Uterine, oviductal, and conceptus responses to heat shock

MISSING IMAGE

Material Information

Title:
Uterine, oviductal, and conceptus responses to heat shock
Physical Description:
xv, 302 leaves : ill., photos ; 29 cm.
Language:
English
Creator:
Malayer, Jerry Rhea, 1957-
Publication Date:

Subjects

Subjects / Keywords:
Animal Science thesis Ph. D   ( lcsh )
Dissertations, Academic -- Animal Science -- UF   ( lcsh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001576407
oclc - 22914738
notis - AHK0260
System ID:
AA00003750:00001

Full Text










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