Cellular, subcellular, and developmental responses of two-cell bovine embryos to a physiologically relevant heat shock

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Title:
Cellular, subcellular, and developmental responses of two-cell bovine embryos to a physiologically relevant heat shock
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xv, 183 leaves : ill. ; 29 cm.
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Rivera, Rocio Melissa
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Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
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Includes bibliographical references.
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by Rocio Melissa Rivera.
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Printout.
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Vita.

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CELLULAR, SUBCELLULAR, AND DEVELOPMENTAL RESPONSES OF TWO-
CELL BOVINE EMBRYOS TO A PHYSIOLOGICALLY RELEVANT HEAT SHOCK














By

ROCIO MELISSA RIVERA

















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

2003



























I would like to dedicate this dissertation to three people who have been very influential in
shaping my life as a person and scientist. First of all, my parents Jos6 D. Rivera and
Zaida Varas who have showed me by example the importance of hard work,
determination, thoroughness, and taking pride in ones work. I thank them for their love
and support, and for giving me many opportunities to develop as a well-rounded person
with a good balance between education, arts, and sports. The third person is my mentor
and friend Dr. Peter J. Hansen. Dr. Hansen has been instrumental in my development as
a scientist. His love for science and the pursuit of knowledge, cheerful disposition, and
love for teaching and mentoring have been an incredible source of inspiration to me.
These qualities have provided fertile soil for me to grow as a scientist and to fall in love
again with science and the excitement of doing research. I will be forever be indebted to
him for believing in me and for his patience, guidance, and encouragement.














ACKNOWLEDGEMENTS


The work presented in this dissertation would have not been possible without the

love and support of many people. First of all I would like to thank God for directing my

plans. Even when I do not understand them, they are always right for me. I am most

grateful to my family for their love and support: Jos6 D. Rivera Ortiz (father), Zaida

Varas Mufioz (mother), Providencia (Tat6n) Mufioz (grandmother), Zaida Rivera Varas

and Saudith Mariela Rivera Varas (sisters), Jos6 Juan (Honty) Rivera Varas (brother),

Walter G6mez (brother-in-law), Paola Cristina Gomez Rivera and Andrea Melisa G6mez

Rivera (nieces), and Cristian Andr6s G6mez Rivera (nephew). I also want to thank all

the friends I made here in Gainesville, especially Josiris DelKarmen Valed6n Miranda

and Gisette G. Seferina, for their love and support. I would also like to thank my little

one, Chiwi, for many happy moments and for always wagging her tail and giving me a

smile.

I am very grateful to my committee members, Dr. Peter J. Hansen (chair). Dr.

William Thatcher, Dr. Charles Guy, and Dr. Kenneth Drury, for all their advice and for

critically reviewing my work. I want to thank Dr. Hansen for providing a great working

environment in which to develop as a scientist, for all his advice, and for financially

supporting the work presented in this dissertation. I also would like to thank Dr.

Thatcher for many words of encouragement and for good conversations about my






iii









research and for the many jokes. Last but not least, I would like to thank Drs. Drury and

Guy who have provided insight into my research findings.

I have been very blessed with many wonderful collaborators during my stay here

at Florida. First I would like to mention Karen Kelley from the ICBR Electron

Microscopy Core Lab. Karen is Ms. Electron Microscope herself, and her willingness to

help goes beyond the call of duty. She was always there to help when I was stuck even

during weekends. I would also like to thank Gabriella Dahlgren from the Department of

Chemistry for collaborating with the oxygen consumption experiment. When I first

decided to do that experiment, I did not know Gabriella. I contacted her by telephone,

and after explaining what we wanted to do, she happily agreed to help even when she

realized that the experiments were going to have to be done in the middle of the night. I

would also like to thank Dr Greg Erdos, scientific director of the ICBR Electron

Microscopy Core Lab, for answering my many questions and providing useful

information. Last but not least, I thank Dr. Alex Angerhofer from the Department of

Chemistry. At one point during my studies, I wished to measure free radical production

by two-cell embryos using electron paramagnetic resonance spin-trap technology. After

learning that there is an expert in such technique at the University of Florida, I contacted

Dr. Angerhofer by email and soon we met and agreed to collaborate. I can not describe

the amount of time and effort that he put into helping me to get this technique to work.

Unfortunately, the machine was not sensitive enough for our experimental model and we

had to abandon the experiment. I remain extremely grateful for all his help.

I am indebted to all the scientists and members of Dr Hansen's laboratory. Some

of the work would have not been possible without their assistance. I thank Dr. Alice de




iv









Moraes, Andrew Majewski, Anthony Galarza, Dr. Carlos Ar6chiga, Catherine Brocas,

Charles Krininger, Dean Jousan, Dr. Fabiola Paula-Lopes, Heather Rosson, Imke Mebes,

Dr. Jennifer Trout, Jeremy Block, Dr. Joel Hernandez Cer6n, Jose Queijeiro, Jose

Osorio, Justine Fitzgerald, Mr. Khaled Mohammed, Dr. Lannet Edwards, Dr. Liuz

Augusto de Castro e Paula, Maria Beatriz Padua, Moises Franco, Dr. Morgan Peltier,

Olga Oc6n, Dr. Paolete Soto, Dr. Ramesh Chandolia, Ren6 Arrazola, Dr. R6mulo

Bafiuelos, Dr. aban Tekin, Dr. Victor Bermudez. Mr. William Rembert, Dr. Yaser Al-

Katanani, and Dr. Zvi Roth. I especially thank 'Zvika" for many great conversations and

always picking my brain and Dr. de Castro for collaboration with the experiment using

low oxygen embryo culture. I also thank the many people in the Department of Animal

Sciences for all their help and support especially Mary Ellen and Dale Hissem and the

people in the Animal Molecular and Cell Biology program that encouraged me during all

these years especially Dr. Karen Moore and Dr. William Buhi.


























V














TABLE OF CONTENTS

ACKN OW LED GEM EN TS ................................................. ........................................ iii

ABSTRACT............................ ..................................................... x

LIST OF ABBREVIATIONS............... ......................................................... xii

PRE FA C E ............................... ..................................................... ........................... xiv

CHAPTER

1 REVIEW OF THE LITERATURE......................................................................... I

Maternal Hyperthermia and Embryonic Loss an Historical Account...................
Concepts Derived From In Vivo Studies ............ .. .............................. 2
Heat Stress Decreases Embryonic Survival ..................................................2..
Changes in Sensitivity to Heat Stress as Pregnancy Advances........................9...
Effects on the Reproductive Tract vs. Embryo............................................10
Summary of Experiments Concerning Effects of Maternal Hyperthermia on the
Early Preimplantation Embryo ........................................ 11
Concepts Derived From In Vitro Studies...............................................................12
Cellular, Molecular, and Ultrastructural Effects of Heat Shock on Cells and
E m bryos ........................................................ .................................................. 15
C ytoskeleton................................................... ............................................. 15
Organelles ............... .......................................... 17
M em branes............................................................................... ...................... 19
Nucleus, Nucleic Acids, Cell Cycle, and Mitosis..........................................22
C ellular Proteins.............................................................. ....................... 24
Free Radicals and Antioxidants ............................................. ...................... 30
H eat Shock-Induced Apoptosis.............................................. ...................... 33
Responses of Preimplantation Embryos to Heat Shock...........................................35
D isruption of Cellular Function............................................. ...................... 35
Thermoprotective Mechanisms in Embryos .................................................. 36
H sp 70 ............................................................................ ....................... 36
Antioxidants....................................................................................... 37
A poptosis ................................................. ............................................ 37
Synopsis Rationale For Thesis.............................................................................39







vi









2 DEVELOPMENT OF CULTURED BOVINE EMBRYOS AFTER EXPOSURE
TO INCREASED TEMPERATURES IN THE PHYSIOLOGIC RANGE ............41

Introduction ..................................................................................... ......... ........... .. 4 1
M aterials and M ethods ............................... ........................ ............................... 43
M aterials...................................................................................................... 43
In Vitro Production of Embryos........................................ .............................. 43
Heat Shock During Fertilization ................................................................... 44
Heat Shock at the One-Cell Stage........................................ ........................ 45
Heat Shock at the Two-Cell Stage ....................................... ........................ 45
Effect of Type of BSA on Response to Heat Shock ......................................46
Effect of Oxygen Concentration on Response to Heat Shock .......................46
Rectal Temperatures of Heat-Stressed Lactating Dairy Cows........................ 47
Exposure of Embryos to a Pattern of Temperatures Similar to Those
Experienced by Heat-Stressed Cows.............................. ............................49
Statistical Analysis .................. ........................................................... 49
R esu lts ................................................................................... ............................. 50
Effect of Heat Shock During Fertilization...................................................50
Heat Shock at the One-Cell Stage................................................................50
Heat Shock at the Two-Cell Stage ...................................... ......................... 53
Effect of Oxygen Concentration on Response to Heat Shock .......................53
Rectal Temperatures of Heat-Stressed Lactating Dairy Cows.......................56
Exposure of Embryos to a Pattern of Temperatures Similar to Those
Experienced by Heat-Stressed Cows.................................. ...................... 56
Discussion ........................................ ...................................... 59

3 ALTERATIONS IN ULTRASTRUCTURAL MORPHOLOGY OF TWO-CELL
BOVINE EMBRYOS PRODUCED IN VITRO AND IN VIVO FOLLOWING
A PHYSIOLOGICALLY-RELEVANT HEAT SHOCK .....................................64

Introduction ............... .......................................... 64
M aterials and M ethods........................................................................................... 66
M aterials................ ......................................... 66
Production of Em bryos ................................................................ ................ 67
Inhibition of Blastocyst Development by Heat Shock at the Two-Cell Stage..68
Effects of Heat Shock on the Ultrastructure of Two-Cell Embryos Produced
In V itro ........................................................................................................ 69
Effects of Heat Shock on the Ultrastructure of Two-Cell Embryos Produced
In V ivo and Treated Ex Vivo ......................................... ......................... 69
Electron M icroscopy ............................................................ ........................ 70
Morphometric Analysis of Electron Micrographs .........................................71
Statistical A nalysis ............................................................... ........................ 71
R esults ........................... ................................................................................ 72
Inhibition of Embryonic Development by Heat Shock at the Two-Cell
S tag e .......................................................................................................... ... 7 2
Heat-Shock Induced Changes in Ultrastructure of Two-Cell Embryos
Produced In Vitro .......................... .................. .... .................... 72


vii









Heat-Shock Induced Changes in Ultrastructure of Two-Cell Embryos
Produced in Vivo and Treated Ex Vivo ..................................................80
D iscussion ............................... ........................................................................ 80
4 POSSIBLE INVOLVEMENT OF FREE RADICALS AND MITOCHONDRIAL
DAMAGE IN THE DELETERIOUS ACTIONS OF A PHYSIOLOGICALLY-
RELEVANT HEAT SHOCK IN TWO-CELL BOVINE EMBRYOS ................89

Introduction .................................... .....................................89
M aterials and M ethods ........................................................... ....................... 91
In Vitro Production of Embryos...................................................................91
H eat Shock Treatm ent..................................................................................... 92
Effects of Oxygen Tension on Inhibition of Development Caused by Heat
S h o ck ..................................................................................... ..................... 9 3
Effects of Heat Shock on Glutathione Content...............................................93
Effects of Heat Shock on Oxygen Consumption .............................................94
Effects of Heat Shock on ATP Content .........................................................96
Effects of Heat Shock at the Two-Cell Stage on Subsequent Development to
Day Three Post-Insem ination........................................... ........................ 97
Statistical A nalysis.................................................................. ........................ 98
R esu lts ..................................................................................................................... 9 9
Effects of Oxygen Tension on Inhibition of Development Caused by Heat
S h o ck ...................................................................................... .................... 9 9
Effect of Heat Shock on Glutathione Content..............................................99
Effect of Heat Shock on ATP Content.........................................................99
Effect of Heat Shock on Oxygen Consumption.............................................99
Effects of Heat Shock at the Two-Cell Stage on Subsequent Development
to Day Three Post-Insemination.............................................................104
D iscussion ...................................................... ................................................ 107

5 ACTIONS OF HEAT SHOCK ON REORGANIZATION OF
MICROFILAMENTS AND MICROTUBULES IN TWO-CELL BOVINE
E M B R Y O S ....................................................... ................................................ 112

In tro d u ctio n .................................................................................. .................... 1 12
M aterials and M ethods ................... ...........................................................1...... 13
M aterials..................................................... ............................................... 113
Production of Em bryos ................................................................................. 114
Effects of Microfilament and Microtubule Depolymerization on Heat
Shock-Induced Movement of Organelles..................................................114
Effects of Microtubule Stabilization on Heat Shock-Induced Movement of
O rgan elles ........................................................................ ...................... 115
Electron Microscopy and Morphometric Analysis of Electron
M icrograp hs.......................................................................... .................... 116
Localization of Microfilament by Fluorescence Microscopy......................1...16
Statistical A nalysis ............... ...........................................................1... 17
R esults ...............................................................................117



viii









Effects of Microfilament and Microtubule Depolymerization on Heat
Shock-Induced Changes in Ultrastructure................................................. 117
Effects of Microtubule Stabilization on Heat-Induced Movement of
Organelles............................................ 123
Microfilament Reorganization in Heat-Shocked Cells................................ 127
D iscussion ................................................................................. ..................... 133

6 GENERAL DISCUSSION............... ...................................................... 142

APPENDIX PHOTO GALLERY OF TWO-CELL BOVINE EMBRYOS ...............154

R E FE R E N C E S ........................................................ .................................................. 162

BIOGRAPHICAL SKETCH ................ .......................................................... 183






































ix















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

CELLULAR, SUBCELLULAR, AND DEVELOPMENTAL RESPONSES
OF TWO-CELL BOVINE EMBRYOS TO A PHYSIOLOGICALLY RELEVANT
HEAT SHOCK

By

Rocio Melissa Rivera

December, 2003


Chair: Peter J. Hansen
Major Department: Animal Sciences

Exposure of preimplantation embryos to elevated temperatures blocks

development. Temperatures used to demonstrate heat shock effects on development have

been higher, however, than body temperatures typically experienced by heat-stressed

cows. The goal of this dissertation was to determine the mechanism by which heat shock

in the physiological range blocks development.

Initial findings demonstrated that development was disrupted by a short-term heat

shock of 41.0C or by continuous exposure to a 24-h pattern of temperatures similar to

those experienced by heat-stressed cows. Ultrastructural changes induced in embryos by

exposure to 41.0 or 43.00C for 6 h were examined to determine mechanisms for

inhibition of development. Heat shock of 41.00C caused movement of organelles

towards the center of the blastomere and an increase in the percent of mitochondria





x









exhibiting a swollen morphology. Exposure to 43.0C (a non-physiological temperature)

caused more widespread ultrastructural damage such as chromatin precipitation.

Experiments evaluated consequences of mitochondrial disruption caused by heat

shock. Exposure to 41.0C appears not to have an immediate effect on oxidative

phosphorylation because heat shock did not immediately alter oxygen consumption or

ATP content. It was observed that heat shock at the two-cell stage first caused a block in

development at the eight-cell stage. Thus, damage to mitochondria or other structures at

the two cell-stage first become critical to survival as the embryo enters the 8-16 cell

stage.

An experiment was designed to test whether the heat shock-induced movement of

organelles is a result of cytoskeletal rearrangement. At 41.00C, movement of organelles

from the periphery of the cell was blocked by rhizoxin, an agent causing microtubule

depolymerization, and latrunculin B, a microfilament depolymerizer. Thus, disruption of

both microfilaments and microtubules is involved in organellar movement. Only

rhizoxin blocked organellar movement at 43.0'C, suggesting that microtubule damage at

43.00C causes redistribution of organelles even when microfilaments are depolymerized.

Microtubules are also involved in mitochondrial swelling because rhizoxin blocked

effects of heat shock on mitochondrial swelling.

In conclusion, physiologically-relevant heat shock disrupts development of two-

cell embryos and this action is mediated, at least in part, by disruptions of the

mitochondria and cytoskeleton. Heat shock does not cause an immediate block to

development in the two-cell embryo but prevents development past the eight-cell stage.






xi















LIST OF ABBREVIATIONS

3H Tritium
"S Sulfur-35
3T3 cells mouse, Swiss albino, fibroblast
ADP Adenodisne Diphosphate
AIF Apoptosis-Inducing Factor
Apaf-1 Apoptosis activating Factor 1
ATP Adenosine Triphosphate
Bim Bcl-2-Interacting Mediator of cell death
BSA Bovine Serum Albumin
BSO Buthionine Sulfoximine
Ca2+- Calcium
CaC12 Calcium Chloride
CI Chloride
CHO Chinese Hamster Ovary Cells
CO2 Carbon dioxide
d day
DMSO Dimethyl Sulphoxide
DNA Deoxyribonucleic Acid
DNTB 5,5-dithiobis(2-nitrobenzoic acid)
EDTA Ethylenediaminetetraacetic Acid
EFAF BSA Essentially Fatty Acid Free Bovine Serum Albumin
eIF-4F Eukaryotic Initiation Factor-4F
ER Endoplasmic Reticulum
FBS Fetal Bovine Serum
FSH Follicle Stimulating Hormone
Gl Gap 1
G2 Gap 2
GnRH Gonadotrophin Releasing Hormone
GSH Glutathione
h hour
H' Hydrogen
HCO3 Bicarbonate
HCT116 cells Human Colon Carcinoma cells
HeLa human, Black, cervix, carcinoma, epitheloid
HEPES Hydroxyethylpiperazine Ethanesulfonate
HL60 cells Human, Caucasian, peripheral blood, leukemia
hpi hours post-insemination
HSBP Heat Shock Binding Protein
HSE Heat Shock Element



xii









HSF Heat Shock Factor
Hsps Heat shock proteins
IgGi Immunoglobulin 1
K' Potassium
KC1 Potassium Chloride
KCN Potassium Cyanide
KH2PO4 Potassium Phosphate Monobasic
KOH Potassium Hydroxide
KRB Krebs' Ringer Buffer
KSOM Potassium Simplex Optimized Medium
KSOM BEI Potassium Simplex Optimized Medium Bovine Embryo I
KSOM BE2 Potassium Simplex Optimized Medium Bovine Embryo 2
M Mitosis
MgSO4 Magnesium sulfate
MPT Mitochondrial Permeability Transition
mRNA messenger Ribonucleic Acid
Na+ Sodium
NaCI Sodium Chloride
NADPH Nicotinamide-Adenine Dinucleotide Phosphate, reduced
NaPO4 Sodium Phosphate
NRK cells rat, Osbom-Mendel, kidney, fibroblast
02 Oxygen
OCC Oocyte Cumulus Complex
PBS Phosphate Buffered Saline
PBS+PVP Phosphate Buffered Saline + Polyvinyl Pyrrolidone
PBS+S Phosphate Buffered Saline + Serum
PARP poly(ADP-ribose)
PGF2a Prostaglandin F 2-alpha
PROC GLM General Linear Model Procedure
PTP Permeability Transition Pore
RNA Ribonucleic Acid
ROS Reactive Oxygen Species
S DNA replication phase
SAS Statistical Analysis System
SEM Standard Error of the Mean
SIS Soft Imaging System
TALP Tyrodes Albumin Lactate Pyruvate
TCM-199 Tissue Culture Medium-199
V-79 hamster, Chinese, lung, fibroblast
VDAC Voltage Dependent Anion Channel
z-DEVD-fmk N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone








xiii














PREFACE


Heat stress can reduce fertility in cattle (Dunlap and Vincent, 1971; Turner, 1982;

Ealy et al., 1993; Ryan et al., 1993) and other species (Shah, 1956; Alliston and Ulberg,

1961; Tompkins et al., 1967). Many methods based on reducing the magnitude of heat

stress have been applied under farm conditions to ameliorate the hyperthermia

experienced by the animal as a result of heat stress. Some of these include the addition of

cooling systems to the barns, such as sprinklers, fans, and high-pressure foggers (Bray

and Buclkin, 1997). However, fertility in the summer months, determined as 90 day non-

return rate, is still less than 20% (Al-Katanani et al., 1999). Further approaches to

improve fertility in dairy cattle may be possible as more is learned about the

physiological mechanisms by which heat stress reduces fertility. It is known that the

reduction in fertility observed as a result of heat stress is multifactorial in nature. For

example, heat stress affects endocrine secretion of reproductive hormones (Gwazdauskas

et al., 1973; Roman-Ponce et al., 1981; Badinga et al., 1993; Trout et al., 1998), follicular

development (Badinga et al., 1993; Wolfenson et al., 1995), blood flow to the

reproductive tract (Roman-Ponce et al., 1978), and oocyte competence (Al-Katanani et

al., 1999). Several studies have also shown that the early mammalian embryo is very

susceptible to elevated temperatures (Dutt, 1963; Elliot et al., 1968; Elliot and Ulberg,

1971; Ealy et al., 1993; Edwards and Hansen, 1997; Krininger et al., 2002). Taken






xiv









together, these observations suggest that the mechanisms of decreased fertility in the

summer could be the result of disruption of embryonic development due to hyperthermia.

Susceptibility to hyperthermia experienced by early embryos is reduced as they

advance through development (Ealy et al., 1993; Edwards and Hansen, 1997). For

example, Ealy and coworkers (1993) showed that embryonic loss was greatest in cows

that were heat-stressed on the first day after insemination when compared to cows heat-

stressed on Days 3 or 5 after insemination (Day 0 = day of estrus). In addition, Edwards

and Hansen (1997) demonstrated that two-cell bovine embryos produced in vitro were

less tolerant to heat shock than in vitro-produced compact morulae (corresponding to Day

I and Day 5 post-insemination, respectively). These data imply that it is during the first

few cleavage divisions when embryos are most susceptible to the deleterious effects of

elevated temperatures. Limited information has been gathered as to the mechanisms by

which heat shock affects the early bovine embryo.

The goal of this dissertation is to evaluate whether heat shock in the physiologic

range can disrupt embryonic development and to determine the basis for this effect at the

cellular and sub-cellular levels. Demonstrating effects of physiologically-relevant heat

shock on embryonic development would lend credence to the idea that infertility caused

by exposure of cows to heat stress is, at least in part, a result of direct effects of maternal

hyperthermia on embryonic development. Identifying the mechanisms by which heat

shock causes these effects may lead to new strategies for improving fertility in the heat-

stressed cow.








XV














CHAPTER 1
REVIEW OF THE LITERATURE


Maternal Hyperthermia and Embryonic Loss an Historical Account

It has long been recognized that elevated temperatures adversely affects fertility

of females. As mentioned in the preface, the temporary infertility caused by heat stress is

a multifactorial problem that involves alterations in several physiological systems

including endocrine secretion of reproductive hormones (Roman-Ponce et al., 1981;

Badinga et al., 1993), follicular development (Badinga et al., 1993; Wolfenson et al.,

1995) blood flow to the reproductive tract (Roman-Ponce et al., 1978) and oocyte

competence (Al-Katanani et al., 1999). To this mix of pathologies should be included

inhibition of development and death of the preimplantation embryo caused by exposure

to elevated temperatures. Indeed, the early preimplantation embryo is more sensitive to

elevated temperature than most cells: temperatures as low as 39.0-41.0C can inhibit

embryonic development (Gwazdauskas et al., 1992; Dutt, 1963; Ealy et al., 1992). This

section will use a historical approach to describe the experimentation that has led to the

concept that elevated temperature is a key disruptor of embryonic development.

Subsequent sections will develop concepts about how elevated temperature disrupts cells

in general and preimplantation embryos in particular.






2


Concepts Derived From In Vivo Studies

Early research on the effects of heat stress on fertility in females led to an

enhanced understanding of the role of this environmental stressor in the establishment

and maintenance of pregnancy. Among the critical concepts demonstrated were the ideas

that 1) heat stress decreases embryonic survival, 2) the effects of heat stress decline as

pregnancy advances, and 3) actions of heat stress involve disruption to both the embryo

and to the reproductive tract environment in which it resides.

Heat Stress Decreases Embryonic Survival

One of the first reports that fertility can be reduced by elevated temperatures dates

back to a paper from Cordelia Ogle (1933) in mice. In that study, mice were caged

under controlled environments at 16-20*C, 21-27C, or 31-33*C. It was noticed that the

females raised at 31-330C had the lowest litter sizes.

From the early 1960's to the mid 1970's there was an explosion of information on

effects of heat stress on early pregnancy in sheep (Alliston et al., 1961; Alliston and

Ulberg, 1961; Dutt, 1963), mice (Aldred et al., 1961; Elliot et al., 1968; Elliot and

Ulberg, 1971; Bellvy, 1972), pigs (Warnick et al., 1965; Tompkins et al., 1967; Edwards

et al., 1968; Omtvedt et al., 1971; Wildt et al., 1975), and cows (Dunlap and Vincent,

1971). In all of these species, with the exception of the pig, clear evidence was provided

that exposure of females to heat stress early in pregnancy leads to embryonic death.

In the sheep for example, lambing rate was significantly decreased for ewes that

were placed in rooms maintained at 330C for 24 h after mating (Dutt, 1963). That the

decrease in lambing rate was due to embryonic loss was shown in another study in which

ewes were maintained in a room at high temperature (33C) for 8 days (from five days






3


from the expected time of estrus until three days after mating) and embryos recovered at

Day 6 to 7 after mating. The majority of the embryos in the heat-stressed females were

classified as abnormal and had halted development at the 32-cell stage (Alliston et al.,

1961).

In one study, Elliot et al. (1968) evaluated the effects of exposure of females to

24-h of heat stress (34*C) beginning at -16 h after mating (1600 h on the day that a

copulatory plug was observed) on subsequent embryonic development and pregnancy

outcome. A portion of the females were sacrificed -50 h after mating and the rest on Day

10 of gestation. Embryos collected at -50 h after mating were classified and cultured for

three days. At the time of collection, there were more two-cell embryos in the heat-

stressed females than in controls (22% vs. 3%) and a corresponding reduction in embryos

at more advanced stages (34 vs. 51% eight cell embryos for heat stressed vs. control).

None of the two-cell embryos continued development after culture. For embryos

collected at later stages, fewer became blastocysts after culture when retrieved from heat-

stressed females (45 vs. 62% for heat-stressed vs. control, respectively). On Day 10,

there was a 21% decrease in the number of implantation sites in the heat-stressed group.

These results confirm the idea that heat stress early in development can reduce embryonic

development and suggests that in the mouse, at least, one major block to development

occurs at the two-cell stage.

In 1971, Elliot and Ulberg reported a study that characterized effects of maternal

heat stress during the first cleavage division on subsequent embryonic development.

Females were assigned to be heat stressed (33*C) for a period of 24 h beginning

approximately 16 h post-mating or remain at control temperature (21 0C). Embryos






4


collected from heat-stressed females had a reduced number of cells when collected at -60

h post-mating when compared to embryos collected from control females. In addition,

fewer successful pregnancies resulted when females were heat-stressed (47% vs. 81% for

heat-stressed and control females, respectively). Figure 1-1 shows the percent of

embryos found at each stage of development at -60 h post-mating. As the graph

demonstrates, 80% of the embryos from heat-stressed females were halted in

development before the eight-cell stage while only 23% of embryos from control females

were less than eight cells in number.

Similar to results of Elliot and Ulberg (1971), Bellv6 (1972) showed that heat

stress of female mice at 34.5*C for 24 h beginning approximately 16 h after mating

caused extensive retardation of embryonic development (Figure 1-2). As compared to

embryos from controls, there was an increased proportion of two-, three-, and four-cell

embryos and a decreased proportion of eight-cell embryos recovered at ~60 h post-

mating.

The first study documenting an effect of maternal heat stress on embryonic

survival in cattle was by Dunlap and Vincent (1971) who studied the influence of post-

breeding thermal stress on conception rates in beef cattle. In this experiment, beef heifers

were exposed to 32.20C for 72 h following breeding. None of the heat-stressed heifers

calved while 48% of the control group maintained at 21.10C calved. The next study

demonstrating effects of maternal heat stress on embryonic survival was reported by

Putney et al. (1988). In this study, superovulated heifers were used as a model to

determine the effects of heat stress during the first seven days of pregnancy on embryonic






5








80

70

60

50

2 40

30

20

10

0 --
1-cell 2-cell 4-cell 8-cell
Embryonic stage at -60 hr post-mating




Figure 1-1. Summary of the effects of heat stress on percent of embryos at the 1-, 2-,
4-, and 8-cell stage of development at -60 h post-insemination. Females were heat
stressed (330C) or left as control (21 'C) for a period of 24 h beginning approximately
16 h post-mating. Data presented in this graph represent the average of three
individual experiments performed by Elliot and Ulberg (1971). Open bars represent
embryos from control females (n = 49) and grey bars represent embryos from heat-
stressed females (n = 51).






6







80

70

60

50

0 40

30

20

10


2-cell 3-cell 4-cell 8-cell
Embryonic stage at -60 h post-mating


Figure 1-2. Effects of a 24 h heat stress beginning at approximately 16 h post-
insemination on percent of embryos at the 1-, 2-, 4-, and 8-cell stage. Embryos were
recovered from females at -60 h post-insemination. Data presented in this graph are
taken from Bellv6 (1972). Open bars represent embryos from control females (n = 96)
and grey bars represent embryos from heat-stressed females (n = 96). Note that numbers
are approximations as they were estimated from a graph in the original paper.






7


development. Heifers were superovulated and artificially inseminated. Beginning at 30 h

after estrus, heifers were either maintained at 20.00C or exposed to a heat stress for 6

days that consisted of daily exposure to 30.0C for 16 h and 42.0C for 8 h. Embryos

were recovered at Day 7 after insemination. Heat stress reduced the percent of embryos

having good to excellent quality from 58% in the control group to 45% in the heat-

stressed group. Furthermore, the percent of retarded and abnormal embryos increased as

a function of increased rectal temperatures.

Collective reports regarding effects of heat stress in the pig are difficult to

interpret because of contradictory data. In addition, experiments with this species are

more varied as to duration of heat stress as well as the time during early pregnancy when

heat stress was applied. In general, however, results generally point to a reduction in

survival of preimplantation embryos as a result of maternal hyperthermia. Nevertheless,

the pig seems much more resistant to heat-stress associated embryonic mortality than

some other species.

In an early experiment, Warnick et al. (1965) found that exposure to heat stress of

32.3"C for the first three days after mating did not affect the number of live embryos on

Day 25 of pregnancy. More prolonged heat stress, for 25 days, did reduce the number of

live embryos observed at Day 25 of pregnancy. The study of Warnick et al. (1965) is

illustrative of the severe heat stress required to disrupt embryonic development in the pig.

Others have observed similar results. Tompkins et al. (1967) failed to find an effect of a

24 h heat stress of 35.0*C given on Day 1, 3, or 5 after breeding on embryonic survival at

Day 35 of pregnancy. Although not statistically significant, that data showed a 30%

reduction in viable embryos when heat stress was applied on Day 1. When the heat stress






8


temperature was increased to 36.7C and the duration of heat stress extended to five days

beginning Day 1 of gestation, there was only a slight reduction in embryonic death

despite the death of four of nine heat-stressed sows. Edwards et al. (1968) observed that

heat stress reduced the number of viable embryos on Day 33 post-breeding but the heat

stress was exposure to 38.9C for 17 h per day for the first 15 d post-breeding. In this

study, too, some animals died as a result of heat stress. Likewise, Omtvedt et al. (1971)

observed that a heat stress regimen of 17 h daily at 37.80C for 8 days resulted in a 43%

reduction in pregnancy rate on Day 33 post-breeding.

Even when heat stress reduces embryonic survival in pigs, not all animals

experience embryonic death. Wildt et al. (1975) exposed pigs to 40.4*C for 2 h each day

for 12 days beginning on Day 2 post-breeding. While overall heat stress increased

embryo mortality from 35% to 63%, loss of embryos was not seen in all animals. Of the

ten pigs exposed to heat stress, four experienced an average embryonic loss of 93%

whereas the remaining animals had embryonic mortality similar to controls.

Taken together, it would appear that the pig is not a good general model for

understanding early embryonic death in response to heat stress. Perhaps the early porcine

embryo is not as susceptible to the effects of elevated temperature as embryos of other

species. Another possibility is that the pig has better ability for thermoregulation as

compared to some other species. In the work of Wamick et al. (1965), Omtvedt et al.

(1971) and Wildt et al. (1975), pigs tended to adapt to heat stress so that body

temperature became progressively closer to controls as the experiment progressed.






9


Changes in Sensitivity to Heat Stress as Pregnancy Advances

The first report to indicate that the maternal system becomes more tolerant to the

effects of heat stress as pregnancy progresses was provided by Femandez-Cano (1958)

using the rat as a model. A change in maternal body temperature from 37 to 390C on

Day 1 and 2 of pregnancy increased the number of degenerated embryos from 2.5% in

control rats to 65% in heat stressed rats. In contrast, hyperthermia during Days 3-4 or 6-7

of pregnancy reduced the percent of degenerated embryos to 30 and 15%, respectively.

Similar results were obtained in the mouse by Aldred et al. (1961). In this study,

mice were subjected to 40C for 1, 3, or 5 h on Day 1 following mating, for 5 h on Day 2

after mating, or for 5 h on Day I and 2. The number of viable and reabsorbed embryos

was determined as a percent of the number of corpora lutea following autopsy on Day 17.

Their data show that embryonic loss due to heat stress on Day 1 increased as the duration

of heat stress increased (embryonic loss control = 14%, 1 h = 15%, 3 h = 17%, 5 h =

19%). Also, there were less severe effects of 5 h of heat stress on Day 2 (embryonic loss

= 10%) than when females were stressed on Day 1 and 2 for 5 h each (embryonic loss =

33%).

Results in accordance with findings in rodents were obtained in the sheep by Dutt

(1963). Ewes were exposed to high temperatures from Day 0-1, 3, or 5 after breeding

until Day 24 of pregnancy and then returned to control temperature until lambing. The

proportion of ewes lambing was 85% for control ewes and 10, 35 and 40% for ewes heat-

stressed beginning on Day 0, 3, and 5, respectively. When a subset of ewes were

analyzed on Day 3 after mating, it was found that 38.5% of embryos in heat-stressed

ewes were classified as abnormal versus only 3.7% of embryos from control ewes.





10


In the cow as well, the magnitude of the disruption in embryonic survival caused

by heat stress decreases as pregnancy advances. Such a conclusion was derived from the

work of Ealy et al. (1993), who exposed superovulated cows to a single day of heat stress

at 1, 3, 5, or 7 d after insemination and then measured embryo survival at Day 8. To

induce heat stress, cows were placed in an unshaded lot from 0800 h to 1500 h. The

percent of embryos classified as live based on vital dye staining was 70% for control

cows and 55, 68, 65, and 89% for cows heat-stressed on Days 1, 3, 5, or 7, respectively.

The percent of embryos that were blastocysts was reduced from 49% in the control cows

to 34% for cows heat-stressed at Day 1. Heat stress at other days had no effect on

percent blastocyst (60, 57, and 64% for Day 3, 5, or 7, respectively).

Effects on the Reproductive Tract vs. Embryo

The reduction in embryonic survival caused by maternal heat stress administered

after fertilization could be a consequence of direct actions on the embryo or indirect

actions caused by alterations in the reproductive tract environment. Using the rabbit as a

model, Shah (1956) was the first to conduct an experiment to study the embryo-uterine

relationship during heat-induced pregnancy failure. It was concluded that effects of heat

stress were mediated by changes in the uterine environment because embryos from heat-

stressed females that were transferred to a non-stressed female could establish pregnancy.

A similar experiment was performed in ewes by Alliston and Ulberg (1961).

Three experimental groups were used ewes raised in a control environment (housed at

21C) that received an embryo from a female in a control environment, ewes raised in a

control environment that received an embryo from a female exposed to heat stress

(housing at 320C), and ewes raised in a heat-stress environment (housed at 320C). Heat





11


stress was performed from 5 d before estrus until the time of transfer. Therefore, effects

on the oocyte and embryo were possible. Embryo transfers were performed around 72 h

post-insemination (approximately at the eight-cell stage) and pregnancy was determined

on Day 25-30 post-mating. Pregnancy rates for the three groups were 56% (control

embryos in control ewes), 10% (heat-stressed embryos in control ewes) and 24% (control

embryos in heat-stressed ewes). Thus, heat stress appears to affect both the embryo and

reproductive tract with the former effects being more severe.


Summary of Experiments Concerning Effects of Maternal Hyperthermia on the
Early Preimplantation Embryo


The experiments described in this section provide abundant evidence that the

processes leading to embryonic survival during early pregnancy can be disrupted by

exposure of pregnant females to heat stress. Moreover, the female is particularly

sensitive to effects of heat stress in the first few days of pregnancy. Experiments with

reciprocal embryo transfer lead to the conclusion that heat stress can compromise

embryonic survival by affecting the embryo directly and by altering the reproductive tract

environment. One problem with the reciprocal transfer approach is that a reduction in

pregnancy rate in control animals receiving embryos from heat-stressed females could be

result of direct damage to embryo or effects on the reproductive tract to cause permanent

disruption of the embryo that cannot be corrected by transfer into the reproductive tract of

a non-stressed female. Further evidence that the preimplantation embryo is susceptible to

heat stress requires use of cultured embryos to study effects of heat shock on the embryo

independent of alterations in the reproductive tract.





12


Concepts Derived From In Vitro Studies

Rather than examine the effects of heat stress (i.e., the sum of the environmental

conditions that determine an animal's heat balance and which drive it into hyperthermia),

experiments with cultured embryos have focused on heat shock (a chronic or acute

elevation in temperature applied to a cell, organ, tissue, or organism). In vivo,

mammalian or avian cells become exposed to heat shock when body temperature rises

because of fever or heat stress. Heat shock can be induced in cultured cells and tissues by

exposure to culture conditions involving elevated temperature. Such experiments with

cultured embryos are necessary for two reasons; 1) to determine direct effects of elevated

temperature on embryonic development and 2) to determine the mechanisms involved in

the disruption of embryonic function caused by heat stress.

Heat shock directly applied to the embryo has been reported to decrease

development in many species including mouse (Gwazdauskas et al, 1992; Ar6chiga et al.,

1995; Edwards et al., 1997; Ar6chiga and Hansen, 1998), cow (Ealy et al., 1995; Edwards

and Hansen, 1996; Ju et al., 1999; Al-Katanani and Hansen, 2002; Block et al., 2002;

Krininger et al., 2002; Paula-Lopes and Hansen, 2002a; Paula-Lopes et al., 2003a,b),

rabbit (Alliston et al, 1965), and pig embryos (Kojima et al., 1996). Studies both in vivo

(Fernandez-Cano, 1958; Alliston and Ulberg, 1961; Alliston et al., 1961; Dutt, 1963;

Elliot et al., 1968; Elliot and Ulberg, 1971; Dunlap and Vincent, 1971; Bellv6, 1972;

Putney et al.,1988; Ealy et al., 1993) and in vitro (Gwazdauskas et al, 1992; Ealy et al.,

1995; Edwards and Hansen, 1997; Ar6chiga and Hansen, 1998; Al-Katanani and Hansen,

2002; Block et al., 2002; Krininger et al., 2002; Paula-Lopes and Hansen, 2002a; Paula-





13


Lopes et al., 2003a,b) show that temperatures as low as 39.0-41.0C render the embryo

unable to continue development.

Preimplantation embryos are susceptible to elevated temperature from the one-

cell stage though the blastocyst stage of development (Ealy and Hansen, 1994; Ealy et al.,

1995; Edwards et al., 1997; Ar6chiga and Hansen, 1998; Ju et al., 1999; Al-Katanani and

Hansen, 2002; Block et al., 2002; Krininger et al., 2002; Paula-Lopes et al., 2003b).

The previous statement should not be interpreted to mean that all stages of

embryonic development are equally sensitive to heat shock. There is evidence in several

species that heat shock effects are reduced as development advances. The most severe

effects of heat on developmental competence of embryos usually occur before the eight-

cell stage and more specifically during the first two cleavage divisions (Gwazdauskas et

al., 1992; Edwards et al., 1997; Krininger et al., 2002). From the eight-cell stage

onwards, preimplantation mammalian embryos gain the ability to withstand the

deleterious effects of heat (Ealy et al., 1995; Edwards and Hansen, 1997; Ar6chiga and

Hansen, 1998; Ju et al., 1999; Krininger et al., 2002). For example, Edwards and Hansen

(1997) showed that a heat shock of 41.00C for 12 h obliterated development of two-cell

bovine embryos to the blastocyst stage while only slightly reducing development of four-

to eight-cell embryos and having no effect on compacted morulae. Similarly, Krininger

et al. (2002) showed that two-cell bovine embryos were severely affected by an elevation

in temperature than embryos at Day 5 post insemination that were 16-cells or greater.

Moreover, in the mouse (Ar&chiga and Hansen, 1998), a heat shock of 41.0C for 1 h at

the two-cell stage reduced the percent of embryos developing to blastocyst from 98% in

control to 75%. On the other hand, when a similar heat shock was applied at the morula






14


stage there was no difference in the percent of embryos becoming blastocysts (95 and

90%, for control and heat shocked embryos, respectively). In addition, Muller et al.

(1985) observed differences in the sensitivity to heat shock between one-cell and

blastocyst stage mouse embryos with the former being extremely sensitive and the later

being resistant. Taken together, these data would suggest that some of the differences

observed in pregnancy responses when heat stress is applied on different days after

conception (Fernandez-Cano, 1958; Dutt, 1963; Ealy et al., 1993) reflect, at least in part,

changes in embryonic responses to heat shock.

In addition to the developmental differences in resistance to heat shock, several

studies have demonstrated that there is a genetic component controlling embryonic

responses to heat shock. For instance, Paula-Lopes et al. (2003b) demonstrated that the

deleterious effect of heat shock at Day 5 after insemination on blastocyst formation and

the number of cells per embryo was less pronounced for Brahman embryos than embryos

of the Holstein or Angus breeds. Similarly, Hemrndez-Cer6n et al. (2003) found that

embryos from the Romosinuano (a heat tolerant breed of Bos taurus genetic origin) and

Brahman (heat tolerant breed of Bos indicus genetic origin) were more tolerant of heat

shock applied to embryos > 8-cells than embryos from the Angus breed (a heat sensitive

breed of Bos taurus genetic origin). When heat shock was applied at the two to four cell

stage, however, there were no differences in resistance to heat shock between Brahman

and Holstein embryos (Krininger et al., 2003). Thus, genetic differences may require

advanced development, perhaps in conjunction with embryonic genome activation.

There is also evidence that genetic resistance to heat shock is inherited maternally (Block

et al., 2002). In that study, heat shock > 9-cell stage reduced the percent of embryos






15


formed from insemination of Holstein oocytes with Angus sperm developing to

blastocyst from 56 to 30% while causing a slight increase in development for embryos

formed from insemination of Brahman oocytes with Angus sperm (42 vs. 56% for control

vs. heat-shocked, respectively). In contrast to this effect of breed of oocyte, there was no

difference in resistance to heat shock for embryos derived from Holstein oocytes

regardless of whether spermatozoa used for fertilization were from Brahman or Angus

bulls. These results were interpreted to mean that either 1) genes determining

thermotolerance are paternally imprinted or 2) are dependent upon a preformed factor in

the oocyte.

A question that arises from all these studies is; what is the cellular mechanism

whereby heat shock renders the preimplantation embryo unable to continue development?

Limited information has been gathered on the direct effects of heat shock at the cellular

level on embryos. Therefore, the approach will be taken to review the literature on the

effects of heat shock on other cell types and, when possible, describe information

collected thus far on the mammalian preimplantation embryo.


Cellular, Molecular, and Ultrastructural Effects of Heat Shock on Cells and
Embryos

Cytoskeleton

One of the most noticeable effects of heat shock on cells are changes in cell

shape. Several studies have demonstrated that, upon heat shock, one or many of several

cell shape changes can occur, as for example, cell flattening, cell rounding, and

membrane blebbing (Coss and Linnemans, 1996). These changes are a result of






16


cytoskeletal reorganization (Glass et al., 1985; Shyy et al., 1989; Coss and Linnemans,

1996; Wang et al., 1998).

The cytoskeleton is a cellular component which is common to the plasma

membrane, cytoplasm, and nucleus. The cytoskeleton plays important roles in 1)

regulation of cell structure (by supporting the cell membrane), 2) cell movement, 3)

cytokinesis, 4) binding of cell components such as enzymes and messenger RNA, and 5)

redistribution of cell organelles through the actions of molecular motors (Gallicano et al.,

1994; Brown, 1999; Lodish et al., 2000; Kamal and Goldstein, 2000).

This dynamic and extensive intracellular network is composed of three types of

fibers, namely, 1) microfilaments, 2) intermediate filaments, and 3) microtubules

(Lodish, 2000). Microfilaments are composed of actin subunits, microtubules of tubulin

subunits, and the intermediate filaments of cell-specific proteins (i.e. cytokeratins,

vimentin, desmin, lamins) which are grouped into five classes (types I to V; Hermann and

Aebi, 2000). Microfilaments and microtubules are highly dynamic systems (Coss and

Linnemans, 1996) which show a steady-state equilibrium between the polymerized

structures and large unpolymerized pool of subunits. It is for this reason that the

organization of these networks can be quickly altered as a result of polymerization or

depolymerization. Although dynamic changes occur in intermediate filaments also, the

pool of unpolymerized subunits is small (Eriksson et al., 1992) and it has been speculated

that phosphorylation and dephosphorylation cycles control their dynamic properties.

Exposure of primary cultures of mouse epithelial cells to elevated temperatures

resulted in change in the organization of keratin filaments and actin filaments while

having no effect on microtubules (Shyy et al., 1989). Similar results were observed in 9L






17


cells where microtubules were only slightly affected by heat shock while cytoplasmic

microfilaments and intermediate filaments collapsed (Wang et al., 1998). Rearrangement

of microfilaments as a result of heat shock have been observed by lida et al. (1986) who

demonstrated that heat shock of 42.0-43.0C causes disintegration of normal actin and

induced formation of intranuclear actin paracrystals and actin rods. Detection of the

intermediate filament protein vimentin revealed that heat shock of 45.0C for 30 min

caused the intermediate filaments to rearrange from an elaborate lattice work in the

cytoplasm to a cage-like form around the nucleus (Cheng and Lai, 1994). Similar

collapse of intermediate filaments as a result of heat shock have been observed in the

chicken fibroblast (Collier et al., 1993) but not in NRK cells in which intermediate

filaments are very resistant to elevated temperatures (Ohtsuka et al., 1993).

In contrast to the lack of effect on the microtubules in the previously cited works,

heat shock can disrupt microtubules in Chinese hamster ovary (CHO) cells (Coss et al.,

1996) and mouse 3T3 cells (Lin et al., 1982). Further, it has been documented that

microtubules isolated from calf brain (body temperature of 38.5C) will disassemble at

41.00C and will denature at 43.0*C (Turi and Lu, 1981). From these results, it is obvious

that the effects of heat shock on the different cytoskeletal components vary depending on

cell type (for a review see Coss and Linnemans, 1996).

Organelles

Another major effect of heat shock in cells is the induced redistribution of

organelles to the perinuclear region as well as changes to organelle morphology (Shyy et

al., 1989; Cole and Armour, 1988; Welch and Suhan, 1985; Wang et al., 1998; Funk et

al., 1999). These observations are very consistent among different cell types and appear






18


to be due to cytoskeletal rearrangement (Welch and Suhan, 1985; Coss and Linnemans,

1996; Wang et al., 1998). Using both electron microscopy and immunological methods,

Welch and Suhan (1985) characterized a number of morphological changes occurring in

rat embryo fibroblast cells after a heat shock of 42.0*C for 3 h. They demonstrated

mitochondrial mobilization to the vicinity of the nucleus as a result of heat shock as well

as a number of structural changes within the mitochondria. Specifically, the

mitochondria appeared swollen, the cristae more prominent, and the intracristae spaces

appeared enlarged. These authors suggested that this relocalization of mitochondria may

be due to a collapse and aggregation of the intermediate filaments around the nucleus of

heat-treated fibroblasts. Funk et al. (1999) demonstrated by means of electronic light

microscopy, confocal laser scanning microscopy, and electron microscopy, that heat

shock caused mitochondria to become swollen, to change from a rodlike shape into an

annular-shaped organelle and to experience a reduction in motility. Similar results were

observed by Wang et al. (1998) who demonstrated that in normal cells, mitochondria are

mostly long and straight and evenly distributed in the cytoplasm. Upon heat shock, these

filamentous mitochondria changed their morphology rapidly and became shortened and

swollen and migrated towards the perinuclear region where they became mostly granular.

After heat shock, the Golgi apparatus dissociated into numerous small vesicles. In

addition, Welch and Suhan (1985) observed that the Golgi complex as well as the

endoplasmic reticulum (ER) appeared to be fragmented and had formed vesicles which

could be found throughout the perinuclear region.

Of particular interest is the mitochondrial swelling observed as a result of heat

shock. Mitochondria are very important organelles as they produce the energy for the






19


cell in the form of ATP, are tightly integrated in cellular calcium signaling, serve as stress

indicators for the cell, and play a crucial role in apoptosis as one of the central signaling

systems for activation of execution caspases in programmed cell death (Duchen, 1999;

Funk et al., 1999). As mentioned previously, heat shock has profound effects on this

organelle. Funk et al. (1999) showed that after heat shock the mitochondrial membrane

potential was depressed in most mitochondria as monitored with the fluorescent probe

JC-1. The loss in membrane potential as a result of heat shock can have devastating

results for the mitochondria and the cell as it could result in the release of apoptotic

proteins from the intermembrane space. Hyperthermia has been noted to impair electron

transport along the respiratory chain (Floridi et al., 1987) resulting in a decrease in the

level of ATP and phosphorylation efficiency (Findly et al., 1983; Calderwood, 1987;

Macouillard-Poulletier de Gannes et al., 1998). Since mitochondria undergo

temperature-dependent uncoupling during increases in temperature (Floridi et al., 1987;

Flanagan et al., 1998; Kourie, 1998), it is likely that heat-shocked cells would have a

different rate of oxygen consumption than controls cells. In fact, Macouillard-Poulletier

de Gannes et al. (2000) showed that the respiratory properties of microglial cells are

compromised during and immediately after heat shock as measured by and immediate

and strong drop in oxygen consumption. In addition, Calderwood et al. (1985) showed

that ATP content of cells decreased as a result of heat shock.

Membranes

One of the effects of heat shock on cells is altered physiology of cellular

membranes (Lepock, 1982; Gerner et al., 1980; Arancia et al., 1989; Bates et al., 1996;

Coss and Linnemans, 1996). Changes as a result of heat shock include accumulation of






20


electron-dense particles, discontinuous plasma membranes, loss ofmicrovilli, and lifting

of the plasma membrane from the underlying cytoplasm (Bowler, 1987; Arancia et al.,

1989). In addition, other modifications observed to occur during heat shock are changes

in plasma membrane fluidity, changes in membrane potential, and the loss of activity of

membrane-bound proteins (Coss and Linnemans, 1996). Changes in membrane potential

have been observed in plasma membranes of hamster lymphocytes exposed to heat

(Marcocci and Mondovi, 1990). Heat shock generally enhances membrane permeability

and thus increases the flux of ions and small molecules (Gemer et al., 1980; Marcocci

and Mondovi, 1990; Kuhl and Rensing, 2000) so that transmembrane gradients of ions

and metabolites are disturbed (Marcocci and Mondovi, 1990). For example, exposure of

cells to heat shock causes the plasma membrane to become leaky to polyamines (Gemer

et al., 1980). In addition, heat shock of 43.0*C results in increases in intracellular

calcium ions due to an influx into the cytoplasm from both internal stores and the

extracellular medium (Itagaki et al., 1998). It has been suggested that the stress-induced

disturbances of Ca and thiol homeostases (specifically glutathione: GSH) affect the

cortical cytoskeletal network of isolated hepatocytes and, subsequently, the integrity of

the plasma membrane, leading to bleb formation (Jewel et al., 1982).

Changes in temperature will disrupt both the lipid and the protein moieties that

make up membranes. An increase in temperature will increase the molecular motion of

lipids, due to an increase in kinetic energy, which results in an increase in fluidity (Lee

and Chapman, 1987). In an unsaturated fatty acid requiring mutant of Escherichia coli,

there is evidence for a correlation between membrane microviscosity (reciprocal of

fluidity) and sensitivity to hyperthermia (Dennis and Yatvin, 1981). However, increasing






21


microviscosity of ascites and V79 cell membranes did not influence the response of cells

to heating (Yatvin et al., 1983). In 1980, Gemer et al. proposed a model for thermal

killing in which removal of cholesterol molecules (a molecule involved in cell membrane

fluidity) via the action ofpolyamines (ubiquitously occurring polycations that have been

demonstrated to sensitize cells to heat) makes the plasma membrane more fluid and leaky

resulting in the inability of the cell to exert normal osmotic controls. Further, heat shock

does not alter levels of the major phospholipids such as phophatidylcholine,

phophatidylserine, and phosphatidylethanolamine (Bates et al., 1996).

There is also evidence to suggest that membrane proteins, possibly those

involved in intracellular pH regulation and lipid-protein interactions, can be disrupted by

heat shock (Coss and Linnemans, 1996). The regulation of intracellular pH is a vital

aspect of cell homeostasis (Dale et al., 1998). Major intracellular processes are highly

sensitive to intracellular pH including protein synthesis, cell metabolism, Ca2+

homeostasis, mitochondrial function, gene expression, cytoskeletal regulation, and cell

death (Puceat, 1999; Dale et al., 1998). The ion transport mechanism is crucial in

maintaining intracellular pH in all cell types. Interestingly enough is the fact that in

monkey kidney Vero cells, heat shock has been shown to decrease the activity of the Na+-

independent HCO3'/Cl exchanger (Oehler et al., 1998). Further, heat shock has been

demonstrated to result in acidification of cells (Oehler et al., 1998; Kiang and Tsokos,

1998).

Although little is known regarding effects of heat shock on membrane functions

in preimplantation embryos, it is likely that cells of these organisms are less likely to

adjust to disruptions in membrane function than many other cells. Observations by Baltz






22


et al. (1991) showed that none of the known intracellular pH regulatory mechanisms

found in other mammalian cells (i.e. Na+/H+ antiport, Na/ H' pumps, K+/H+ pumps) are

active in two-cell stage mouse embryos during recovery from acidosis. They concluded

that recovery from acid loads in the two-cell mouse embryo is a passive process

following the electrochemical gradient across the plasma membrane (Baltz et al., 1991;

Baltz, 1993). Recovery from alkaline loads in two-cell mouse embryos, however,

involves the HCO3 /Ci exchanger (Baltz et al., 1991).

Nucleus, Nucleic Acids, Cell Cycle, and Mitosis

The nucleus contains thermolabile proteins that denature and aggregate as a result

of exposure of hydrophobic domains during heat shock (Lepock et al., 2001). The

aggregated protein in the nucleus can be observed by electron microcopy as regions of

electron-dense material (Swanson et al., 1995). Further, Welch and Suhan (1985)

showed that upon heat shock, fibrous containing, rod-like structures appeared within the

nucleus. These inclusion bodies appeared to be packed with thin filaments. Using

meromyosin decoration studies, these authors determined that the inclusion fibers

contained actin. This study suggests that disruption of the nuclear matrix involves

cytoskeletal rearrangement.

Upon heat shock, nuclear chromatin loses it's normal appearance (Swanson et al.,

1995), nucleoli became less condensed and show signs of unraveling, and aggregation of

denatured proteins at the nuclear matrix are observed (Welch and Suhan, 1985;

(Hildebrandt et al., 2002). It is well established that DNA synthesis, as measured by

incorporation of precursors such as [3H]thymidine, is rapidly inhibited by heat shock

(Marcocci and Mondovi, 1990; Warters and Roti Roti, 1982). Exposure of HeLa and






23

Chinese hamster V79 cells to heat shock results in the inhibition of replicon initiation and

DNA replicative elongation and inhibition of DNA repair synthesis (Marcocci and

Mondovi, 1990). Warters and Roti Roti (1982) have suggested that the inhibition of

DNA synthesis is a result of heat-induced denaturation of replicative enzymes since DNA

polymerase a and 0 are both inhibited by heat shock. Furthermore, heat shock reduces

[3H]uridine incorporation in HeLa cells (Burdon, 1987) indicating reduced transcription

as a result of elevated temperatures.

In most cases, heat shock causes antiproliferative effects (Huang et al., 1999;

Kuhl and Rensing, 2000). Differential effects of heat have been observed during the

different phases of the cell cycle. The cell cycle is composed of four phases that proceed

successively in most growing cells. These are three cycles that make up the interphase -

G1 (the first gap before DNA synthesis occurs), S (phase where DNA replication

occurs) and G2 (the second gap after DNA synthesis) and the M phase (or mitosis)

during which time occurs condensation of the duplicated chromosomes, their alignment

at the metaphase plate, separation of the sister chromatids, and the segregation into two

daughter cells (Lodish et al., 1995). The heat shock-induced rearrangement of the

cytoskeleton, especially of the tubulin system interferes, with the normal distribution of

the genetic material to the daughter cells and arrests the cell cycle (Huang et al., 1999).

In a study using mouse oocytes, for example, (Fiorenza and Mangia, 1992), mild heat

shock conditions during oocyte maturation disrupted the process of bivalent chromosome

disjunction, blocking oocytes at the metaphase I stage. In addition, heat shock induced

the appearance of abnormal chromosome morphology and number. Even though the

authors did not look at effects of heat shock directly on the cytoskeleton, the unequal






24


chromosome segregation suggests cytoskeletal and especially microtubule damage. Heat

shock of CHO cells also caused alterations to the centrosomes resulting in multipolar

mitotic spindles, delay in prophase-metaphase, and formation of multinucleated cells

which were non-clonogenic (Vidair et al., 1993). Heat shock of HeLa cells at 41.5C

resulted in asymmetric segregation of chromatid clusters and premature reformation of

the nuclear membrane resulting in multiple micronuclei formation (Swanson et al., 1995).

In mammalian cells, heat shock leads to a transient arrest of cells at mainly the

two cell cycle check-points, the G1/S and G2/M transitions. The first check-point in late

Gl registers cell size, nutritional sate, and DNA damage. The second check-point is

located in the order within G2 and M and it registers completion of DNA synthesis in G2

and preparatory steps for mitosis (Kuhl and Rensing, 2000). As mentioned earlier, DNA

synthesis is rapidly inhibited by heat shock and results in DNA strand breaks. Disrupting

the initiation of replication and mitosis represent the most important points of no return

(Kuhl and Rensing, 2000). Sensitivity to heat shock is dependent on cell type and cell-

cycle phase and depends on the severity of the stress. For example, Nover (1991)

explained that there is an apparent difference to sensitivity to thermal killing between

cells in the Gl/GO phases (more resistant) vs. those in G2/S phase (more sensitive).

However, Coss et al. (1996) and Wachsberger and Coss (1990) showed that heat-induced

cytoskeletal alterations play a role in the death of cells heated in GI phase, but had no

significant influence on the death of cells heated in the S phase in CHO cells.

Cellular Proteins

Heat shock rapidly increases the activation of a specific set of proteins, the heat

shock proteins, which are tightly involved in the stress response. However, other protein






25


synthesis is normally inhibited by heat shock (Burdon, 1987; Kuhl and Rensing, 2000;

Hilderbrandt et al., 2002) by inducing translational changes in cells. In Drosophila cells,

the transcriptional regulation of heat shock protein genes is promoted while the

translation of pre-existing mRNAs is repressed (Burdon, 1987). In mammalian cells,

however, no sequestration of pre-existing mRNAs occurs but rather there is decrease in

polysomes. Burdon (1987) explains that two possible reasons for the inhibition and loss

of polysomes include a) inactivation of elIF-4F function, and b) dephosphorylation of

ribosomal protein S6. In addition, heat shock induces degradation of damaged proteins

by the ubiquitin-proteosome and lysosomal pathways (Kuhl and Rensing, 2000;

Hilderbrandt et al., 2002) and losses of stable RNA occur as a result of increases in

lysosomal ribonuclease activity (Burdon, 1987).

The changes in cellular physiology after exposure to elevated temperature have

been referred to as the heat shock (or stress) response. The heat shock response is of

fundamental importance to the cell's survival and is one of the most highly conserved

genetic systems known. This response was discovered in 1962 by Ritossa, who found

that heating salivary cells of Drosophila induced areas of localized transcription that

correlated to increased synthesis of several families of proteins. These proteins were

termed heat shock proteins (Hsps) due to their initial discovery in cells exposed to

elevated temperatures. In addition to the increase in Hsps, the heat shock response also

involves cytoskeleton reorganization, alteration in protein phosphorylation, and, at high

temperatures, suppression of general protein synthesis (Coss and Linnemans, 1996). The

importance of the heat shock response for cellular survival after heat shock has been

demonstrated in many cells through experimental manipulation to cause induced






26


thermotolerance. In this phenomenon, cells are made resistant to the lethal effects of a

severe heat shock by pre-exposure to a mild, sub-lethal elevation in temperature (De

Maio, 1999). The phenomenon of induced thermotolerance involves increased

production of Hsps.

Heat shock proteins, which are present in the cytoplasm, mitochondria,

endoplasmic reticulum, and nucleus, have been divided into families that are named

principally on the basis of the approximate molecular weight (kDa) of the protein. Heat

shock protein families include Hspl00, Hsp00, Hsp70, Hsp60, Hsp40, and a large family

of small heat shock proteins (sHsp) that range from 12-30 kDa (Knowlton, 1994).

Inhibition of synthesis of Hsp70 and Hsp27 (Jakubowicz-Gil et al., 2002) by

administration of antisense mRNA has been shown to prevent induced thermotolerance in

specific cell types. In addition, transfection of rat cells with recombinant human Hsp70

conferred resistance to heat (Li et al., 1991) and constitutive high levels of Hsp27 give

elevated intrinsic thermal resistance in rodent cells (Landry et al., 1989; Lavoie et al.

1993). Hsp27 (Arrigo, 1998) and Hsp32 are known to confer protection against other

stressful stimuli (Bechtold and Brown, 2003). Antioxidant systems may also be involved

in induced thermotolerance since inhibition of GSH synthesis prevented induced

thermotolerance (Mitchell et al., 1983; Konings and Penninga, 1985; Skibba et al., 1989).

In general, Hsps function in the cell as molecular chaperones (Becker and Craig,

1994; Morimoto et al., 1992; Buchner, 1999), i.e., they function by mediating non-

covalent associations and modifications of other proteins through associative interactions.

It is now clear, in fact, that Hsps are not simply stress proteins but function as part of the

normal machinery of the cell in the absence of cellular stress. Proteins in the Hsp90






27


family form heterodimers with other proteins to participate in formation of clathrin-

coated pits during endocytosis, stabilization of steroid receptor and regulation of elF2

kinase (Caplan, 1999). The Hsp70 family (Li and Mivechi, 1999), which are functional

ATPases, can be divided into four types of proteins, namely Bip, found in the

endoplasmic reticulum and also called glucose-regulated protein 70 since glucose

concentration rather than heat shock is the primary regulator of transcription;

mitochondrial Hsp70; Hsc70, a constitutive form that is expressed in high amounts in the

absence of heat shock which can also be enhanced by heat shock; and Hsp70, the stress-

inducible form that is present in low amounts in the absence of heat shock and whose

synthesis is greatly increased during heat shock (Lee et al., 1992; Cheng and Lai, 1994;

Macouillard-Poulletier de Gannes et al., 1998; Wang et al., 1998; Bechtold and Brown,

2003). Proteins of the Hsp70 family play a major role in protein folding. Among their

actions, Hsc70 participates in ensuring that proteins are folded into an active

conformation as they are being synthesized (Li and Mivechi, 1999). Both Bip and

mitochondrial Hsp70 assist proteins to cross plasma membranes by facilitating unfolding

and then refolding after membrane translocation. The Hsp60 (also known as

chaperonins) are involved in Hsp70 and 90 interactions. The Hsp40 (Hdj-1) protein

assists with ATP hydrolysis functions of Hsp70 (Michels et al., 1997) by stabilizing the

ADP bound state of Hsp70 and thereby stimulating Hsp70 nucleotide hydrolysis, and by

coupling nucleotide binding and hydrolysis to release of the substrate in a folded state

(Minami et al., 1996).

Apart from their actions during normal cellular physiology, Hsps are involved in

protecting the cell from harmful stimuli. The most studied Hsp family is Hsp70. Hsc70






28


is constitutively expressed with very little heat inducibility while Hsp70 is highly

inducible by stress but is rarely present in unstressed cells (Widelitz et al., 1987; Wang et

al., 1998; Macouillard-Poulletier de Gannes et al., 1998). During heat shock, Hsp70 is

involved in recognizing denatured proteins and refolding them (Kiang and Tsokos, 1998).

In addition, Hsp70 has been shown to have antiapoptotic properties (Mosser et al., 2000).

Hsp32 is thought to contribute to cell recovery from stress through stimulating production

of bilirubin, a strong antioxidant (Dalton et al., 1999; Bechtold and Brown, 2003).

In addition, many Hsps have a close relationship with the cytoskeletal

components of the cell (Liang and MacRae, 1997). Several Hsp70 proteins have been

characterized as microtubule-associated proteins (MAPs) and attach polymerized tubulin

at the carboxy-terminal end. In addition, Hsps modulate actin assembly. Many studies

have demonstrated that upon heat shock the inducible form of Hsp70 localizes to the

cytoskeleton (Lee et al., 1992; Sanchez et al., 1994; Liao et al., 1995; Cheng and Lai

1994; Liang and MacRae, 1997; Wang et al., 1998). Thus, localization of Hsp70 to these

sites could be a an important mechanism by which Hsp70 aids in cell recovery from heat

shock. Furthermore, Hsp27 also have been shown to interact with the actin cytoskeleton

during heat shock (Lavoie et al., 1993).

Elevation in temperature causes a rapid induction of Hsps (Lee et al., 1992;

Cheng and Lai, 1994; Macouillard-Poulletier de Gannes et al., 1998; Wang et al., 1998;

Bechtold and Brown, 2003). In the mammalian cell, Hsp70 is maintained in an inactive

state by the transcription factors heat shock factor I and 2 (HSF1 and 2; Morimoto et al.,

1992; Sistonen et al. 1994; Pirkkala et al. 2001). These transcription factors are

synthesized constitutively in a latent form and undergo changes in respond to stressful






29


stimuli (HSF1) or to developmental and differentiation cues (HSF2) that allow the factors

to stimulate transcription. For the case of heat shock, protein denaturation in the cell

results in exposure of protein motifs in the hydrophobic regions of the denatured protein

that is recognized by Hsp70. As a result, Hsp70 binds to the protein by a mechanism

dependent upon ATP hydrolysis and interacts with the protein to refold it. At the same

time, HSF1 is released from Hsp70 and forms homotrimers. The trimerized HSFI

translocates to the nucleus where it binds to heat shock elements (HSE) and, upon

phosphorylation, directs transcription of the Hsp70 gene and other genes containing an

HSE (Morimoto et al., 1992). Following transcription, the trimeric forms of HSF

dissociate from the DNA and are converted back into non-active monomers (Morimoto et

al., 1992).

The HSE is found in the promoter region of many genes induced by heat shock

and consists of 5'-nGAAn-3' pentanucleotide repeats which usually occur in strings of

four or more (Barros et al., 1992). Other regulatory elements also exist in the promoter

regions of Hsps genes (Morimoto et al., 1992; De Maio, 1999). The Hsp70 gene also has

a TATA box and a CAAT box upstream of the HSE (Morimoto et al., 1992).

Furthermore, heat shock genes tend to lack introns or may possess them in small size and

number. At least in Drosophila, the promoter region of the Hsp70 gene seems poised to

induce rapid transcription upon heat shock. Transcriptional factors are bound and PolI is

engaged and has transcribed 20-40 bases before it stops. After HSF binds to the HSE and

gets phosphorylated Poll is activated and transcription of the gene occurs.

The chaperone complement in a cell determines the speed and duration of the heat

shock response. Attenuation of the response is mediated by molecular chaperones Hsp70






30


and Hdjl (Hsp40; Minami et al., 1996) through binding to HSF and prevention of

trimerization. Accordingly, overexpression of Hsp70 can represses the activation of

HSF-1. Thus, HSF-1 and Hsp70 form a negative-feedback loop whereby the amount of

transcriptional activation of the Hsp70 gene by HSF-1 is dictated by the amounts of free

Hsp70. When Hsp70 utilization increases during heat shock, HSF-1 trimerization is

facilitated and increased transcription of Hsp70 results. Heat shock binding protein 1

(HSBPl) is another protein that negatively regulates stress-induced HSFI DNA binding

and transcriptional activity (Satyal et al., 1998). HSBP1 selectively binds the HSF1

trimer and converts it to a monomer. Furthermore, association of HSBP1 with HSF

trimers and with Hsp70 coincides with attenuation of the heat shock response (Satyal et

al., 1998).

Free Radicals and Antioxidants

Oxygen radicals and other reactive oxygen species (ROS) are formed as the result

of incomplete reduction of molecular oxygen (Sies and Cadenas, 1985; Halliwell and

Gutteridge, 1990). The major species of free radicals in cells are superoxide ion,

hydrogen peroxide, and hydroxyl radical, which represent the result of reduction of 02

with one, two or three electrons respectively. Reactive oxygen species are generated

within a cell through several biochemical pathways including oxidative phosphorylation

and actions of enzymes such as NADPH oxidase, xanthine oxidase, and cycloheximide

(Guerin et al., 2001). It has been estimated that about 1-2% of the oxygen consumption

of a cell is incompletely oxidized to form ROS (Fulbert and Cals, 1992).

Many conditions that disrupt cellular homeostasis enhance the production of

reactive oxygen species (ROS; Flanagan et al., 1998; Ozawa et al., 2002). Because of





31


their strong electrophilic properties and propensity to react with most molecules within

the cell, ROS are potent mediators of cellular injury (Kourie, 1998; Jabs, 1999;

Leonarduzzi et al., 2000; Shackelford et al., 2000; Dizdaroglu et al., 2002; Halliwell,

2003). Cellular pathologies caused by ROS include damage to nucleic acids, which can

disrupt transcription, translation and DNA replication (Dizdaroglu et al., 2002), oxidation

and crosslinking of proteins, and lipid peroxidation (Wells et al., 1997). Lipid

peroxidation is a particularly-harmful response to free radicals since in the presence of

iron, the products of lipid peroxidation can generate additional hydroxyl radicals in a

chain reaction that consumes unsaturated fatty acids in membranes (Dargel, 1992).

Changes in lipids and membrane proteins induced by ROS can result in changes in

intracellular calcium concentrations (Dalton et al, 1999) and ion transport mechanisms

(Kourie, 1998).

Flanagan et al. (1998) demonstrated that heat shock increases the flux of cellular

free radicals in a non-transformed rat intestinal epithelial cell line. Their work supports

the hypothesis that heat shock-induced cellular damage may be mediated by the oxidative

stress resulting from increased generation of ROS. Elevated temperatures also activate

enzymes which are involved in the generation of free radicals. For example, heat shock

increases the conversion of xanthine dehydrogenase to the oxidase form, an important

source of oxygen-derived free radicals (Skibba et al., 1989). Studies by Skibba et al.

(1989) and Powers et al. (1992) showed that the hyperthermic toxicity to the liver is the

result of oxidative stress brought about by conversion of xanthine oxidase in

hyperthermic liver perfusions. In addition, liver levels of GSH were significantly

lowered following perfusion at hyperthermic temperatures. Further, heat shock has been






32


reported to increase phospatidilinositol turnover and to release arachidonic acid, the

substrate for cyclooxygenase and lipooxygenase which are possible sources of ROS

(Stevenson et al., 1986).

Given the abundant production of ROS in cells (-1-2% of oxygen consumed;

Fulbert and Cals, 1992), it is not surprising that cells possess several biochemical systems

to remove free radicals. In general, antioxidant systems can be divided into those that

react directly with free radicals to reduce the molecule to water and to enzymes that

facilitate transfer of electrons from donor molecules to achieve reduction of the free

radical. Among the former are water soluble antioxidants such as GSH and ascorbic acid

that are functional in the cytoplasm, and lipid-soluble antioxidants such as vitamin E

(Kaikkonen et al., 2001) and 3-carotene (Zhang and Oaye, 2000) that function in lipid

membranes. Among the enzymes involved in antioxidant defense are superoxide

dismutase (Harris, 1992), which converts superoxide to hydrogen peroxide, catalase and

GSH peroxidase (Arthur, 2000), which reduce hydrogen peroxide to water. Glutathione

is a tripeptide with sequence y-glu-cys-gly in which the hydrogen on the sulfhydryl of

the cysteine is used as an electron donor (Meister, 1983). Reduced GSH can be

regenerated from oxidized GSH by the enzyme GSH reductase and NADPH.

Several studies have demonstrated a beneficial effect of antioxidants on survival

or recovery of cells after heat shock. Addition of the antioxidant taurine blocked the

heat shock-induced killing of lymphocytes (Malayer et al., 1992). In another study,

Kuninaka et al. (2000) showed that suppression of mitochondrial manganese superoxide

dismutase in HCT 116 colon cancer cells by antisense RNA increased sensitivity to

hyperthermia in a dose-dependent manner. When HeLa cells are pretreated with diethyl






33


dithiocarbamate to deplete cells of super oxide dismutase or with diethymaleate, to

deplete cells of non-protein thiols, the inhibitory effects of heat were more pronounced

(Evans et al., 1983; Freman et al., 1985).

Heat Shock-Induced Apoptosis

Apoptosis is a form of programmed cell death essential for normal development

and homeostasis as well as in the removal of damaged cells. Among the features of

apoptotic death are the generation of large condensed chromatin bodies and degradation

of DNA into nucleosomal units of 200 base pairs fragments, condensation of cytoplasm,

generation of evoluted membrane segments, cell fragmentation and shrinkage, and

disintegration of mitochondria (Fiers et al., 1999; Gulbins et al., 2000). Apoptosis

initiation may be brought about by many physiological and pathophysiological stimuli

including heat shock (Luchetti et al., 2002; Ko et al., 2000; Paula-Lopes and Hansen,

2002a,b).


Apoptosis involves the activation of caspases that cleave a variety of subcellular

targets to cause the cellular features of apoptosis described above (Gulbins et al., 2000).

The degradation of DNA into 200 bp nucleosomal units that is the hallmark of apoptosis

involves activation of caspase 3 from an inactive proenzyme form and the result

proteolysis of an inhibitor to caspase-induced DNAse as well as the DNA repair enzyme

PARP (poly(ADP-ribose) polymerase; Eamshaw et al., 1999). One of the two major

mechanisms whereby caspases become activated is through increased permeability of the

mitochondria to release pro-apoptotic proteins such as cytochrome c, apoptosis-inducing

factor (AIF), and smac/Diablo from the inter-membrane space of the mitochondria (Ichas

and Mazat, 1998; Duchen, 1999; Waterhouse et al., 2003). When mitochondria are






34


damaged by harmful stimuli, the proton gradient is lost and the mitochondrial matrix

undergoes swelling (Ichas and Mazat, 1998). This swelling phenomenon is known as

permeability transition and results in outer mitochondrial membrane rupture and release

of proteins normally housed in the inter-membrane space (Duchen 1999; Waterhouse et

al., 2003). Cytochrome c induces caspase activation by forming a complex called the

apoptosome with a protein called Apaf-1 (apoptosis activating factor 1; Jiang and Wang,

2000). Upon formation of the apoptosome Apafl undergoes a conformational change

which leads to the autoproteolysis and self activation of activate procaspase-9. Mature

caspase-9 cleaves procaspase-3 to form caspase-3, the main executioner of apoptosis

(Gottlieb, 2000).

Environmental insult, such as heat shock, will lead to the initiation of the

apoptotic cascade through actions directly on the membrane. Acid sphingomyelinase will

be activated by heat shock and. in turn generate ceramide from sphingomyelin, which

through actions on the stress activated protein kinase/c-Jun N-terminal kinase

(SNAP/JNK) activates caspases Heat shock induces apoptosis in many cells (Boreham et

al., 1997; Matsumo et al., 1997; Buzzard et al., 1998; Jakubowicz-Gil et al., 2002). For

example, Jakubowicz-Gil et al. (2002) showed that heat shock at 42.0*C for 1 h increased

induction of apoptosis in HeLa cells as estimated by comet assay. In addition, heat shock

induces changes in the mitochondrial membrane potential and cytochrome c release

resulting in apoptosis (Ko et al., 2000). Heat-induced chromatin changes characteristic of

apoptosis have been characterized in HL60 human hemopoietic cells (Luchetti et al.,

2002). Heat shock of 43.0*C for 1 h induced the characteristic oligonucleosomic

fragmentation as determined by DNA gel electrophoresis. In addition, chromatin clusters






35


at the nuclear periphery, frequently showing dense masses protruding towards the

cytoplasm were observed.


Responses of Preimplantation Embryos to Heat Shock

Disruption of Cellular Function

While many experiments have demonstrated that exposure to elevated

temperature can inhibit development of preimplantation embryos (Ealy and Hansen,

1994; Ealy et al., 1995; Edwards et al., 1997; Ar6chiga and Hansen, 1998; Ju et al., 1999;

Al-Katanani and Hansen, 2002; Block et al., 2002; Krininger et al., 2002; Paula-Lopes et

al., 2003a,b) there is little experimental evidence to describe the mechanisms by which

this inhibition is affected. An early study by Elliot and Ulberg (1971) indicated that heat

shock can compromise formation and function of nucleoli. Four- and eight-cell mouse

embryos were collected from control or heat-stressed females and cultured in the

presence of [3H]uridine for 1 h. Embryos collected from heat-stressed females had fewer

stained nucleoli than did those from controls, indicating a reduced RNA synthetic ability.

Furthermore, there were fewer nucleoli in heat-stressed embryos than in control embryos.

There are also data that heat shock can reduce protein synthesis in embryos. In

particular, exposure of mouse (Edwards et al., 1995) and bovine embryos to a heat shock

of 42.0C decreased total intracellular protein synthesis as determined by monitoring

incorporation of [35S]methione into trichloacetic acid-precipitable protein. In bovine

embryos, the effects of heat shock depended on stage of development (Edwards and

Hansen, 1997). While heat shock reduced protein synthesis from the two-cell stage

through blastocyst stage of development, it had no effect on protein synthesis by






36


expanded blastocysts and increased protein synthesis by hatched blastocysts (Edwards

and Hansen, 1997).

At least in the mouse, actions of heat shock on embryos involve free radicals.

Maternal heat stress significantly increased hydrogen peroxide concentration of two-cell

embryos (Ozawa et al., 2002) while intracellular concentrations of GSH were reduced by

heat shock in two-cell embryos (Ozawa et al., 2002) and morulae (Ar6chiga et al., 1995).

Adverse effects of heat shock on mouse embryonic development can be

minimized by various antioxidants (Malayer et al., 1992; Ar6chiga et al., 1994). For

example, some thermoprotection was provided to mouse embryos by addition to culture

medium of taurine (Malayer et al., 1992), GSH (Ar6chiga et al., 1994, 1995) and vitamin

E (Ar6chiga et al., 1994). In contrast, bovine embryos in general are not protected from

effects of heat shock by antioxidants. While taurine and GSH provided some

thermoprotection to blastocysts (Ealy et al., 1992), there was no protective effect of GSH,

GSH ester, taurine or vitamin E (Ealy et al., 1995; Paula-Lopes et al., 2003a) on embryos

at earlier stages of development. Taken together, these results indicate that free radicals

are a more important source of cellular damage in mouse embryos than is the case for

bovine embryos.

Thermoprotective Mechanisms in Embryos

Hsp70

Heat shock proteins are one of the first proteins to be synthesized constitutively in

the early preimplantation embryo (Christians et al., 1995). In addition, heat shock can

induce synthesis of Hsp70 in mouse (Edwards et al., 1995) and bovine embryos (Edwards

and Hansen, 1996; Edwards et al., 1997; Chandolia et al., 1999a; Kawarsky and King,






37


2001). Data are contradictory, however, as to how early in development heat shock can

first induce Hsp70 synthesis. Edwards and Hansen (1996), Edwards et al. (1997), and

Chandolia et al. (1999a) all reported a large increase in the synthesis of Hsp70 following

heat shock in the two-cell bovine embryo, a stage at which the embryo's genome is

largely inactive (Thompson, 2000). The increase in Hsp70 synthesis was due to

transcription since inhibitors of transcription reduced the amount of Hsp70 mRNA

(Chandolia et al., 1999a). In contrast, Kawarsky and King (2001) did not observe

induction of Hsp70 synthesis until the eight-cell stage. In porcine embryos, there was no

effect of heat shock on synthesis of Hsp70 or Hsp90 in compact morula or blastocysts

(Kojima et al., 1996).

Antioxidants

As described earlier, heat shock appears to increase free radical production in

embryos, at least in mice (Arechiga and Hansen, 1995; Ozawa et al., 2002). Although

inhibition of GSH synthesis by buthionine sulfoximine (BSO) did not alter the

susceptibility of morula-stage mouse embryos to heat shock (Ardchiga et al., 1998), it did

block the induced thermotolerance phenomenon (Ar6chiga et al., 1995). Moreover,

induction of GSH synthesis by S-adenosyl-L-methionine decreased the deleterious effects

of heat shock (Ar6chiga et al., 1995).

Apoptosis

Recent studies demonstrate that early bovine embryos are capable of undergoing

heat-induced apoptosis (Krininger and Hansen, 2002; Paula-Lopes and Hansen, 2002a,b).

The response of embryos to heat shock is developmentally regulated since embryos less

than the 8-16 cell stage were not capable of undergoing apoptosis in response to heat






38


shock (Paula-Lopes and Hansen, 2002a). There are also developmental changes in

induction of apoptosis by exposure to arsenic (Krininger and Hansen, 2002) and tumor

necrosis factor-a (Soto et al., 2003), with 2-cells being incapable of apoptosis and

embryos at Day 5 after insemination undergoing apoptosis in response to these stimuli.

When induced, not all blastomeres of the bovine embryo undergo apoptosis.

After 41.0*C for 9 h, for example, 15% of cells from a Day 5 embryo were apoptotic.

While apoptosis is obviously fatal to the cell undergoing it, it is possible that a limited

amount of apoptosis in response to a harmful stimulus is beneficial to the function of a

tissue or, in this case, embryo. This argument has been advanced for the preimplantation

embryo exposed to heat shock and experimental evidence obtained to support it. First,

embryos that are not capable of apoptosis (< 8-16 cells) are more susceptible to inhibition

of development by heat shock than embryos capable of apoptosis (Edwards et al., 1997).

Furthermore, inhibition of heat shock-induced apoptosis by incubation with the caspase

inhibitor z-DEVD-fmnk not only blocked apoptosis in Day 5 bovine embryos but also

exacerbated the block to development cause by heat shock (Paula-Lopes and Hansen,

2002b).






39







Synopsis Rationale for Thesis

Although there is a great deal of information on the effects of heat shock on

cellular systems, information is scarce on the effects of heat shock on early mammalian

embryo. Most of the work performed to understand the biochemical, cellular and

ultrastructural effects of heat shock on cells have involved temperatures that would be

lethal in most mammals. For example, temperatures in the range of > 43.00C have been

used to study the cytoskeleton (Wachsberger and Coss, 1990), functional morphology of

organelles (Funk et al., 1999), and mitochondrial function (Macouillard-Poulletier de

Gannes et al., 2000). In contrast, embryonic loss in females due to heat stress occurs

when maternal rectal temperatures are in the range of 40.0-41.00C in cattle (Dunlap and

Vincent, 1971; Putney et al., 1988; Ealy et al., 1993), 39.0-40.3C in mice ( Elliot et al.,

1968; Elliot and Ulberg, 1971;Bellv6, 1972), 40.00C in rats (Femandez-Cano, 1958) and

40.0C in sheep (Alliston and Ulberg, 1961; Alliston et al., 1961; Dutt, 1963). Hence,

many of the conclusions regarding the mechanism by which heat shock damages cells

derived from studies using temperatures at 43.00C or above may not be relevant to the

situation for the early mammalian embryo. Accordingly, the objective of this dissertation

is to characterize some of the cellular, subcellular and developmental responses of the

two-cell bovine embryo (i.e., when the embryo is most sensitive to heat shock in the cow)

to a physiologically-relevant heat shock. These results offer the promise of extending our

understanding of cellular consequences of heat shock to the conditions that are actually

experienced commonly by mammalian cells. In addition, identification of the






40


mechanisms by which heat shock disrupts embryonic development may eventually lead

to new approaches for improving embryonic survival during heat stress through

manipulation of cellular function of the embryo.














CHAPTER 2
DEVELOPMENT OF CULTURED BOVINE EMBRYOS AFTER EXPOSURE TO
INCREASED TEMPERATURES IN THE PHYSIOLOGIC RANGE


Introduction

Heat stress can reduce fertility in cattle (Dutt, 1963; Dunlap and Vincent, 1971;

Turner, 1982; Ealy et al., 1993; Ryan et al., 1993) and other species (Shah, 1956; Alliston

and Ulberg, 1961; Tompkins et al., 1967). The mechanism by which heat stress causes

embryonic mortality is multifactorial since heat stress can alter several aspects of

reproductive physiology including blood flow to the reproductive tract (Roman-Ponce et

al., 1978), ovarian steroid concentrations (Gwazdauskas et al., 1973; Roman-Ponce et al.,

1981; Badinga et al., 1993; Trout et al., 1998), and patterns of follicular development

(Badinga et al., 1993; Wolfenson et al., 1995). One possibility is that embryos cannot

survive the elevation in oviductal and uterine temperature coincident with heat stress.

Culture of embryos at elevated temperatures has been reported to reduce embryonic

development (Ulberg and Sheean, 1973; Edwards and Hansen, 1996; Edwards and

Hansen, 1997). Similarly, elevated culture temperatures can compromise oocyte function

(Lenz et al., 1983; Baumgartner and Chrisman, 1987; Edwards and Hansen, 1996) and

fertilization rate (Lenz et al., 1983; Ulberg and Burfening, 1967).

Studies in cattle that have demonstrated deleterious effects of heat shock on

cultured embryos have used temperatures of 41.0* through 43.0*C (Ealy et al., 1992; Ealy

and Hansen, 1994; Edwards and Hansen, 1996; Edwards and Hansen, 1997). However,




41






42


these experimental temperatures are higher than those often experienced by heat-stressed

cows with reduced fertility. For example, Dunlap and Vincent (1971) saw a reduction in

fertility from 48% in control animals to 0% for heifers exposed to 32.2*C for 72 h

immediately following breeding. In that study, however, the average rectal temperature

of heat-stressed heifers was 40.0*C. A heat shock of 40.0*C actually increased

development of cultured bovine embryos (Ryan et al., 1992).

Heat shock in culture also differs from the situation in utero in several other

respects. In vitro, for example, increases in temperature cause decreased CO2 solubility

in culture medium and an increase in medium pH. This confounding of pH with heat

shock has not been considered heretofore in experiments evaluating effects of heat shock

on embryos or, more generally, for cultured cells. Experiments on heat shock have also

been done in a gaseous environment where 02 content is higher than in the oviduct (i.e.

8.7% in rabbit; Fischer and Bavister, 1993). Since effects of heat shock on embryonic

development also involve free radicals (Ealy et al., 1992; Ar6chiga et al., 1995), it is

unknown whether heat shock would affect embryonic development at oxygen

environments similar to those in vivo.

The objectives of the present studies were: 1) to determine whether exposing

bovine oocytes and embryos to temperatures characteristic of body temperatures of heat-

stressed cows would affect embryonic development in vitro, 2) to test if culturing early

bovine embryos in low oxygen concentration would eliminate the adverse effects of heat

shock on embryonic development, and 3) to verify that the detrimental effects of heat

shock were not due by changes in pH as a result of reduced solubility of CO2 at increased

temperatures.






43


Materials and Methods

Materials

Follicle stimulating hormone (FSH), was Folltropin-V from Vetrepharm Canada

Inc. (London, Ontario) and was purchased from AgTech (Manhattan, KS). The culture

media SP-TL, IVF-TL, and HEPES-TL were prepared by Cell and Molecular

Technologies, Inc. (Lavallete, NJ) using recipes described by Parrish et al. (1986). Using

these media, SP-TALP (Tyrodes Albumin Lactate Pyruvate), HEPES-TALP, and IVF-

TALP were prepared as described by Parrish et al. (1986). The CRlaa medium was

prepared as described by Rosenkrans et al. (1993). Bovine serum albumin Fraction V

(Fraction V BSA), essentially fatty-acid free BSA Fraction V (EFAF-BSA), and all other

chemicals were from Sigma (St. Louis, MO).

In Vitro Production of Embryos

Bovine ovaries were obtained from a local abattoir located at a travel distance of

-1.5 h from the laboratory. Ovaries were transported to the laboratory in 0.9% (w/v)

NaCI at room temperature. Ovaries were sliced and oocyte-cumulus complexes (OCCs)

were collected into a beaker containing oocyte collection medium [TCM-199 with

Hank's salts without phenol red and supplemented with 2% (v/v) bovine steer serum

(containing 2 U/ml heparin), 100 lU/ml penicillin, and 0.1 mg/ml streptomycin]. Oocyte-

cumulus complexes were cultured in 50 pl microdrops of oocyte maturation medium

(TCM-199 with Earle's salts supplemented with 100 IU/ml penicillin, 0.01 mg/ml

streptomycin, 2 ptg/ml estradiol, 20 ig/ml FSH, and 0.2 mM sodium pyruvate) in groups

of 10 for 20-23 h. After maturation, OCCs were washed in HEPES-TALP and placed in

groups of- 30 in 600 tl IVF-TALP in 4-well plates. Spermatozoa, purified by Percoll






44


gradient centrifugation (Parrish et al., 1986) and suspended in SP-TALP, were added to

the matured oocytes at a density of~l x 106 spermatozoa/well. Coculture of spermatozoa

and OCCs proceeded for 8-10 h after which putative zygotes were denuded of cumulus

cells by vortexing in a 2.0 ml microcentrifuge tube containing ~0.5 ml HEPES-TALP for

5 min. Putative zygotes were cultured in 50 pl microdrops of CRIaa. On Day 5 post-

insemination, 5 l fetal bovine serum were added to each culture drop.

All cultures were performed at 38.5C in 5% (v/v) CO2 in humidified air unless

otherwise specified. When cultures were performed at elevated temperatures, the CO2

percentage of the gas phase was adjusted to prevent pH changes in medium caused by

decreased solubility of CO2 at increased temperatures. The CO2 percentage needed to

maintain a pH of ~7.4 in the CR1 aa medium was experimentally determined for each

temperature. These were 5.5%, 6.0%, 6.5%, and 7.0% CO2 for 39.5, 40.0, 40.5, and

41.0*C, respectively. The accuracy of the incubators was 0.2*C for one of the

incubators and 0.1C for two of the incubators. The temperatures of all incubators were

routinely calibrated by the use of mercury thermometers.

Heat Shock During Fertilization

Co-culture of spermatozoa and OCCs progressed for 8 h at 38.5, 40.0, or 41.0*C.

After fertilization, putative zygotes were cultured at 38.5C for 8 d. Cleavage rate was

determined on Day 3 post-insemination and development to the blastocyst stage was

determined on Day 8 post-insemination. This experiment was replicated six times using a

total of 179-180 oocytes per treatment.






45


Heat Shock at the One-Cell Stage

After co-culture of spermatozoa and OCCs had taken place (8 hpi), putative

zygotes were cultured in 50 pil microdrops in groups of 27-32 at 38.50C continuously or

were exposed to 40.0 or 41.00C for 3, 6, 9, or 12 h. After this period, all embryos were

cultured at 38.5*C for the duration of the culture. Cleavage rate was determined on Day

3 post-insemination and development to the blastocyst stage was determined on Day 8

post-insemination. The experiment was replicated four times using 117-121 putative

zygotes per treatment.

Heat Shock at the Two-Cell Stage

Two experiments were conducted. In both, embryos at the two-cell stage were

collected from culture drops at 28 h post-insemination and transferred to a new drop of

CRlaa medium (8-25 embryos/drop). For the first experiment, embryos were then

cultured for 3, 6,9, or 12 h at 40.0 or 41.00C or were maintained at 38.5C. Further, a

group of embryos was left undisturbed in the original culture drops (i.e., two-cell

embryos were not separated) to test whether the handling itself would adversely affect

development. After heat shock, embryos were returned to 38.5C for the duration of

culture. Development to the blastocyst stage was determined on Day 8 post-

insemination. Unlike most experiments, the type of BSA used to supplement CRlaa was

Fraction V. The experiment was replicated five times with a total of 54-84 two-cell

embryos per treatment.

The second experiment was conducted similarly except that heat shocks were 39.5

and 40.5*C. Further, the CRlaa was supplemented with EFAF-BSA. The experiment

was replicated four times with a total of 52-68 embryos per treatment.






46


Effect of Type of BSA on Response to Heat Shock

The experiment was designed as a 2 x 2 factorial with two types of BSA (Fraction

V and EFAF) and two temperatures (38.5 or 41.0C). Two-cell embryos were collected

at ~28 h post-insemination and placed in CRlaa supplemented with either EFAF-BSA or

with Fraction V BSA. Embryos were cultured at 38.5*C continuously or at 41.0*C for 12

h and 38.5*C thereafter. Development to the blastocyst stage was determined on Day 8

post-insemination. The experiment was replicated three times with a total of 57-58

embryos per treatment.

Effect of Oxygen Concentration on Response to Heat Shock

This experiment was performed to test whether the effect of heat shock in culture

is exacerbated by a high 02 environment. In order to regulate the gaseous environment of

cultured embryos, a chamber was constructed (Figure 2-1) from a 115 ml vacuum filter

unit from Coming. Tygon tubing (3.2 mm i.d. x 6.4 mm o.d.) with a stopcock fastened to

the end was attached to the hose connector and a 00 size rubber stopper was inserted into

the pour spout. The cellulose nitrate membrane in the filter was punctured in several

places to facilitate movement of gases. Culture plates were gently positioned on top of

the membrane. The lid of the filter system was taped securely with scotch tape and

parafilm was placed around the lid to make the chamber airtight. Gases where injected

for 3 min through the pour spout from a hose connected to the gas cylinder while the

stopcock was in the open position. After injection of gases was completed, the rubber

stopper was fastened to the pour spout and the stopcock was dialed to the closed position.

To prevent contamination during injection of gases, a filter (0.22 pun) was attached to the






47


gas hose. The chambers were shown to be airtight since pH was maintained for 24 h in

the presence of various gas mixtures.

The design was a 2 x 2 factorial with two 02 tensions (5% or 20.95%) and two

temperatures (38.5 and 41.0C). Embryos were collected at the two-cell stage and placed

in fresh microdrops (14-27/drop). Culture plates were then placed into airtight chambers.

The chambers containing the embryos cultured at 38.5*C were injected with a mixture of

gases containing high (20.95%, v/v) or low (5%, v/v) 02 with 5% (v/v) CO2 and a balance

of N2. Chambers containing the embryos cultured at 41.0C were injected with a similar

mixture of gases except that CO2 was 7% (v/v) to maintain a pH of ~ 7.4 and the N2

content was adjusted accordingly. After 12 h of culture, plates were removed from the

chambers and returned to incubators at 38.5C and 5% (v/v) CO2 in air until Day 8 post-

insemination. The experiment was replicated four times with a total of 120-122 embryos

per treatment.

Rectal Temperatures of Heat-Stressed Lactating Dairy Cows

This experiment was performed as a prelude to determining effects of culture

temperatures characteristic of those experienced by heat-stressed cows on embryonic

development. Animals used were primiparous (n = 8) or multiparous (n = 17) lactating

Holsteins (50-150 d in milk except for one cow at 266 d in milk) located in north Florida

(Hague, Florida; 29' 46" N 82' 25" W). Cows were milked three times per Day and

received injections of bovine somatotropin (Posilac, Monsanto, Chesterfield, MO)

according to manufacturer's recommendations. Milk yields on the day of recording

ranged from 17.2 to 33.6 kg/d. Cows were maintained in free-stall barns equipped with a

cooling system utilizing high-pressure foggers. On each of three separate days in August






48





























Figure 2-1. Chamber used to regulate gaseous environment of cultured embryos in low
and high oxygen tensions. The chamber was constructed from a 115 ml-filter system
from Corning. Tygon tubing (3.2 mm i.d. x 6.4 mm o.d.) with a stopcock fastened to the
end was attached to the hose connector and a 00 size rubber stopper was inserted into the
pour spout. The cellulose nitrate membrane in the filter was punctured in several places
to facilitate movement of gases. Culture plates were gently positioned on top of the
membrane. The lid of the filter system was taped securely with scotch tape and parafilm
was placed around on the lid to make the chamber airtight. Gases where injected for 3
min through the pour spout from a hose connected to the gas cylinder while the stopcock
was in the open position. After injection of gases was completed, the rubber stopper was
fastened to the pour spout and the stopcock was dialed to the closed position. To prevent
contamination during injection of gases, a filter (0.22 pm) was attached to the gas hose.
The chambers were shown to be airtight since pH was maintained for 24 h in the presence
of various gas mixtures.






49


1999, a group of 9 cows was examined. Cows were chosen so that three cows were

greater than 75% white in color (white), three cows were between 25-75% white (black

and white) and three cows were less than 25% white (black). At each hour for 24 h,

rectal temperatures were measured using mercury rectal thermometers (precision =

0.1C) and respiration rates were determined. In addition, relative humidity, dry bulb

temperature, and black globe temperature were recorded each hour. For each replicate, a

separate group of 8 or 9 cows in a different free-stall barn was used.

Exposure of Embryos to a Pattern of Temperatures Similar to those Experienced by
Heat-Stressed Cows

Incubators were set at 38.5, 39.5, or 40.5C. The heat shock treatment consisted

of sequential exposure to 38.5C (5 h), 39.51C (5 h), 40.5*C (5 h), and 39.5*C (9 h).

Given the limited number of incubators available, this pattern was found to most closely

mimic the pattern of rectal temperatures experienced by cows in the previous experiment

(see results). Putative zygotes were cultured at either 38.50C continuously (control), heat

shocks from d 0-1 post-insemination (HSDO-1), or heat shock on each day from Day 0-8

post-insemination (HSDO-8). The experiment was replicated four times with a total of

121 to 122 embryos per treatment.

Statistical Analysis

For embryo experiments, percent cleavage and development was calculated for

each microdrop of putative zygotes or embryos. Each experiment was replicated on

several days with one or more microdrop per treatment on each day. Data were analyzed

by least-squares analysis of variance using the PROC GLM procedures of SAS (1989).

All main effects were considered fixed. Embryo number per drop was also included as a

covariate for experiments with two-cell embryos where there was variation in the number






50

of embryos per drop. Percentage data were transformed by arcsine transformation before

analysis. The analysis of transformed data was used for probability values but least-

squares means SEM are presented from analysis of untransformed data. In some

experiments, the SAS option pdiff was used to compare the mean of each treatment with

the mean of the control group. Also, orthogonal contrasts were used to determine

whether the effects of duration of heat shock followed a linear, quadratic or cubic pattern.

Data from rectal temperatures were analyzed by least-squares analysis of

variance. The mathematical model included main effects of coat color, replicate, cow

(replicate x coat color), and time. All interactions were included in the model. Cow was

considered a random effect and other main effects were considered fixed.



Results

Effect of Heat Shock During Fertilization

Bovine oocytes fertilized at 41.0*C had lower (P < 0.01) cleavage rate than

oocytes fertilized at 38.5*C (Figure 2-2, top panel). The proportion of oocytes (P < 0.01)

and cleaved embryos (P < 0.01) that developed to blastocyst was also decreased (P <

0.01) by 41.0*C (Figure 2-2, bottom panel). A heat shock of 40.00C during fertilization

had no effect on cleavage rate and tended to increase (P < 0.06) the proportion of oocytes

forming blastocysts when compared to oocytes fertilized at 38.5C.

Heat Shock at the One-Cell Stage

No reduction in cleavage rate or blastocyst formation was detected for one-cell

embryos cultured at 40.00C for 3, 6, 9, or 12 h or 41.0C for 3 or 6 h. However, a heat

shock of 41.00C for 12 h reduced (P < 0.01) cleavage rate (Figure 2-3, top panel).






51






100
90 T
80
70
> 60
(3) 50
0 40
0 30
20
10
0 __
38.5 40.0 41.0


100
90
U 80
70
0 60 oocyte to blastocyst cleaved embryo to blastocyst
C/ 50
S40
30
-0
20
10 '

38.5 40.0 41.0 38.5 40.0 41.0


Temperature (oC)



Figure 2-2. Effect of temperature during fertilization on the percentage ofoocytes that
cleaved (top panel) and that became blastocyst on Day 8 post-insemination (bottom
panel). Data are least-squares means SEM. Fertilization was for 8 h at the
temperatures indicated. Replications were 6 and number ofoocytes per treatment were
179-180. ** = different from control, P < 0.01.






52








100
90

0 80
( 70
CU 60
5 50
40
30
20
10
0 ......
0 3 6 9 12
100
lm 38o ST
0 90 40 OC
0 80 } 41 (fC
S 70
0 60
0 50
40
C? 30
20
10
0-
0 3 6 9 12

Hours of heat shock



Figure 2-3. Effect of heat shock at the one-cell stage on the percentage of oocytes that
cleaved (top panel) and that became blastocyst on Day 8 post-insemination (bottom
panel). Data are least-squares means SEM. Replications were 4 and number of
oocytes per treatment were 117-121. ** = different from control, P < 0.01.






53


Similarly, development to the blastocyst stage was adversely affected only by heat shocks

of 41.00C for 9 or 12 h (P < 0.01; Figure 2-3, bottom panel).

Heat Shock at the Two-Cell Stage

In the first part of this experiment, 40.00C did not significantly reduce the percent

of two-cell embryos that became blastocyst by Day 8 post-insemination (Figure 2-4, top

panel). In contrast, 41.00 C for 9 and 12 h reduced (P < 0.01) the proportion of embryos

that were blastocysts at Day 8 post-insemination. In the second experiment, neither 39.5

nor 40.5C reduced the rate of blastocyst formation (Figure 2-4, bottom panel). Note that

the proportion of embryos reaching blastocyst was greater for the second experiment, in

which medium was supplemented with EFAF-BSA, than for the first experiment, in

which medium was supplemented using Fraction V BSA. Accordingly, another

experiment was done to verify this effect of BSA type, and to test whether BSA source

would affect responses of heat shock (Figure 2-5, top panel). Two-cell embryos cultured

at 38.5C had a higher rate of blastocyst formation than embryos exposed to 41.0C for

12 h (P < 0.02). In addition, the percent of embryos becoming blastocyst was higher (P <

0.01) for embryos cultured in CRlaa medium containing EFAF-BSA than for those

cultured in CRlaa medium containing Fraction V. There was no temperature x BSA

interaction; however, indicating that the effect of heat shock was similar for the two

media.

Effect of Oxygen Concentration on Response to Heat Shock

Two-cell embryos cultured at 38.5*C had a higher rate (P < 0.01) of blastocyst

formation than embryos exposed to a heat shock of 41.0C for 12 h (Figure 2-5, bottom

panel). Low 02 had a beneficial effect on subsequent development to the blastocyst stage






54









100
Cl) 90 M sas
0 80 [ 41 oc
o
70
S60







0

100

90
20 T0
0 10








050



0.) 30
S10






0
0 3 6 9 12
Hours of heat shock



Figure 2-4. Effect of heat shock at the two-cell stage on the percentage of two-cell
embryos that became blastocyst on Day 8 post-insemination. Embryos were shocked at
various periods beginning at -28 h post-insemination. Data are least-squares means +
SEM. For 400C, there was a cubic effect (P < 0.1) of duration of heat shock. For 41.00C,
there was a linear effect (P < 0.001) of duration of heat shock. Replications were 4 and
number ofoocytes per treatment were 52-68. ** = different from control, P < 0.01.






55

100 -
90 38 C
"* 80 4
Q 70
60
B 50
40
30- -
20
10
0-
EFAF Fract V

BSA Type

100
S38 5SC
90 41 OC
>. 80
0

S60
0 50
= 40
S30 I
20-
10

5 % 20.95%

Oxygen content (v/v)


Figure 2-5. Top panel. Effect of culture conditions on responses of two-cell embryos to
heat shock. Effect of type of bovine serum albumin (BSA) used to supplement the
culture medium on subsequent development to the blastocyst stage. Results are the
percentage of two-cell embryos that became blastocyst on Day 8 post-insemination. The
percent of embryos becoming blastocyst was different (P < 0.02) between embryos
cultured at 38.50C vs. 41.00C for 12 h and between embryos cultured in medium
containing essentially fatty acid free (EFAF) BSA vs. Fraction V BSA (P < 0.01). There
was, however, there was no temperature x BSA type interaction. Data are least-squares
means SEM. Replications were 3 and number ofoocytes per treatment were 57-58.
Bottom panel. The effect of O2 concentration (5% or 20.95%) during heat shock of two-
cell embryos on subsequent development to the blastocyst stage. Results are the
percentage of two-cell embryos that became blastocyst on Day 8 post-insemination.
Percent blastocyst was affected by incubation temperature (P < 0.01) and 02
concentration (P = 0.07). Data are least-squares means SEM. Replications were 4 and
number of oocytes per treatment were 120-122.






56


(P = 0.07) even though embryos were in the presence of low 02 for only 12 h at the two-

cell stage. However, culturing two-cell embryos in low 02 tensions did not decrease the

effect of heat shock on embryonic development as indicated by the lack of oxygen x

temperature interaction. Heat shock reduced development from 53.5 to 27.0% at 20.95%

02 and from 69.4 to 29.9% in 5% 02.

Rectal Temperatures of Heat-Stressed Lactating Dairy Cows

The average peak air temperature and peak black globe temperature (Figure 2-6,

top panel) on the days when the experiment was performed were 34.5* and 33.7*C,

respectively. Mean rectal temperatures (Figure 2-6, bottom panel) ranged from a low of

38.6C (0800 h) to a high of 40.5C (1700 h). Mean respiration rates ranged from a low

of 71.8 breaths per minute (000 h) to a high of 105 breaths per minute (1200 h; data not

shown). Coat color affected (P< 0.001) rectal temperatures of cows (Figure 2-6, bottom

panel), with predominantly white cows having lower rectal temperatures that

predominantly black cows.

Exposure of Embryos to a Pattern of Temperatures Similar to Those Experienced
by Heat-Stressed Cows

Results from the previous experiment were used to design a pattern of culture

temperatures that would mimic the 24-h variation in body temperatures (Figure 2-8, top

panel). Applying heat shock in this pattern during the first 24 h after insemination did

not affect embryonic development to the blastocyst stage (Figure 2-8, bottom panel). In

contrast, application of this pattern of fluctuating temperatures for 192 h (from d 0-8 after

insemination) reduced embryonic development to the blastocyst stage (P < 0.05).







57










o 36.0
Dry bulb lemp
0 Black globe temp
^ 34.0

32.0

30.0

.S 28.0

E 26.0
C
2 24.0

LU 22.0 -


41.0
S SEM=0 19 Black
40.5 Bk & Whi

40.0

CL 39.5
E
39.0

38.5

38.0
0700 1100 1500 1900 2300 0300 0700

Time of day



Figure 2-6. Rectal temperatures of lactating dairy cows under heat stress conditions in
North Florida. The top panel shows dry bulb and black globe temperature while the
bottom panel shows rectal temperatures for cows that are predominantly black (n=9),
about equally black and white (n=8), and predominantly white (n=8). Rectal temperature
was affected by coat color (P < 0.001). Data are least-squares means SEM. The black
bar shows the period from sunset to sunrise.







58




41.0

40.5

40.0

39.5

S39.0

38.5

38.0
7am 11am 3pm 7pm 11pm 3am 7am

Time of day

100
90
80
70
60 oocytes to blastocyst cleaved embryo to blastocyst
M 50
40





Control HSDO-1 HSD0-8 Contro HSDO-1 HSDO-8

Treatment






Figure 2-7. Effect of exposing cultured embryos to a pattern of temperatures similar to
that experienced by heat stressed cows. Top panel: closed circles represent rectal
temperatures of heat-stressed, lactating dairy cows in North Florida (least-squares +
SEM). Open circles represent culture temperatures for heat-shocked embryos. Bottom
panel: Percent of oocytes and percent of cleaved embryos becoming blastocyst for
embryos exposed to 38.50C continuously (Control), heat shock for 24 h beginning at the
end of fertilization (HSDO-1) or heat shock throughout the culture period (HSDO-8).
Replications were 4 and number of oocytes per treatment were 121-122. = different
from control P < 0.05.





59


Discussion

The results of this study indicate that embryonic development can be disrupted by

heat shock of 41.0*C applied during fertilization and at the one- and two-cell stage of

development. Further, exposing early embryos to temperatures similar to those

experienced by heat-stressed dairy cows can reduce development to the blastocyst stage.

Thus, under certain circumstances, embryonic development is likely to be compromised

by the direct actions of elevated temperature on the oocyte and embryo. The finding that

short-term heat shock (_ 12 h incubation) only affected development at very high

temperatures (41.0*C) may mean that certain causes of heat-associated infertility

associated with mild maternal hyperthermia is the result of effects other than direct

actions of elevated temperature on embryonic survival.

Many previous reports have indicated deleterious effects of elevated temperature

on oocytes and early embryos (Gwazdauskas et al, 1992; Ealy et al., 1995; Edwards and

Hansen, 1997; Ar6chiga and Hansen, 1998). In those experiments, however, temperature

was confounded with changes in pH because CO2 solubility in the medium decreases as

temperature increases. Here it is shown that, even when controlling for pH changes,

decreased development follows when either oocytes, one- or two-cell embryos are

exposed to elevated temperature. Moreover, this effect of heat shock is not simply an

artifact caused by high oxygen concentration in culture. Embryos are most commonly

cultured in air, which has an 02 content of about 20.95%, which is higher that what is

found in the oviduct and/or the uterus (< 10%; 26). Given the fact that free radical

formation can increase as a result of exposure to high 02 tensions (Fowler and

Callingham, 1978; Goto et al., 1993) and elevated temperatures (Flanagan et al., 1998), it






60

is possible that the detrimental effects of heat shock in cultured embryos involves free

radical formation. In addition, heat shock of cultured embryos decreases glutathione, a

free radical scavenger (Ar6chiga et al., 1995) and adverse effects of heat on embryonic

development can be minimized by various antioxidants (Ealy et al., 1992; Malayer et al.,

1992; Ar6chiga et al., 1994; Ar6chiga et al., 1995). Accordingly, it was tested whether

low 02 tension would reduce the detrimental effect of heat on two-cell embryos,

presumably by lowering free radical production on two-cell embryos. There was no

temperature by 02 interaction, suggesting that the deleterious effects of heat shock did

not depend upon a high 02 environment.

Several studies have demonstrated superior development of bovine embryos

cultured at 5% 02 versus in air (Dumoulin et al., 1995; Takahashi et al., 1996; Fujitani et

al., 1997; Lim et al., 1999). In this study, a beneficial effect of low 02 was seen when

embryos received low 02 for only 12 h at the two-cell stage. The modified media filters

used here offer a practical method for controlling oxygen concentration in embryo culture

to increase blastocyst yield.

The type of BSA used to supplement the culture medium also had an effect on

development of embryos to the blastocyst stage. In particular, embryos developed better

in EFAF-BSA than in Fraction V BSA. Despite their superior development, embryos in

EFAF-BSA were not more resistant to heat shock. The superior development of embryos

in EFAF-BSA could be due in part to the removal of fatty acids and other impurities from

Fraction V BSA. The Fraction V-EFAF BSA difference may also have reflected simple

batch-to-batch variation (McKieman and Bavister, 1992) rather than any purification

process-related effect.






61


In the experiment in which rectal temperatures of cows was determined, cows that

were mostly white had lower rectal temperatures than cows that were mostly black. This

is in accordance with previous observations that white cows exposed to intense solar

radiation had lower rectal and surface temperatures than black cows (Hansen, 1990). In

addition, King et al. (1988) reported that predominantly-white cows bred in warm months

had a shorter interval from calving to conception than did predominantly-black cows. In

contrast, Godfrey and Hansen (1996) failed to observe any effect of coat color on

reproduction of Holstein cows in the Caribbean.

As others have shown (Ulberg and Burfening, 1967; Lenz et al., 1983), heat shock

during fertilization reduced not only cleavage rate but the ability of cleaved embryos to

develop. Again, a high temperature (41.0C) was necessary to achieve this effect. The

fact that 41.00C affected fertilization rates may have been due in part to effects on the

oocyte (Lenz et al., 1983; Baumgartner and Chrisman, 1987; Edwards and Hansen, 1997;

Putney et al., 1988; Rocha et al., 1998) or spermatozoa (Lenz et al., 1983; Chandolia et

al., 1999b). Heat shock could also affect syngamy or the first cleavage division. These

processes involve microtubule and microfilament assembly (Kim et al., 1997) and

cytoskeletal elements collapse and aggregate following heat shock (Welch and Suhan,

1985). Embryos formed from fertilization at 41.0C had reduced developmental

potential, perhaps because damage to the cytoskeleton or other intracellular organelles

persists. In addition, embryos formed via fertilization with heat-damaged spermatozoa

had lower developmental potential (Ulberg and Burfening, 1967). Interestingly, exposure

of oocytes and spermatozoa to 40.0*C during fertilization did not affect cleavage rate but

did increase subsequent embryonic development. Perhaps, 40.0*C was too mild to






62


damage gametes or embryos and the increased thermal energy at 40.0C increased

embryonic metabolic rate.

The present data do confirm that heat shocks such as experienced by cells of heat-

stressed cows can compromise embryonic development. In vivo, heat-stressed cows

typically experience daily fluctuations in body temperature that persist for many days.

Results from the last experiment indicated that exposure of embryos to fluctuating

temperatures similar to rectal temperatures of cows experiencing heat stress could

decrease embryonic development in vitro if occurring for several days. Thus, a longer,

more chronic heat shock may reduce embryo survival through direct actions of elevated

temperature on embryo. One important note is that the temperatures chosen to perform

this experiment were averages of rectal temperatures of cows experiencing heat stress and

that uterine temperatures are -~0.2C higher than rectal temperatures (Gwazdauskas et al.,

1973). Thus temperatures experienced by embryos can be higher than used here.

Despite the fact that heat shock can disrupt development, a striking finding was

that embryonic development was resistant to disruption by heat shocks of 39.5-40.5 for

up to 12 h. Thus, it is possible that infertility associated with mild hyperthermia (Dunlap

and Vincent, 1971) involves rather more chronic effects of heat shock exposure on

embryos or is the result of other changes in oocyte, reproductive tract, or embryonic

function independent of direct effects of heat shock on embryos.

In conclusion, embryonic development can be disrupted by short-term (9 and 12

h) heat shock applied during fertilization and at the one- and two-cell stage but only when

the heat shock is severe (> 41.00C). Further, exposing embryos throughout the 8 days of

in vitro culture to a pattern of temperatures similar to those experienced by heat stressed






63


lactating dairy cattle affects their development to the blastocyst stage. In addition, the

effects of heat shock are not due to artifacts caused by change in pH or high oxygen

concentration.






64







CHAPTER 3
ALTERATIONS IN ULTRASTRUCTURAL MORPHOLOGY OF TWO-CELL
BOVINE EMBRYOS PRODUCED IN VITRO AND IN VIVO FOLLOWING A
PHYSIOLOGICALLY-RELEVANT HEAT SHOCK


Introduction

Development of the cleavage-stage embryo is very susceptible to disruption by

elevated temperatures (Alliston et al., 1965; Ulberg and Burfering, 1967; Edwards and

Hansen, 1996; Edwards and Hansen, 1997; Chapter 2). Temperatures that have little

effect on survival of most cells can be lethal to preimplantation embryos. For example,

development of mouse embryos was inhibited by exposure to 39.0*C beginning at the

one-cell stage (Gwazdauskas et al., 1992) and development of bovine two-cell embryos

was decreased by exposure to 41.0C for as little as 4.5 h (Krininger et al., 2002). In

contrast, temperatures in this range have no effect on survival of other cells (Rattan,

1998). Hypersensitivity of the preimplantation embryo to elevated temperatures is a

transient phenomenon: effects of heat shock on development of cultured bovine embryos

are reduced as embryos advance in development past the two- to four-cell stage (Edwards

and Hansen, 1997; Krininger et al., 2002). The susceptibility of the embryo to elevated

temperature is likely one of the major causes for the decrease in fertility and increase in

embryonic mortality caused by heat stress in cattle (Dunlap and Vincent, 1971; Ealy et

al., 1993; Ryan et al., 1993), sheep (Dutt, 1963), rabbits (Shah, 1956), and mice (Ozawa

et al., 2002).






65


The cellular changes caused by elevated temperature that lead to a block in

embryonic development are not well understood. In other cells, heat shock can cause

cytoskeletal rearrangement (Coss and Linnemans, 1996) changes in mitochondrial

membrane potential and permeability (Funk et al., 1999), organelle disruption and re-

localization (Welch and Suhan, 1985; Wang et al., 1998), increased membrane fluidity

(Coss and Linnemans, 1996; Wang et al., 1998), and membrane blebbing (Jewel et al.,

1982). Most work on effects of heat shock on cultured cells involves temperatures higher

than those that comprise embryonic survival or are experienced by heat-stressed females.

Whereas most studies on the effects of heat shock on mammalian cells have used

temperatures of 43.00-45.0C, temperatures that would be lethal to most mammals,

maternal hyperthermia of 40.0*-41.00C compromises pregnancy rate in cattle (Dunlap

and Vincent, 1971; Turner, 1982; Putney et al., 1988; Ealy et al., 1993). The

hypersensitivity of the early preimplantation embryo to heat shock as compared to

embryos at later stages (Edwards and Hansen, 1997; Krininger et al., 2002) and to other

cells implies that heat shock might be causing different types of cellular pathologies than

those caused by the severe heat shocks required to disrupt most cell types.

Experiments on effects of heat shock on bovine embryos have depended largely

on the in vitro-produced embryo as a model. It is not known, however, if bovine

embryos that develop in vivo respond to the deleterious effects of elevated temperatures

similarly as for in-vitro produced embryos. Embryos produced in vitro differ in several

ways from embryos that develop in utero. For example, they can exhibit retarded

development rate (Holm et al., 2002), increased content of lipid (Crosier et al., 2001) and

lower survivability after freezing (Enright et al., 2000). Thus, there is a need to verify






66


that the in vitro-produced embryo is an acceptable model for studying effects of heat

shock on embryonic development by testing whether similar types of changes are induced

by heat shock in embryos that were produced in vivo.

The principle objective of this study was to characterize ultrastructural changes in

two-cell embryos caused by exposure to a physiologically-relevant heat shock. Another

objective was to evaluate whether differences exist in the fine structure of two-cell

embryos produced in vitro as compared to embryos produced in vivo and to test whether

heat shock induced similar changes in both types of embryos.



Materials and Methods

Materials

All chemicals, except otherwise specified, were from Sigma (St. Louis, MO).

Reagents for in vitro production of embryos were obtained as described elsewhere

(Chapter 2). FSH was Folltropin-V from Vetrepharm Canada (London, Ontario).

Gonadotropin releasing hormone (GnRH) was Cystorelin (Merial, Iselin, NJ) purchased

from Ag-Tech (Manhattan, KS). Percoll was from Amersham Pharmacia Biotech AB

(Uppsala, Sweden). Penicillin-streptomycin and the culture media SP-TL, Hepes-TL,

and IVF-TL were from Cell and Molecular Technologies (Lavallette, NJ) and were used

to prepare SP-TALP, Hepes-TALP, and IVF-TALP (Parrish et al., 1986). Potassium

simplex optimized medium (KSOM; formulation MR-020-D) was also from Cell and

Molecular Technologies and was used to prepare KSOM-Bovine Embryo 1 (KSOM-

BE1) by the addition of 3 mg/ml essentially fatty acid free Fraction V BSA, 2.5 Ag/ml

gentamicin, essential amino acids (Basal Medium Eagle) and non-essential amino acids






67


(Minimum Essential Medium purchased from Sigma) as described elsewhere (Soto et

al., 2003). Bovine steer serum was from Pel Freez Biologicals (Rogers, AK). The

medium CRlaa was prepared as described (Rosenkrans et al., 1993) and modified by

addition of 3 mg/ml essentially fatty acid free Fraction V BSA, 2.5 pg/ml gentamicin,

and essential and non-essential amino acids. Fetal bovine serum (FBS) was from Atlanta

Biologicals (Norcross, GA). The prostaglandin F2a (PGF2a) was Lutalyse and was

donated by Pfizer Animal Health (Kalamazoo, MO). Glutaraldehyde, osmium tetroxide,

ethanol, acetone, EMbed-812, uranyl acetate, Reynolds' lead citrate, Butvar, and copper

grids were from Electron Microscopy Sciences (Fort Washington, PA).

Production of Embryos

In Vitro. Procedures for oocyte maturation, fertilization, and embryo culture were

as described previously (Chapter 2). Briefly, ovaries were collected at a local abattoir

located at a travel distance of approximately 1.5 h from the laboratory. Oocytes obtained

by slashing the ovary were matured for 21 h and then inseminated with a cocktail of

Percoll-purified spermatozoa from three different Angus bulls; a different group of bulls

was used for each replicate. At 18 to 20 h after insemination, putative zygotes were

denuded of cumulus cells by suspending them in Hepes-TALP medium containing 1000

units/ml of hyaluronidase type IV and vortexing in a microcentrifuge tube. Presumptive

zygotes were then placed in groups of-30 in 50 pl microdrops of KSOM-BEl or

modified CRlaa. All cultures at 38.5*C were performed in an environment of 5% CO2 in

humidified air. The percent CO2 was adjusted in the incubators used for heat shock

treatments (7% and 8% CO2 for 41.0 and 43.0C, respectively) to ensure that

concentration of dissolved CO2 was similar between treatment and to maintain medium






68


pH at -7.4 (Chapter 2). The temperature of all incubators was calibrated routinely to

assure accuracy of treatments.

In Vivo. Estrous cycles of three Angus cows were synchronized by injections,

intramuscular injections, of 100 jpg GnRH on Day 0, 25 mg PGF2a, on Day 7, and 100 pg

GnRH on Day 9. On Day 19, superovulation was induced by i.m. injections of

decreasing doses of FSH each morning and afternoon for 4 days (2 injections of 40 mg

each on the first day, 2 injections of 30 mg each on the second and third day, and two

injections of 20 mg each on the last day). Cows received 25 and 15 mg PGF2a,

intramuscularly, coincident with the 6th and 7h FSH injections, respectively.

Approximately 24 h after the last FSH injection, each cow was inseminated with semen

from a different Angus bull and insemination was repeated 12 h later. Cows were killed

approximately 48 h after the first insemination and embryos immediately flushed from

the oviducts. This study was conducted with the approval of the Institutional Animal

Care and Use Committee of the University of Florida.

Inhibition of Blastocyst Development by Heat Shock at the Two-Cell Stage

In vitro-produced two-cell embryos were collected at ~28 h post-insemination

and cultured in fresh microdrops of KSOM-BEI or modified CRIaa at one of two

temperatures; 38.5C (i.e., homoeothermic body temperature of the cow) or 41.0C

(characteristic temperature for heat-stressed cows; Chapter 2). Embryos cultured at

41.00C were returned to control temperature (i.e. 38.5C) after 6 h. On Day 5 after

insemination, the group of embryos cultured in modified CRIaa medium were

supplemented with 10% FBS (no FBS was added to the KSOM-BEI cultured embryos).






69


Development to the blastocyst stage was evaluated on Day 8 post-insemination. The

experiment was replicated five times with a total of 60-70 two-cell embryos/treatment.

Effects of Heat Shock on the Ultrastructure of Two-Cell Embryos Produced In
Vitro

Two-cell embryos were collected at -28 h post-insemination, placed in fresh

microdrops of KSOM-BEI and cultured for 6 h at one of three temperatures; 38.5C,

41.0C, or 43.00C (severe heat shock). Immediately after treatment, embryos were

processed for electron microscopy. The experiment was replicated 4 times on different

days. For statistical analysis, electron micrographs of 1 embryo (selected at random) per

replicate per treatment were used to take measurements on cellular morphology.

Effects of Heat Shock on the Ultrastructure of Two-Cell Embryos Produced In Vivo
and Treated Ex Vivo

Two-cell embryos were obtained from three superovulated Angus cows.

Immediately after slaughter, the oviducts of each cow were flushed with 10 ml sterile and

pre-warmed (-39.0*C) Hepes-TALP into a sterile Petri dish. Embryos (n=38) were

immediately collected and separated by stage of development (one-cell [n=13], two-cell

[n=24], and 4-cell [n=l]). Only embryos that were at the two-cell stage were used for the

experiment. Two-cell embryos from each cow were divided randomly in two groups and

cultured in microdrops of KSOM-BEI at 38.50C or 41.0*C. After 6 h, embryos were

processed for electron microscopy. For statistical analysis, electron micrographs of 1-2

embryo per cow per treatment were used to take measurements on cellular morphology (n

= 3 for 38.50C and 5 for 41.00C, respectively).






70

Electron Microscopy

Embryos were fixed in 2% (v/v) glutaraldehyde in phosphate-buffered saline

(PBS; 0.01 M sodium phosphate and 0.15 M sodium chloride pH 7.2) at 4.0C

overnight (8-10 h). To ease the handling of embryos during dehydration and embedding,

embryos were washed in PBS, individually placed on a glass slide, and a drop of 3%

(w/v) molten low gelling temperature agar (from Sigma) in PBS was placed on the

embryo while observing through a dissecting microscope. Once the agar was hardened,

the excess was cut-off to leave a square (~- 1mm2) of agar containing the embedded

embryo. While in agar, embryos were post-fixed in 1% (w/v) buffered Os04, dehydrated

in ascending concentrations of ethanol and en bloc stained [2% (w/v) uranyl acetate in

75% (v/v) ethanol] overnight at 4.00C. After further dehydration to 100% ethanol

followed by 100% acetone, embryos were infiltrated with EMbed-812, and ultra-thin

sections (~75 nm thick) placed in Butvar covered, 75 mesh copper grids. Sections were

stained with 2% (w/v) aqueous uranyl acetate and Reynolds' lead citrate before viewing

with a Hitachi H-7000 Transmission Electron Microscope (Gaithersburg, MD). In

addition, semi-thin sections (-500 nm) were collected for light microscopy examinations

of whole embryos. Sections were placed on a glass slide and dried on a hot plate

(-50.0C). A drop of toluidine blue 0 stain [1:1 mixture of 0.2 (w/v)% toluidine blue

and 2% (w/v) borax] was added to the sections and incubated on the hot plate for

approximately 1 min (before the slide dried out). The sections were rinsed in a stream of

distilled water prior to visualization.






71


Morphometric Analysis of Electron Micrographs

Electron micrographs of sections that contained the nucleus of at least one

blastomere were used to take measurements on several aspects of cellular morphology.

In addition, electron micrographs were assessed visually to determine effects of heat

shock on other aspects of cellular morphology. For each embryo, morphometric analysis

was performed using SIS Megaview III camera and software (Soft Imaging System,

Lakewood, CO). The distance from the plasma membrane to the organelle-containing

region of the cytoplasm was measured at 10 random locations from each of four pictures

per embryo (i.e. 40 random measurements/embryo). The distance between the two

membranes comprising the nuclear envelope was measured at five random locations

around the nucleus in each of four pictures per embryo. The average of these

measurements was used for statistical analysis. The percent of mitochondria exhibiting a

swollen morphology was determined by counting the total number of mitochondria as

well as the number of swollen mitochondria in each of four pictures per embryo.

Statistical Analysis

Effect of culture temperature on percent of two-cell embryos that developed to the

blastocyst stage was analyzed by least-squares analysis of variance using the Proc GLM

procedures of SAS (SAS for Windows, Version 8e, Cary, NC). Percent development

was calculated for each replicate. Percentage data were analyzed before and after

transformation by arcsine transformation. The analysis of transformed data was used to

obtain probability values while analysis of untransformed data was used to obtain least-

squares means SEM. Heat shock effects on the distance from the plasma membrane to

the outermost organelles, proportion of mitochondria exhibiting a swollen morphology,






72

and the distance between the two membranes of the nuclear envelope was also analyzed

by least squares analysis of variance using Proc GLM procedures. Except for distance

between the two nuclear membranes, data exhibited heteroscasdicity. Accordingly, data

were transformed by log transformation before analysis. The analysis of log-

transformed data was used to obtain probability values while analysis of untransformed

data was used to obtain means SEM.



Results

Inhibition of Embryonic Development by Heat Shock at the Two-Cell Stage

As shown in Figure 3-1, exposure of two-cell embryos to 41.00C for 6 h reduced

the percent of two-cell embryos that were blastocysts at Day 8 after fertilization (p <

0.001). The reduction in development occurred for both media and the temperature x

medium interaction was not significant.

Heat-Shock Induced Changes in Ultrastructure of Two-Cell Embryos Produced In
Vitro

The cytoplasm of embryos cultured at 38.5*C was occupied primarily by vesicles

and mitochondria that extended from the perinuclear region to the edge of the blastomere

(Figure 3-2A and 3-2B). Vesicles appeared to be, at least in part, Golgi. Indicative of

their immature status, mitochondria displayed a hooded morphology and had few cristae.

Embryos contained few endoplasmic reticulum but annulate lamellae (precursor

organelles of the endoplasmic reticulum) were more evident. Lysosomes were also

evident although in scarce abundance. Occasionally, a nucleolus (Figure 3-4A) was

apparent in the nucleus. The plasma membrane of the blastomere was invested with






73










100
00
80 -

60 -

S 40 -

5 20 -

0
CRI aa KSOM-BE1

Type of Medium


Figure 3-1. Inhibition of embryonic development by heat shock at the two-cell stage.
Open bars represent control embryos while grey bars represent heat-shocked (6 h)
embryos. Data represent least-squares means SEM of five replicates with a total of60-
70 two-cell embryos/treatment.






74






A










C























Figure 3-2. Effects of heat shock on morphology of two-cell embryos produced in vitro
as determined by light microscopy (A, C, E) of 500 nm sections stained with toluidine
blue 0 stain or by transmission electron microscopy (B, D, F). Embryos were cultured
for 6 h at 38.5C (A-B), 41.00C (C-D), or 43.00C (E-F). Arrows show area of the
cytoplasm devoid of organelles. ZP = zona pellucida, PVS = peri-vitelline space, N =
nucleus, mv = microvilli.






75

numerous microvilli and clathrin-coated pits could be observed occasionally (data not

shown).

Upon heat shock, all organelles moved centrally to leave the periphery of the

blastomere devoid of organelles (Figure 3-2C to 3-2F). The distance from the plasma

membrane to the organelle-containing region of the cytoplasm was increased by exposure

to 41.0* and 43.00C (means SEM = 604 132, 2261 444, and 4463 1915 nm for

38.5*, 41.0* and 43.00C; p<0.01). In addition, numerous mitochondria were swollen in

heat-shocked embryos. The percent of mitochondria showing a swollen morphology was

increased (p<0.001; Figure 3-3) by both heat shocks (0 0.25, 15 4, and 74 20% for

38.5, 41.00, and 43.0*C, respectively).

Heat shock at 43.00C caused some ultrastructural changes that were not apparent

in embryos exposed to 41.0*C. Heat shock at 43.00C, but not at 41.0*C, increased

(p<0.001) the distance between the two membranes comprising the envelope nuclear

envelope (Figure 3-4F). Distance between the membranes was 45 5, 51 1, and 156

5 nm for 38.50C, 41.00C, and 43.00C, respectively. After culture at 43.0C, the

organelles were more tightly redistributed towards the perinuclear region and could no

longer be recognized (Figure 3-2E and F and 3-6G to 3-61). In addition, culture at 43.0C

resulted in vacuolization of the nucleoplasm (Figure 3-4C) and cytoplasm (Figure 3-2F

and 3-5C). The cytoplasm of embryos cultured at 43.0C for 6 h was covered with

heterogeneous dense content (Figure 3-6G to 3-61). The chromatin underwent severe

degeneration and had a spotty appearance (Figure 3-4F). Moreover, embryos exhibited

increased numbers of lysosomes (Figure 3-6H) and withdrawal of microvilli (data not

shown).






76
















































Figure 3-3. Heat-shock induced alterations in mitochondrial morphology. Two-cell
embryos produced in vitro were cultured for 6 h at 38.5C (A), 41.0C (B), or 43.0C
(C). MT = mitochondrion, V = vesicle, SMT = swollen mitochondrion.







77













































Figure 3-4. Effects of heat shock on nuclear morphology (A, C, E) and distance between
the two membranes comprising the nuclear envelope (B, D, F). Two-cell embryos
produced in vitro were cultured for 6 h at 38.50C (A-B). 41.00C (C-D), or 43.0C (E-F).
N = nucleus, nu = nucleolus, G = Golgi apparatus, V = vesicle; arrows point at the
nuclear envelope.






78












































Figure 3-5. Disruption of plasma membrane by heat shock. Two-cell embryos produced
in vitro were cultured for 6 h at 38.50C (A), 41.00C (B), or 43.00C (C). mv = microvilli,
ZP = zona pellucida, PVS = peri-vitelline space, V = vesicle, MT = mitochondrion.






79





































Figure 3-6. Representative micrographs demonstrating appearance of cytoplasmic
components in two-cell embryos produced in vitro and cultured for 6 h at 38.5C (A-C),
41.00C (D-F), or 43.00C (G-I). V = vesicle, Ly = lysosomes, ER = endoplasmic
reticulum, SMT = swollen mitochondrion, AL = annulate lamellae, G = Golgi apparatus,
MT = mitochondrion, V = vesicle, mv = microvilli, ZP = zona pellucida, PVS = peri-
vitelline space, mt = microtubule.






80


Heat-Shock Induced Changes in Ultrastructure of Two-Cell Embryos Produced in
Vivo and Treated Ex Vivo

Embryos recovered from superovulated cows and treated ex vivo were

ultrastructurally comparable (Figures 3-7 and 3-8) to embryos produced in vitro (Figures

3-2 to 3-6). Like for embryos produced in vitro, the cytoplasm of embryos produced in

vivo and cultured for 6 h was filled primarily with vesicles and mitochondria. Other

organelles, including endoplasmic reticulum, Golgi apparatus, annulate lamellae, and

lysosomes, were indistinguishable from in vitro embryos. Heat shock of 41.0*C for 6 h

caused the same alterations in ultrastructural morphology as for in vitro embryos. In

particular, the organelles of the embryos treated at 41.0C for 6 h pulled away from the

plasma membrane and towards the nucleus to leave an organelle-free area around the

edge of the blastomere (Figure 3-7). The distance from the edge of the plasma membrane

to the organelle-containing region of the cytoplasm was 743 80 vs. 2553 518 nm, for

embryos at 38.5*C and 41.00C, respectively (p < 0.05). The percent of mitochondria that

exhibited a swollen morphology was also increased by heat shock (percent swollen = 0.7

0.6 vs. 7 2 for 38.5C and 41.0C, respectively; p = 0.07; Figure 3-8). A notable

finding was the fact that lysosomes appeared with more frequency in the embryos

produced in vivo and heat shocked at 41.0C for 6 h than for embryos produced in vitro

and treated under similar conditions (data not shown).



Discussion

The present study demonstrates that disruption of development caused by

exposure of two-cell bovine embryos to a physiologically-relevant heat shock involves

alterations to the cytoskeleton and mitochondria. The observation that there were no














































Figure 3-7. Effects of heat shock on morphology of two-cell embryos produced in vivo
and treated ex-vivo as determined by light microscopy of 500 nm sections stained with
toluidine blue 0 stain (A, C) or by transmission electron microscopy (B, D). Embryos
were cultured for 6 h at 38.50C (A-B) or 41.00C (C-D). Arrows show area of the
cytoplasm devoid of organelles. ZP = zona pellucida, PVS = peri-vitelline space, N =
nucleus, MT = mitochondrion.






82




















Figure 3-8. Heat-shock induced alterations in mitochondrial morphology of two-cell
embryos produced in vivo. Embryos were cultured for 6 h at 38.5'C (A) or 41.0C (B).
MT = mitochondrion, V = vesicle, SMT = swollen mitochondrion, G = Golgi apparatus.






83

differences observed at the ultrastructural level between two-cell bovine embryos

produced in vitro and in vivo at 38.5*C or in their response to hyperthermia indicates that

use of embryos produced in vitro to study heat shock is relevant to the embryo that

develops in vivo, at least with respect to ultrastructural changes.

Much is known about the cellular mechanisms whereby heat shock disrupts cell

proliferation and differentiation. Heat shock can induce morphological alterations to the

cytoskeleton (Coss and Linnemans, 1996) and changes to mitochondrial morphology,

function, and potential (Welch and Suhan, 1985; Cole and Armour, 1988; Wang et al.,

1998; Funk et al., 1999; Maccouillard-Poulletier de Gannes et al., 2000; Willis et al.,

2000). In addition, heat shock causes disruption and redistribution of organelles (Welch

and Suhan, 1985; Wang et al., 1998; Funk et al., 1999), damages the nucleus (Welch and

Suhan, 1985; Gniadecki et al., 2001), induces protein aggregation (Wachsberger and

Coss, 1993), and induces functional and ultrastructural changes in membranes (Jewel et

al., 1982; Lepock et al., 1982; Funk et al., 1999). However, most of the work regarding

cellular effects of heat shock used temperatures above 43.0*C and is not relevant to

effects of much milder heat shocks affecting embryo survival. Temperatures at 43.00C or

above are lethal to many mammals and well above the body temperatures associated with

hyperthermia-induced embryonic death in cattle (i.e. < 41.00C; Dunlap and Vincent,

1971; Putney et al., 1988; Ealy et al., 1993). As the first experiment of this paper

indicates, development of the two-cell bovine embryo is very susceptible to disruption by

mild heat shock (i.e. 41.0*C for 6 h). How such a mild heat shock affects the cellular and

sub-cellular processes orchestrating early embryonic development has not been well

understood and is the topic of this paper.






84

The most obvious alterations to the ultrastructure of two-cell embryos produced in

vitro and in vivo caused by heat shock were the movement of organelles away from the

plasma membrane and the increase in the percent of mitochondria that exhibited a

swollen morphology. The translocation of organelles towards the center of the

blastomere would suggest that heat shock affected the cytoskeleton because this system is

involved in active transport of organelles and other components of the cell using both

microtubule- and actin filament-based systems of transport (i.e. dynein, kinesin; Capco,

1996; Stebbings, 1996). The movement of organelles in the heat-shocked two-cell

embryo is reminiscent of the translocation of melanosomes in the fish melanophore.

Movement of these melanized organelles via aggregation towards the perinuclear region

or dispersion to the periphery of the dorsal skin cells is responsible for rapid background

adaptation. Both microtubules and microfilaments are involved during aggregation and

dispersion of the melanosomes (Skold et al., 2002). It would be of interest to determine

if the aggregation of organelles following heat shock also involved similar patterns of

movement of organelles on microtubules and microfilaments.

While present evidence suggests that heat shock fractures or alters the

cytoskeleton, the specific alterations involved are not known. The specific cytoskeletal

element affected by heat depends on cell type (Coss and Linnemans, 1996) and, in all

likelihood, temperature. Further studies need to be conducted to verify that the

movement of organelles caused by heat shock is a result of cytoskeletal rearrangement

and to determine which elements are involved in this response. An additional question

that arises is whether sensitivity of the cytoskeleton to 41.0C is unique to embryos or a

general characteristic of mammalian cells.






85

It is likely that disruption of the cytoskeleton is a major cause of the inhibition of

development caused by heat shock. The cytoskeleton is a cellular component that is

common to the plasma membrane, nuclear membrane, and cytoplasmic organelles. The

three components of the cytoskeleton (i.e., actin filaments, microtubules and intermediate

filaments) work together to enhance structural integrity, impart cell shape, provide cell

motility, position mRNA and protein (Capco, 1996; Gniadecki et al., 2001), and play an

important role in cell signaling (Hang et al., 1999), mitosis and cytokinesis (Lodish et al.,

2000). During the first three cleavage divisions, the mammalian embryo spends the

majority of the cell cycle in S and M phases (Barnes and Eyestone, 1990). These cell-

cycle stages are differentially affected by elevated temperatures. For instance, exposure

of cells to heat shock during the S phase results in spontaneous premature chromosome

condensation and micronuclear formation (Swanson et al., 1995). Heat shock during the

M phase results in disassembly of the mitotic spindle, failure of cytokinesis, and

polyploidy (Coss et al., 1982; Vidair et al., 1993). Experiments to test whether heat

shock of two-cell embryos results in micronuclei formation and/or alterations in the

ploidy of the blastomeres would provide further evidence for cytoskeleton-mediated

damage of the developmental program.

The second major effect of heat shock observed for both in vitro-produced

embryos and embryos produced in vivo was swelling of mitochondria. Mitochondrial

swelling occurs under several different stressful situations such as calcium overload

(Ichas and Mazat, 1998; Halestrap et al., 2002), oxidative stress (Lemasters, 1999;

Halestrap et al., 2002), ischemia/reperfusion (Ooie et al., 2001), chronic ethanol exposure

(Li et al., 2001), and hyperthermia (Cole and Armour, 1988). Swelling of the