medium under silicon oil for 2 h in 5% C02 at 42 °C, or at 42 °C in
the presence of 50 mM D,L-alanine or at 42 °C in the presence of
50 mM taurine. Embryos were examined at 24 h after heat shock.
Taurine exerted positive effects on development (P < 0.01) and
viability (P < 0.01) of 16-cell embryos after heat shock (Table
8-3) . About 30% of embryos developed to morula-blastocyst stage
when incubated with taurine, whereas none developed in the
absence of amino acid supplementation. D,L-alanine also exerted
a beneficial effect on development of 16-cell embryos after heat
shock (P < 0.06) as about 25% of embryos incubated with alanine
developed to morula-blastocyst stage. All embryos treated with

234
Table 8-2. Effects of alanine and taurine on development and
eosin B staining of 4 to 8-cell embryos at 24 h after heat shock.
U>
CO
o
O
Temperature
42 ° C 42 ° C
42 'C
Development
Stage
No
Treatment
No
Treatment
+alanine
+taurine
Development
Degenerate
3
10
10
14
12%
47%
39%
29%
4 to 8-cell
2
17
15
7
8%
53%
34%
14%
16-cell
21
0
10
24
81%
0%
23%
49%
Morula-Blast.
0
0
2
4
0%
0%
5
8%
Eosin Stainina
Eosin pos.
0
5
7
10
0%
16%
16
20%
Eosin partial
26
17
18
17
100%
53%
41%
35%
Eosin negative
0
10
19
18
0%
31%
43%
37%
Note: Data are expressed as number of embryos in each category
and as percent of total number of embryos in each treatment.
Exposure of 4 to 8-cell embryos to elevated temperature caused
developmental retardation (P < 0.01) and reduced embryo via¬
bility (P < 0.02) .

235
taurine were eosin-negative or partially stained while nearly
50% of untreated embryos were eosin-positive at 24 h after heat
shock. Alanine had no positive effect on embryo viability
however (P = .15).
For comparison of interactions of temperature and treatment
with developmental stage at heat shock, embryos were classified
as degenerate, arrested or as having advanced. There was no
stage x treatment interaction for development, indicating no
difference in the response of 4 to 8-cell and 16-cell embryos to
treatment (Tables 8-2 and 8-3). Viability was affected by a
stage x treatment interaction (P < 0.01) because taurine was more
effective at increasing viability of 16-cell embryos than at the
4 to 8-cell stage.
Discussion
These results extend previous findings of a thermoprotec-
tive effect of alanine (Vidair and Dewey, 1987; Henle et al.,
1988) to bovine peripheral lymphocytes and preimplantation mouse
embryos. In addition, apparent thermoprotective effects of
taurine, which protects cells during oxidative stress (Wright et
al., 1986; Banks et al., 1989), have been demonstrated. Two
other agents reported in the literature to enhance cell survival
during heat shock were tested. Reduced-glutathione (Mitchell et
al. , 1983; Russo et al. , 1984) exerted thermoprotective effects
on bovine lymphocytes, however glycerol (Henle et al. , 1982) was
toxic.

236
Table 8-3. Effects
eosin B staining of
of alanine and
16-cell embryos
taurine on
24 h after
development and
heat shock.
Developmental
Stage
42 'C
-
+alanine
+taurine
Develooment
Degenerate
9
12
14
27%
27%
30%
16-cell
24
20
16
73%
45%
35%
Morula-Blast.
0
12
16
0%
27%
35%
Eosin Stainina
Eosin pos.
18
19
0
55%
43%
0%
Eosin partial
15
25
44
45%
57%
96%
Eosin neg.
0
0
2
0%
0%
4%
Note: Data are expressed as number of embryos in each category
and as percent of total number of embryos in each treatment.
Taurine exerted positive effects on viability (P < 0.01) and
development (P < 0.01) of 16-cell embryos after heat shock at
42 'C. D,L-alanine also exerted a beneficial effect on
development of 16-cell embryos after heat shock (P < 0.06).
Alanine had no positive effect on embryo viability however (P =
. 15) .

237
Thermoprotective effects of amino acids in the present
experiment were consistent over several tests of heat-induced
cell killing of bovine lymphocytes. Effect of alanine was
transient, concentration-dependent and not due to increased
osmolarity. The effect was also not dependent upon
stereospecificity of the amino acid as the L- and D-isomers were
equally effective.
As expected, elevated temperature at both 42°C and 45°C
resulted in impaired embryonic development and embryonic
viability in all experiments (Elliott and Ulberg, 1971; Ulberg
and Sheean, 1973). Alanine and taurine generally enhanced
embryonic development and viability. Nonetheless, heat-shock
effects were not offset completely, as development and viability
were never equal to responses at 38°C. At 42°C, 50 mM alanine
enhanced embryonic development compared to untreated embryos at
42 °C in 3 of 5 experiments. Alanine was able to enhance embryo
viability in 1 of 4 experiments. At 45°C, alanine enhanced
embryonic development in 2 of 3 experiments and maintained embryo
viability in 2 of 2 experiments compared to heat-stressed embryos
without alanine addition. Addition of 50 mM taurine to embryo
cultures incubated at 42°C resulted in improved development,
compared to untreated embryos at 42"C, in 2 of 2 experiments;
taurine enhanced viability in 1 of 2 experiments. Four to 8-cell
stage and 16-cell stage embryos in the present experiment did not
appear to differ in their response to hyperthermia.

238
Sensitivity of early embryos to hyperthermia may lie in
their inability to mount a cellular response to external
stressors such as heat shock. Mammalian cells exhibit a
characteristic response pattern to heat shock, including the
production of a set of proteins, called heat-shock proteins, that
are believed to exert stabilizing effects within the stressed
cell (Ashburner, 1982; Pelham, 1984; Welch and Mizzen, 1988).
Mouse embryos are capable of constitutive production of members
of the family of 70-kDa heat-shock proteins at the 2-cell stage
(Bensaude et al., 1983) but cannot increase heat-shock protein
synthesis in response to thermal stress until the
morula-blastocyst stage (Wittig et al., 1983). Thus, 4 to 8-
cell and 16-cell stages do not differ in their capacity to mount
such a response.
Embryonic development is dependent upon temporal coordina¬
tion of a number of processes, many of which may be disrupted by
hyperthermia, as referred to earlier. In addition, cleavage-
stage embryos are more likely at a given time to be in S or G2-M
stages of the cell-cycle when heat-shock sensitivity may be
greatest (Read et al., 1983; Rice et al., 1984 a,b). Heat shock
terminates DNA synthesis (Ashburner, 1982; Warters and Henle,
1982) and results in failure of DNA repair mechanisms in the cell
(Henle and Dethlefson, 1978; Warters and Roti Roti, 1982),
possibly due to accumulation of excessive protein in the nucleus
(Armour et al. , 1988) . Failure of unstressed cells to complete
replication of DNA during the S phase of the cell cycle results

239
in increased strand breakage during the next replication event
(Laskey et al., 1989) an effect seen in heat-shocked cells
(Warters and Roti Roti, 1982) . Failure of DNA replication during
subsequent rounds of cell division after heat shock would result
in mortality in early embryos. Relative resistance to heat shock
of lymphocytes, especially in the presence of alanine and
taurine, may lie in their placement within the cell cycle at G0
or G1. Maximal thermotolerance is manifested at G1. Association
of the 70-kDa heat-shock protein with proteins in the nucleolus
varies in a cell-cycle dependent fashion (Milarski et al. , 1989) ,
stabilization of nucleolar components during heat-shock being a
proposed function for this species (Pelham, 1984) .
Mitogen-induced lymphocytes produce heat-shock proteins,
particularly during the G0 through G, transition preceeding DNA
synthesis (Kazmarek et al., 1987; Haire et al., 1988), a result
of translation of preexisting mRNA (Colbert et al. , 1987) .
The preimplantation mouse embryo from 1-cell through the
blastocyst stage exhibits uptake of radiolabeled amino acids
(Wittig et al., 1983 ; Bensaude et al., 1983 ; Kaye et al., 1982;
Sallens et al., 1981) including alanine and taurine (Sallens et
al. , 1981). Amino acid uptake involves a facilitated transport
system (Kaye et al. , 1982) with coordinate efflux of methionine.
There is development of Na+-dependent active transport at or
before the blastocyst stage (Kaye et al. 1982). In addition,
taurine is abundant in the mammalian reproductive tract (Lorincz
and Kuttner, 1968) .

240
Vidair and Dewey (1987) found that CHO cells in a
nutrient-deprived state were extremely sensitive to heat shock
and that several hydrophilic, neutral amino acids, including
alanine, would reduce this sensitivity. This effect was similar
to that found in the present experiments? millimolar
concentrations were required, the effect was time and
concentration dependent and did not require conversion of the
amino acid into protein. Henle et al. (1988) found that alanine
exerted this protective effect upon CHO cells in the absence of
any nutrient deprivation-induced hyper-sensitivity. Sequential
addition and removal of the supplemental alanine around the time
of heat shock demonstrated that alanine was a thermoprotective
agent, i.e. it must be present during the heat shock, rather than
an inducer of thermotolerance whose effect remains for a time
after it is removed. Both Henle et al. (1988) and Vidair and
Dewey (1987) found that alanine thermoprotection was evident
within 5 min of alanine addition to cell cultures. Vidair and
Dewey (1987) suggested that because amino acids were required in
such large amounts and were not involved in formation of
proteins, that stabilization of cellular components, such as
proteins, was their likely role. This type of effect, i.e.
nonspecific aggregation around a protein molecule, is similar to
that suggested for the 70-kDa heat-shock protein (Lewis and
Pelham, 1985) .
Taurine has been implicated in several intracellular
functions, including as an antioxidant (Wright et al., 1986).

241
The intracellular oxidative-reductive state and heat shock may
be related since oxidative stress also induces heat-shock
proteins (Li and Werb, 1982; Lee et al., 1983 ; Drummond et al.,
1987) and heat shock induces increases in antioxidant enzymes
such as superoxide dismutase (Omar et al. , 1987). Additionally,
cellular responses to hydrogen peroxide exposure are similar to
heat-shock responses and development of tolerance to these two
stressors are, to a large degree, interchangeable (Li and Werb,
1982; Lee et al., 1983; Spitz et al., 1987). The beneficial
effect of reduced-glutathione (Mitchell et al., 1983; Russo et
al., 1984) during heat shock is also based on this apparent
overlap of the two stress-response systems.
In conclusion, both alanine and taurine exerted positive
effects upon viability of bovine peripheral lymphocytes during
in vitro heat shock. These effects were concentration dependent,
time dependent in the case of alanine and not dependent on
incorporation of the amino acids into new protein. Development
of preimplantation embryos is dependent upon temporal
coordination of numerous processes. Stabilization of
intracellular and intranuclear components during heat shock by
agents such as alanine and taurine may be possible once an
understanding of intracellular homeostatic activities and
molecular-level interactions of alanine and taurine with other
cell components has been achieved.

CHAPTER 9
EFFECTS OF EXOGENOUS ALANINE UPON EMBRYONIC
SURVIVAL DURING MATERNAL HYPERTHERMIA
AT TWO STAGES OF PREIMPLANTATION DEVELOPMENT
Introduction
Maternal hyperthermia during early pregnancy results in
significant embryonic loss in mammals, especially in the period
immediately after fertilization (Alliston and Ulberg, 1961;
Dutt, 1963; Dunlap and Vincent, 1971; Elliott and Ulberg, 1971;
Gwazdauskas, 1973; Putney et al., 1988a), but including sensi¬
tivity throughout preimplantation development (Omtvedt et al. ,
1971; Monty et al., 1982; Wise et al., 1988; Biggers et al.,
1987). Comparison of relative effects of hyperthermia
immediately after fertilization with effects during later
preimplantation development indicates that sensitivity is
greatest during the first 1 or 2 cell divisions (Dutt, 1963;
Tompkins et al. , 1967) . Indirect comparison based on results of
studies conducted at early (Dutt, 1963; Dunlap and Vincent, 1972;
Stott and Weirsma, 1976; Putney et al., 1988a) and late preim¬
plantation development (Dutt and Jobara, 1976; Stott and
Weirsma, 1976; Biggers et al., 1987; Wise et al., 1988) also
demonstrates greater sensitivity, in terms of effects on
conception rate, in the early cell divisions.
242

243
One possible cause for greater sensitivity of early embryos
to elevated temperature is the failure, before activation of the
embryonic genome, of maternal mRNA-directed protein synthesis to
generate the cellular responses to heat shock common to euka¬
ryotic organisms, such as heat-shock protein synthesis (Nover,
1984; Welch et al., 1988). Heat-shock proteins are highly
conserved families of proteins having molecular weights of from
25-kDa to 110-kDa. While their functions remain to be completely
elucidated, they are believed to exert various stabilizing
effects during cellular stress (Lewis and Pelham, 1985) and have
been correlated with cellular thermotolerance (Li and Werb,
1982 ; Landry et al., 1982). Recently, Landry et al. (1989) have
demonstrated induction of thermotolerance in murine cells
transfected with a plasmid-construct encoding a human 27-kDa
heat-shock protein and Riabowel et al. (1988) have found that
thermotolerance cannot be induced in cells receiving intracel¬
lular injections of antibodies against the 70-kDa heat-shock
proteins.
One objective of the present study was to determine whether
preimplantation embryos capable of producing heat-shock proteins
in response to elevated temperature are less likely to be killed
by maternal hyperthermia than embryos not capable of induced
synthesis. Induction of heat-shock protein synthesis in
response to hyperthermia is apparently not possible until the
early blastocyst stage in mouse embryos (Wittig et al., 1983)
although they are capable of constitutive synthesis of members

244
of the 70-kDa heat-shock protein family at the two-cell stage
(Bensaude et al., 1983). A second objective was to evaluate
whether administration of alanine in vivo would reduce hyper¬
thermia-induced infertility. Alanine has been shown to protect
cells during hyperthermia in vitro (Vidair and Dewey, 1987; Henle
et al. , 1988) , including preimplantation mouse embryos (Chapter
8) .
Materials and Methods
Materials
Mice, C57BL/10J and ICR-random bred, were obtained from
Jackson Labs (Bar Harbor, ME) and from Harlan Sprague-Dawley
(Indianapolis, IN), respectively. Follicle-stimulating hormone
(FSH) and human chorionic gonadotropin (hCG) were purchased from
Calbiochem (La Jolla, CA.). D, L-alanine was purchased from Sigma
(St. Louis, MO.) and D, L- [ 1-14C] alanine (specific activity =52.6
mCi/mmole) was from ICN Biomedical (Irvine, CA.). Components of
buffer used for tissue solubilization were described previously
(Chapter 2) . Materials used to collect mouse embryos in vitro
were described in Chapter 8.
Effect of stage of embryonic development and alanine on fertility
following hyperthermia
The effect of developmental stage and alanine treatment upon
mortality of preimplantation embryos during maternal hyper¬
thermia was determined by subjecting female mice to elevated
temperature for 6 h immediately following an intraperitoneal
(i.p.) injection 75 mg D, L-alanine or saline, on either day 1 or

245
day 3 after mating. Female ICR-random bred mice (6 weeks old) on
a 14:10 light:dark cycle in an environmentally-controlled
chamber at 21 °C to 2 4°C were induced to ovulate by i.p. injection
of 2 IU FSH followed 48 h later by 2 IU hCG. Females were then
exposed to C57BL/10J males overnight and examined the following
morning for the presence of a vaginal plug. At 42 h or 90 h after
hCG injection, females were injected i.p. with 75 mg D,L-alanine
dissolved in sterile .9% (w/v) saline or an equal volume (1 ml)
saline alone. The animals were then randomly assigned to either
remain in thermoneutral surroundings (21°C to 24°C; 50% RH) or
to be subjected to elevated temperature (37'C to 39 °C; 65% RH) in
a similar environmentally-controlled chamber for 6 h (ie., 42-
48 h after hCG or 90-96 h after hCG) . Numbers of females in each
treatment are shown in Table 9-1. All mice were housed in the
same room during the experiment except during this 6 h period.
Rectal temperatures of mice were measured following 6 h heat
stress (n = 12) or 6 h in thermoneutral environment (n = 12) . At
day 15 of pregnancy, females were killed by cervical dislocation
and reproductive tracts were removed. Fetuses and their
placentae were removed individually with membranes intact,
counted and weighed. Ovaries were examined and numbers of
corpora lútea (CL) were recorded.
Timing of embryo development
A group of ICR-random bred females was superovulated by
injection of 5 IU FSH, followed 48 h later by 5 IU hCG. Females
were then exposed to ICR-random bred males overnight and examined

246
the following morning for the presence of a vaginal plug. A
second group of ICR-random bred females was superovulated as
described and exposed to C57BL/10J males overnight. Animals were
killed by cervical dislocation at 45 h after hCG (ICR x ICR, n =
10;C57BL x ICR, n = 4) or 93 h after hCG (ICR x ICR, n = 13 ; C57BL
x ICR, n = 3). Their reproductive tracts were collected and
flushed as described previously (Chapter 8) and the stage of
development of embryos recorded.
Distribution of alanine in vivo
To determine the pattern of distribution of alanine injected
i.p. , nonpregnant ICR-random bred females (6 weeks old) received
7 5 mg D, L-alanine plus 2.5 (jlCí D, L- [ 14C] alanine dissolved in 1 ml
sterile saline. This dose, 75 mg, was used after preliminary
studies determined that very high doses of alanine (> 300 mg)
initially planned were toxic. While no LD50 was determined,
injection of D, L-alanine in concentrations of 360 mg were nearly
always fatal, doses of 150 mg were occasionally fatal and did
induce some symptoms of toxicity. Following injection, mice (n
= 3) were killed by cervical dislocation at 5, 15, 30, 60, 180 and
360 min after injection and biopsy samples taken immediately.
Samples of blood, brain tissue, kidney, liver, skeletal muscle,
uterus and oviducts were collected, weighed and placed in 1 ml
ice-cold tissue solubilization buffer [50 mM Tris-HCl, pH 7.6,
which contained 1 mM PMSF, ImM EDTA and 2% (w/v) CHAPS] and
frozen at -20'C. After thawing, samples were sonicated [Fisher
Dismembrator Model 300 (Fisher Scientific, Orlando, FL) using

247
the intermediate tip at 50% relative output) in two 15 sec
bursts, 30 sec apart. Samples were centrifuged (12,000 x g, 30
sec) and 100 /¿I aliquots of supernatants were counted by scintil¬
lation spectrometry.
Statistical analysis
Presence of [UC]alanine in tissues, litter size, conceptus
weight, numbers of embryos per CL, and ovulation rate were
analyzed by least-squares analysis of variance using General
Linear Models procedure of the Statistical Analysis System (SAS,
1985). The model used in the analysis of [uC]alanine distribu¬
tion included effects of time, animal nested within time, tissue
type and their interactions. The model used to analyze litter
size, embryos per CL and conceptus weight included effects of
time, temperature, treatment and their interactions. The
percent pregnant in each group at day 15 was analyzed by the
CATMOD procedure of SAS. The model included effects of time,
temperature, treatment, and their interactions.
Results
Effect of Stage of Development and Alanine During Hyperthermia
Rectal temperatures of females exposed to hyperthermic
treatment was 39.3 + .14°C compared to 37.4 + .23°C in homeo-
thermic mice. There were no differences among treatment groups
in proportions of bred animals that were pregnant at day 15
(Table 9-1) . For animals with pregnancies at day 15, litter size
was unaffected by elevated temperature or by alanine treatment

248
(Table 9-1) . There was considerable variation in ovulation rate
between treatments however (Table 9-1), which may have obscured
results. When embryo numbers were expressed as numbers of
embryos present per CL, survival of embryos was decreased by
hyperthermia (P < 0.02) . The degree of depression did not depend
on the developmental stage at which hyperthermia occurred ( i . e. ,
no temperature by stage interaction) and was not affected by
alanine (Table 9-2) . Mean weight of individual conceptuses from
dams exposed to elevated temperature was lower (P < 0.06) (Table
9-3) .
Timing of Embryo Development
When bred females were killed by cervical dislocation at 43
to 45 h after hCG and their reproductive tracts flushed, embryos
were present in the oviducts as expected. Of the ICR x ICR
embryos, 88% were 2-cells and the remaining 12% were at the 4-
cell stage. The C57BL x ICR embryos were also at 2-cell (90%) and
4-cell (10%) . When the females were killed at 93 h after hCG and
their reproductive tracts flushed, embryos had entered the
uterine horns. At this time, 78% of the ICR x ICR embryos present
and 74% of the C57BL x ICR embryos were blastocysts, and the
remainder were morulae.
Distribution of Alanine In Vivo
The distribution of [uC]alanine in body tissues of mice was
affected by a tissue type x time interaction (P < 0.01) (Figure
9-1). Blood contained the greatest amount of [uC]alanine,
showing an immediate rise in [UC] content and declining

249
Table 9-1. Effect of intraperitoneal injection of D,L-alanine
on litter size following maternal hyperthermia.
Environmental Developmental Treatment
Temperature Stage Saline Alanine
Homeothermic 2-4 cell
blastocyst
Hyperthermic 2-4 cell
blastocyst
9.4 ± 1.3
9.8 ± 1.4
11.7 ± 1.0b
12.1 ± 1.1
3
II
â–  o
n = 11
14/3 ld
11/16
(45%)e
(69%)
10.8 ± 1.3
8.2 ± 1.3
13.7 ± 1.1
13.3 ± 1.0
n = 12
n = 12
12/23
12/21
(52%)
(57%)
9.5 ± 1.4
8.3 ± 1.3
14.4 ± 1.1
13.3 ± 1.0
n = 12
n = 13
12/31
13/26
(39%)
(50%)
8.9 ± 1.6
8.5 ± 1.7
15.7 ± 1.3
13.8 ± 1.3
n = 8
n = 8
8/27
8/27
(30%)
(30%)
Note: Data are expressed as least-square means ± sem.
aLitter size.
bOvulation rate.
cNumber of litters per treatment.
dProportion of treated animals pregnant at day 15.
ePercent pregnant at day 15.

250
Table 9-2. Effect of intraperitoneal injection of D,L-alanine
on embryos per CL following maternal hyperthermia.
Treatment
Environmental Developmental Saline Alanine
Temperature Stage (no. embryos/CL)
Homeothermic
2-4 cell
.76 ± .08
.82 ± .09
blastocyst
.86 ± .09
.66 ± .08
Hyperthermic
2-4 cell
.65 ± .09
.61 ± .08
blastocyst
.58 ± .10
.64 ± .11
Note: Data are expressed as least-square means ± sem. Embryonic
mortality was affected by temperature (P < 0.02) .

251
Table 9-3. Effect of intraperitoneal injection of D,L-alanine
on mean weight of individual conceptus following maternal
hyperthermia.
Treatment
Environmental Developmental
Temperature Stage
Saline
(wet weight,
Alanine
g/conceptus)
Homeothermic
2-4 cell
.80 ± .07
.74 ± .08
blastocyst
.80 ± .07
.97 ± .07
Hyperthermic
2-4 cell
.67 ± .97
.59 ± .07
blastocyst
.87 ± .09
.73 ± .19
Note: Data are expressed as least-square means ± sem. Mean
weight of conceptuses affected by temperature (P < 0.06) and time
(P < 0.02) of treatment.

252
thereafter. The oviduct sequestered significant amounts of
radiolabeled amino acid, maintaining the highest level of any
tissue through the first hour after infection and then declining
to low levels by 3 h after injections. As expected, brain tissue
contained little radiolabeled amino acid due to effects of the
blood-brain barrier. Other tissues, such as liver and kidney,
contained moderate amounts of [UC]alanine over most of the 6 h
period as they worked to metabolize and excrete the excess amino
acid.
The injection contained 75 mg D,L-alanine plus 5.5 x 10’6 dpm
[KC]alanine (2.5 nCi) ; equal to 13.6 x 10’6 mg alanine/dpm. The
oviducts contained about 200 dpm [UC] per mg tissue, which is
equivalent to 2.7 ^q alanine/mg tissue. The concentration of
alanine in the oviduct was about 2.7 /ig/ml or 33.8 mM, within the
range of effective concentration in vitro.
Discussion
Exposure of early pregnant females to elevated temperature
induced an increase in rectal temperature of 2°C. This hyper¬
thermic state did not reduce litter size when embryos were 2- to
4-cells or blastocysts. There was considerable variation in
ovulation rate between groups, however. This variation likely
obscured effects of hyperthermia since random variation occurred
before imposition of treatment. When results were normalized to
constant ovulation rate, i.e., number of embryos per CL, it was
found that elevated temperature reduced embryonic survival.

253
14 C (cpm/mg tissue)
BRAIN
EmD uterus
LIVER
â–¡ OVIDUCT
dH SKEL, M
Hi BLOOD
KIDNEY
Figure 9-1. Incorporation of D, L-[1-14C]alanine into tissues
following intraperitoneal injection. There was a tissue type x
time interaction (P < 0.01).

254
Additionally there was a negative effect of hyperthermia on
individual conceptus weight. Warnick et al. (1965) found that
gilts exposed to elevated temperature exhibited considerable
variation in litter size also, and that elevated temperature
during the first 3 days after breeding caused no reduction in
litter size but did result in a significant reduction in the
number of embryos per CL.
As previously stated, one possible cause of sensitivity to
elevated temperature during early, cleavage-stage development
is the failure of maternal mJRNA-directed protein synthesis to
generate responses to heat shock such as synthesis of heat-shock
proteins (Nover, 1984; Welch et al., 1989), which are believed
to exert various stabilizing effects during cellular stress. At
the 2-cell stage, mouse embryos constitutively produce members
of the 70-kDa family of heat-shock proteins (Bensaude et al.,
1986) , but embryos apparently do not respond to heat stress with
induction of heat-shock protein synthesis until the blastocyst
stage (Wittig et al., 1983). In the present experiment there
were no interactions of hyperthermia with stage of development,
indicating that under these conditions the proposed ability of
embryos to produce heat-shock proteins in response to elevated
temperature lends no particular advantage to the blastocyst in
escaping effects of hyperthermia. These data suggest that other
changes occurring as a result of heat stress, such as effects on
conceptus protein secretion (Chapter 5) , oviduct secretory
function (Chapters 2 and 4), and blood flow (Ford, 1982) may

255
cause embryonic mortality even in the presence of a functional
heat-shock response.
The present experiment had several other interesting
results, including the activity of the oviduct in seguestering
alanine injected into the peritoneal cavity. The oviduct
contained more alanine per unit mass than any other tissue except
blood and the molar concentration of alanine was within the range
of effective concentrations in vitro (Chapter 8) . This elevated
concentration lasted for a longer period in the oviduct as well,
although not for the entire 6 h period necessary to maintain high
concentrations throughout the hyperthermic treatment.
Failure of D,L-alanine to exert effects in vivo may stem
from several possibilities. The dose given in the present
experiment was sufficient to create the high concentrations
utilized in vitro to cause thermoprotection, but only for a short
time (Vidair and Dewey, 1987; Henle et al., 1988; Chapter 8) . A
proposed mechanism for members of the 7 0-kDa group of heat-shock
proteins is nonspecific aggregation about macromolecules to
stabilize their structure in the face of denaturing heat stress
(Lewis and Pelham, 1985). This may also possibly be the action
of the high concentrations of neutral amino acids which act as
thermoprotectants during heat shock (Vidair and Dewey, 1987;
Henle et al., 1988; Chapter 8). The dose calculated to create
such a concentration throughout the body, 3 60 mg, was lethal when
given as a bolus injection. Furthermore, the elevated concen¬
tration induced in the present experiment was transient and did

256
not last 6 h. It remains to be determined whether a timed-
release system that creates a tolerable and sustained elevation
in amino acid concentration might have the desired effect
without toxicity. Carryover effects of hyperthermia, such as
chromosomal damage (Warters and Roti Roti, 1982), for which a
thermoprotectant cannot guard against may also result in
embryonic loss. In addition, heat shock terminates DNA synth¬
esis (Ashburner, 1982; Warters and Henle, 1982) and results in
failure of DNA repair mechanisms in the cell (Henle and Dethlef-
son, 1978; Warters and Roti Roti, 1982) , possibly due to accum¬
ulation of excessive protein in the nucleus (Armour et al.,
1988). Failure of unstressed cells to complete replication of
DNA during the S phase of the cell cycle results in increased
strand breakage during the next replication event (Laskey et al. ,
1989) an effect seen in heat-shocked cells (Warters and Roti
Roti, 1982). Failure of DNA replication during subsequent rounds
of cell division after heat shock would result in disruption of
the developmental program, even if temporary relief is afforded
by thermal protective agent such as alanine.
In conclusion, the induction of hyperthermia in pregnant
mice in the present experiment reduced the number of embryos per
CL. A bolus injection of D,L-alanine was not sufficient to
enhance survival of embryos during a 6 h period of maternal
hyperthermia. The method of delivery of the alanine or the dose
may have been suboptimal, or the embryonic loss may have been due
to carryover effects of hyperthermia. There was no effect of

257
stage of development on the capacity to survive hyperthermia, and
thus the capability to produce heat-shock proteins alone is
probably not sufficient to allow embryonic survival during
maternal hyperthermia.

CHAPTER 10
GENERAL DISCUSSION
Reproductive performance of female cattle can be affected by
hyperthermia in three major ways. Behavioral changes and reduced
activity levels result in failure to display estrus or to display
estrus over a shorter time period. Secondly, during hyperthermia
there are changes in maternal endocrine function, metabolic
activities and alterations of uterine blood flow. Together these
changes may perturb oviductal and uterine function which undergo
steroid-driven changes in activity in preparation for, and to
maintain, pregnancy. Thirdly, direct thermal effects on the
oviduct, uterus and developing conceptus may disrupt temporal
coordination of the numerous events necessary for successful
establishment and maintenance of pregnancy.
Data presented in this dissertation demonstrate direct,
local thermal effects upon uterine endometrium and oviductal
tissues of the cow. The greatest effects on protein secretion
were seen in the oviduct and uterine horn ipsilateral to the
corpus luteum. Direct effects of elevated temperature may
disrupt the important interactions between the early embryo and
oviduct as well as the synchrony between steroid-driven uterine
changes and embryonic development. In addition, the apparent
asymmetry of the bovine uterus with respect to uterine-ovarian
258

259
blood supply and distribution of progesterone from the corpus
luteum may affect sensitivity to direct thermal stress. The
impact of heat shock-induced alterations of secretion rates of
individual peptides upon events of early pregnancy will not be
known for certain until the functional importance of a greater
number of these peptides is revealed. Endometrial tissues in
vitro exhibited thermotolerant characteristics, including
synthesis of 70-kDa and 90-kDa heat-shock proteins. Reproduc¬
tive tract tissues of Brahman and Holstein cows responded to
elevated temperature with production of heat-shock proteins in
a similar fashion. Effects of elevated temperature on protein
secretion, particularly on the ipsilateral oviduct, indicate
some variation between the breeds in acute responses. Prolonged
heat shock resulted in no difference in protein synthesis and
secretion between Zebu and Bos taurus.
In contrast to the subtle effects of in vitro heat shock upon
maternal tissues, the exposure of preimplantation mouse embryos
to elevated temperature in vitro resulted in high mortality. The
embryo remains sensitive to hyperthermia throughout preimplanta¬
tion development. Synthesis and secretion of protein by bovine
conceptuses collected at day 17 of pregnancy was depressed by
elevated temperature in vitro. Secretion of the antiluteolytic
signal, bTP-1, by embryos exposed to elevated temperature in
vitro was significantly reduced also. The ability of bTP-1
itself to function under hyperthermic conditions in vitro
appears unaffected.

260
Maternal uterine endometrium collected at day 17 after estrus
secreted elevated amounts of PGF in response to direct thermal
stress. Increased release of PGF in response to heat shock is
not a common response of prostaglandin producing tissues.
Prostaglandin F release by bovine endometrium at day 17 after
estrus may be particularly sensitive to heat shock due to its
normal activity of synthesis and secretion of luteolytic pulses
of PGF unless it is somehow inhibited from doing so. The direct
effect of heat shock was not to cause disinhibition due to loss
of activity of bCSPs. The cause may lie in the membrane composi¬
tion of the endometrium at day 17 after estrus.
Later, during fetal growth, perturbation of maternal
metabolism, endocrine activity and blood flow cause major
hyperthermia-induced effects, i.e., fetal stunting. Data
presented here suggest that local, direct effects of hyper¬
thermia at the uterine-placental interface affects secretion of
PGE2, especially in the maternal caruncular and intercaruncular
endometrium. Vasoregulation in the endometrial vascular bed is
affected by catecholamines, steroids and prostaglandins.
Disruption of the balance of these three effectors, such as
through depression of PGE2 secretion by direct thermal stress,
may adversely affect uterine blood flow. Since the majority of
maternal and fetal blood flow is through the placentomal tissue,
the impact of local effects of heat-shock on PGE2 secretion by
intercaruncular endometrium and chorioallantois may be minimal.
Ovine tissues also exhibited characteristics of thermotolerance

261
and enhanced production of 70-kDa and 90-kDa heat-shock pro¬
teins.
Alanine and taurine were effective in reducing heat-induced
killing of bovine lymphocytes and mouse preimplantation embryos.
The effect of alanine was short-lived and required millimolar
concentrations of amino acid, similar to that reported by others.
Amino acid supplementation resulted in improvement of embryonic
response to heat shock, though not to levels equal with homeo-
thermic development. Taurine is generally associated with
oxidative stress, although it appears to have considerable
potential as a thermoprotectant. Sensitivity of the early embryo
may lie in its rapid rate of progress through the cell cycle,
heat shock during S phase might result in chromosomal abnor¬
malities fatal to the embryo during subsequent development.
Maternal heat stress during preimplantation mouse embryo
development resulted in lower numbers of embryos per corpus
luteum. Temporal coordination of in vivo heat shock with
embryonic capacity to produce heat-shock proteins in response to
stress demonstrated no benefit in terms of maintenance of
embryonic viability. Perhaps perturbations on the maternal
system, i.e., blood flow changes, endocrine effects, are too
great in their negative effects to be overcome by a heat-shock
response in the embryonic cells. Intraperitoneal injection of
D,L-alanine caused increased concentrations of alanine to appear
in the oviduct, but had no effect on embryo survival during
subsequent maternal hyperthermia. A more efficient way to

262
elevate body concentrations of alanine may prove more effective.
The capacity to synthesize heat-shock proteins in response to
elevated temperature is probably not sufficient to confer
thermotolerance upon the embryo.
In conclusion, direct thermal effects were observed to
disrupt activities of endometrium, oviduct and conceptus tissues
in a manner generally consistent with observations of the effects
of maternal hyperthermia upon establishment and maintenance of
pregnancy. These effects were observed at multiple sites and at
multiple stages of pregnancy. Direct effects of heat shock act
in combination with alterations in maternal endocrine, blood
flow and metabolic alterations during periods of hyperthermia to
depress rates of pregnancy in animals. Direct effects of heat
shock may play a more important role in incidences of acute
exposure to elevated temperature at some critical stage of
conceptus-maternal interaction causing damage in the absence of
major alterations in maternal physiology.

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