physiological effects and mechanisms of action of eicosapentaenoic acid on pgf2 production in ...

121 Pages·2005·2.28 MB·English

STUDIES ON THE EFFECTS AND MECHANISMS OF ACTION OF
EICOSAPENTAENOIC ACID ON PGF PRODUCTION IN CULTURED BOVINE
2α
ENDOMETRIAL CELLS

By

CRISTINA CALDARI-TORRES

A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2005
Copyright 2005

by

Cristina Caldari-Torres
Le dedico esta tesis a Mami, Abuela y Lola por ser el ejemplo de lo que una mujer hecha
y derecha debe ser.
ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Lokenga Badinga, for allowing me to be a
part of his laboratory. I am grateful for all his help and support, and for teaching me how
to conduct research and encouraging me to perform well academically. I would also like
to thank my committee members, Dr. Daniel C. Sharp and Dr. Charles R. Staples, for
attending the committee meetings and for their advice on how to improve my work.
I also extend many thanks to Liz S. Greene for her help with laboratory techniques,
and for her support as a lab mate and as a friend. I would like to thank Dr. Alan Ealy for
allowing me the use of his computer. Special thanks go to Carlos J. Rodríguez-Sallaberry
for helping me improve as a researcher and for his patience, friendship and
companionship. I am grateful to him for being a great source of strength and for showing
me the right way when I most needed it.

iv
TABLE OF CONTENTS

page
ACKNOWLEDGMENTS.................................................................................................iv
LIST OF FIGURES..........................................................................................................vii
ABBREVIATION KEY....................................................................................................ix
ABSTRACT......................................................................................................................xii
CHAPTER
1 INTRODUCTION........................................................................................................1
2 LITERATURE REVIEW.............................................................................................4
Estrous Cycle................................................................................................................4
Regulation of the Estrous Cycle............................................................................5
Characteristics of the Bovine Estrous Cycle.........................................................5
Phases of the Estrous Cycle...................................................................................6
Stages of the Estrous Cycle...................................................................................7
Anestrus.................................................................................................................8
Ovarian Follicular Dynamics in Ruminants...............................................................10
Hypothalamic-pituitary-ovarian axis...................................................................11
Follicular Waves in Cattle...................................................................................12
Follicular Growth Factors....................................................................................13
Ovulation.............................................................................................................15
Development and Function of the Corpus Luteum in Ruminants..............................16
Promoters of Angiogenesis..................................................................................17
Endocrine Regulators of CL Function.................................................................19
Autocrine and Paracrine Regulators....................................................................19
Luteolysis in Domestic Ruminants.............................................................................21
Hormonal Regulation of Uterine PGF Synthesis.............................................23
2α
Intracellular Signaling.........................................................................................24
Blood Flow and Vascular Changes.....................................................................25
Morphological Changes.......................................................................................26
PGF Inhibition of P Synthesis.........................................................................26
2α 4
Immune-Mediated Events...................................................................................27
Tissue Metalloproteinases...................................................................................28
Apoptosis.............................................................................................................28
v
Pregnancy Establishment in Ruminants.....................................................................28
Placentation in Ruminants...................................................................................30
Pregnancy and the Immune Response.................................................................30
PGHS-2 Gene Expression...................................................................................32
Shift in PG Production from PGF to PGE ......................................................33
2α 2
Effects of Polyunsaturated Fatty Acids on Reproduction in Cattle............................34
Roles of Peroxisome Proliferator-Activated Receptors in Reproduction...................36
Roles of Mitogen-activated Protein Kinases in Reproduction...................................38
3 STUDIES ON THE EFFECTS OF EICOSAPENTAENOIC ACID ON PGF
2α
PRODUCTION IN CULTURED BOVINE ENDOMETRIAL CELLS....................41
Introduction.................................................................................................................41
Materials and Methods...............................................................................................43
Materials..............................................................................................................43
Cell Culture and Treatment.................................................................................43
PGF Radioimmunoassay..................................................................................45
2α
RNA Isolation and Analysis................................................................................45
Statistical Analyses..............................................................................................46
Results.........................................................................................................................47
Discussion...................................................................................................................48
Summary.....................................................................................................................50
4 STUDIES ON THE MECHANISMS OF ACTION OF EICOSAPENTAENOIC
ACID ON PGF PRODUCTION BY CULTURED BOVINE ENDOMETRIAL
2α
CELLS........................................................................................................................58
Introduction.................................................................................................................58
Materials and Methods...............................................................................................59
Cell Culture and Treatment.................................................................................59
PGF Radioimmunoassay..................................................................................60
2α
RNA Isolation and Analysis................................................................................61
Statistical Analyses..............................................................................................61
Results.........................................................................................................................62
Discussion...................................................................................................................63
Summary.....................................................................................................................66
5 GENERAL DISCUSSION.........................................................................................81
LIST OF REFERENCES...................................................................................................88
BIOGRAPHICAL SKETCH...........................................................................................108

vi
LIST OF FIGURES
Figure page

3-1 Experimental manipulations to examine the effect of eicospaentaenoic acid
(EPA) on prostaglandin F (PGF ) response to phorbol-12,13-dibutyrate
2α 2α
(PDBu) in bovine endometrial (BEND) cells..........................................................51
3-2 Experimental manipulations to study the effect of linoleic acid (LA) on
prostaglandin F (PGF ) response to eicosapentaenoic acid (EPA) in bovine
2α 2α
endometrial (BEND) cells........................................................................................52
3-3 Effect of eicosapentaenoic acid (EPA) on prostaglandin F (PGF ) response to
2α 2α
phorbol-12,13-dibutyrate (PDBu) in bovine endometrial (BEND) cells.................53
3-4 Effect of eicosapentaenoic acid (EPA) on prostaglandin endoperoxide synthase
(PGHS-2) mRNA response to phorbol-12,13-dibutyrate (PDBu) in bovine
endometrial (BEND) cells........................................................................................54
3-5 Effect of eicosapentaenoic acid (EPA) on peroxisome proliferator-activated
receptor δ (PPARδ) mRNA response to phorbol-12,13-dibutyrate (PDBu) in
bovine endometrial (BEND) cells............................................................................55
3-6 Effects of increasing n-6/n-3 fatty acid ratios on prostaglandin F (PGF )
2α 2α
response to phorbol-12-13-dibutyrate (PDBu).........................................................56
3-7 Effects of increasing n-6/n-3 fatty acid ratios on prostaglandin endoperoxide
synthase (PGHS-2) response to phorbol-12,13-dibutyrate (PDBu).........................57
4-1 Experimental manipulations to determine if eicosapentaenoic acid (EPA)-
mediated inhibition of prostaglandin F (PGF )secretion involves peroxisome
2α 2α
proliferator-activated receptor δ (PPARδ) activation in bovine endometrial
(BEND) cells............................................................................................................67
4-2 Experimental manipulations to determine if peroxisome proliferator-activated
receptor δ (PPARδ) inhibition may abolish eicosapentaenoic acid (EPA) effect
on prostaglandin F (PGF ) secretion in bovine endometrial (BEND) cells........68
2α 2α
4-3 Experimental manipulations to examine the role of mitogen-activated protein
(MAP) kinases in eicosapentaenoic acid (EPA) regulation of prostaglandin F
2α
(PGF ) production in bovine endometrial (BEND) cells........................................69
2α
vii
4-4 Effect of a peroxisome proliferator-activated receptor δ (PPARδ) agonist (L-
165,041) on prostaglandin F (PGF ) secretion in bovine endometrial (BEND)
2α 2α
cells...........................................................................................................................70
4-5 Effect of a peroxisome proliferator-activated receptor δ (PPARδ) agonist (L-
165,041) on prostaglandin endoperoxide synthase (PGHS-2) mRNA abundance
in bovine endometrial (BEND) cells........................................................................71
4-6 Effect of a peroxisome proliferator-activated receptor δ (PPARδ) agonist (L-
165,041) on PPARδ mRNA response to PDBu in bovine endometrial (BEND)
cells...........................................................................................................................72
4-7 Effect of sulindac sulfide on prostaglandin F (PGF ) response to
2α 2α
eicosapentaenoic acid (EPA) in bovine endometrial (BEND) cells.........................73
4-8 Effect of sulindac sulfide on prostaglandin endoperoxide synthase (PGHS-2)
mRNA response to eicosapentaenoic acid (EPA) in bovine endometrial (BEND)
cells...........................................................................................................................74
4-9 Effect of sulindac sulfide on peroxisome proliferator-activated receptor δ
(PPARδ) mRNA response to eicosapentaenoic acid (EPA) in bovine
endometrial (BEND) cells........................................................................................75
4-10 Effect of sulindac sulfone on prostaglandin F (PGF ) response to
2α 2α
eicosapentaenoic acid (EPA) in bovine endometrial (BEND) cells.........................76
4-11 Effect of sulindac sulfone on prostaglandin endoperoxide synthase (PGHS-2)
mRNA response to eicosapentaenoic acid (EPA) in bovine endometrial (BEND)
cells...........................................................................................................................77
4-12 Effect of sulindac sulfone on peroxisome proliferator-activated receptor δ
(PPARδ) mRNA response to eicosapentaenoic acid (EPA) in bovine
endometrial (BEND) cells........................................................................................78
4-13 Effect of SB203580 and PD98059 on prostaglandin F (PGF ) response to
2α 2α
eicosapentaenoic acid (EPA) in bovine endometrial (BEND) cells.........................79
4-14 Effect of SB203580 and PD98059 on prostaglandin endoperoxide synthase
(PGHS-2) mRNA response to eicosapentaenoic acid (EPA) in bovine
endometrial (BEND) cells........................................................................................80
5-1 Proposed model for eicosapentaenoic acid (EPA) and linoleic acid (LA) regulation
of prostaglandin F (PGF ) biosynthesis in bovine endometrial (BEND) cells…86
2α 2α

5-2 Signaling cascade leading to synthesis and activation of prostaglandin
endoperoxide synthase (PGHS-2) in bovine endometrial (BEND) cells………….87

viii
ABBREVIATION KEY

9k-PGR PGE -9-keto reductase
2
AA Arachidonic acid
AP-1 Activator protein-1
BEND cells Bovine endometrial cells
CA Corpus albicans
CL Corpus luteum
CRE cAMP response element
CREB cAMP response element binding protein
DAG 1,2-diacylglycerol
DGLA dihomo-γ-linolenic
DPA docosapentaenoic acid
E Estrogen
2
EPA Eicosapentaenoic acid
ERK Extracellular-regulated kinase
ET-1 Endothelin-1
FGF Fibroblast growth factor
FSH Follicle-stimulating hormone
FO Fish oil
GH Growth hormone
ix
GnRH Gonadotropin-releasing hormone
GLA γ-linolenic acid
IGF Insulin-like growth factor
IGFBP Insulin-like growth factor-binding protein
IGFR Insulin-like growth factor receptor
IFN-τ Interferon τ
IP Inositol-1,4,5-triphosphate
3
JAK/STAT Janus kinase signal transducer and activator
of transcription
LA Linoleic acid
LH Luteinizing hormone
LNA Linolenic acid
LXR Liver X receptor
MAP kinase Mitogen-activated protein kinase
MCP-1 Monocyte chemoattractant protein-1
MHC Major histocompatibility complex
NE Norepinephrine
OT Oxytocin
P Progesterone
4
PDBu Phorbol-12,13-dibutyrate
PG Prostaglandin
PGE Prostaglandin E
2 2
PGF Prostaglandin F
2α 2α
x