Oocyte Quality: Molecular Constituents Altered in the Oocyte Due to Various Environmental Factors

282 Pages·2016·12.2 MB·English

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Lindsay Cox
Utah State University
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OOCYTE QUALITY: MOLECULAR CONSTITUENTS ALTERED IN THE OOCYTE
DUE TO VARIOUS ENVIRONMENTAL FACTORS
by
Lindsay Cox
A dissertation submitted in partial fulfillment
of the requirements for the degree
of
DOCTOR OF PHILOSOPHY
in
Reproduction and Development

Approved:

____________________________ ____________________________
S. Clay Isom, Ph.D. Abby Benninghoff, Ph.D.
Major Professor Committee Member

____________________________ ____________________________
Korry Hintze, Ph.D. Irina Polejaeva, Ph.D.
Committee Member Committee Member

____________________________ ____________________________
Lee Rickords, Ph.D. Dirk Vanderwall, D.V.M., Ph.D.
Committee Member ADVS Department Head

_________________________________
Mark McLellan, Ph.D.
Vice President for Research and
Dean of the School of Graduate Studies

UTAH STATE UNIVERSITY
Logan, Utah
2016
ii

Copyright © Lindsay Cox 2016
All Rights Reserved
iii

ABSTRACT

Oocyte Quality: Molecular Constituents Altered in the Oocyte Due to Various
Environmental Factors
by
Lindsay Cox, Doctor of Philosophy
Utah State University, 2016
Major Professor: Dr. S. Clay Isom
Department: Animal, Dairy, and Veterinary Sciences
An estimated 1.6 million American couples struggle with infertility. Some causes
for poor fertility can be clearly defined but in many instances, subfertility is unexplained.
Poor oocyte quality is now considered to be a main contributing factor for many causes
of infertility. Good oocyte quality is crucial for many processes including embryo
development and maintaining pregnancy. There is a possibility that any alterations to the
oocyte can have long lasting effects on embryo development and the health of the
offspring. The oocyte is very sensitive to any perturbations to its surrounding
environment. Transcripts for apoptosis inhibitors and epigenetic modifiers were found to
be significantly more abundant in in vivo-matured oocytes compared to oocytes that were
matured in vitro. RNA degradation and chromatin remodeling pathways may also be
perturbed in in vitro-matured oocytes. While examining the effects of maternal age on the
oocyte, there are age-related differences in gene expression in equine cumulus-oocyte
complexes. Differences in gene expression may lead to a decrease in oocyte
iv

developmental competence. Age related alterations to gene expression in the equine
cumulus-oocyte complexes might be caused by increased rates of oxidative stress and
subsequent DNA damage. These alterations could directly impact many processes within
the oocyte. Higher incidences of apoptosis may be possible in the cumulus cells from
aged mares, which would directly impact the developmental competence of the oocyte.
Lastly, oocyte quality may be impacted by western dietary consumption patterns, which
could lead to many genes being differentially expressed in oocytes. Alterations to the
abundance of these genes have been shown to lead to effects that are commonly seen
with metabolic syndrome, such as glucose intolerance, insulin resistance, obesity and
diabetes. The results of this work will ultimately provide insight into the effects
environmental influences have on the oocyte at the molecular level.

(268 pages)
v

PUBLIC ABSTRACT

Oocyte Quality: Molecular Constituents Altered in the Oocyte Due to Various
Environmental Factors
Lindsay Cox
An estimated 1.6 million American couples struggle with infertility. Some causes
for poor fertility can be clearly defined but in many instances, subfertility is unexplained.
The cause for many of these infertility issues is now thought to be due to defects in the
quality of the woman’s eggs. The egg primarily determines whether a pregnancy will be
achieved and maintained. Any alterations to the egg will have effects on both pregnancy
and possibly the health of the future child. The egg is very sensitive to any alterations to
the environment it is surrounded by in the ovary. Environmental factors, such as
maturation culture conditions, maternal age, and a total western diet that reflects the poor
eating habits of the United States have all been shown to lead to alterations in various
genes. While looking at the effects the environment in which the egg undergoes
maturation has on the quality of the egg, it was demonstrated that genes that are crucial
for proper egg development are affected when maturation occurs in the laboratory, rather
than within the body. Another study demonstrated that maternal age does lead to age-
related alterations to genes, which could explain for the decrease in egg quality due to
age. Lastly, it has been shown that poor nutrition can have an impact on many genes
within the eggs. Alterations to these genes have previously been shown to lead to many
similar effects as those seen with obesity and diabetes. We hope these findings will help
to provide insight into the effects environmental influences can have on the oocyte.
vi

ACKNOWLEDGMENTS

First and foremost, I would like to thank Dr. Clay Isom for giving me the
opportunity to work under his tutelage and for all the support he has provided over the
years. I would also like to thank my committee members, Drs. Abby Benninghoff, Lee
Rickords, Irina Polejaeva, and Korry Hintze for their assistance through the entire
process. We would like to thank the USU Veterinary support staff (Dr. Rusty Stott and
Sarah Behunin) for their help in collecting all of our IVV oocyte samples and the USU
farm staff for their management of the pig facilities. We would also like to thank
Theurer Meats (Lewiston, UT) for allowing us to obtain pig ovaries in order to collect
IVM oocytes. And finally, we would like to acknowledge the University of Utah
Genomics Core and the University of Missouri Bioinformatics Core for their assistance
in the RNA sequencing efforts. I would like to thank Dr. Dirk Vanderwall and his team
for all of their contributions in collecting the mare oocytes.
I want to give a special thanks to my family for their encouragement and moral
support as I worked to complete my degree. I could not have done it without all of you.
vii

CONTENTS

Page
ABSTRACT ....................................................................................................................... iii
PUBLIC ABSTRACT ......................................................................................................... v
ACKNOWLEDGMENTS ................................................................................................. vi
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................ xi
CHAPTER
1. REVIEW OF LITERATURE .................................................................................... 1
Folliculogenesis ........................................................................................... 1
Oocyte development .................................................................................... 4
Role of cumulus granulosa cells ................................................................ 18
Fertilization/oocyte activation ................................................................... 20
Environment and oocyte quality ............................................................... 24
Assessing oocyte quality ........................................................................... 39
Impact of the project ................................................................................. 41
References ................................................................................................. 43

2. GENE EXPRESSION ANALYSIS OF IN VIVO- AND
IN VITRO-MATURED PORCINE METAPHASE II OOCYTES ....................... 54

Abstract ..................................................................................................... 54
Introduction ............................................................................................... 55
Materials and Methods .............................................................................. 59
Results ....................................................................................................... 71
Discussion ................................................................................................. 75
Tables and Figures .................................................................................... 84
References ................................................................................................. 99

3. EXPRESSION PROFILES OF SELECT GENES IN
viii

CUMULUS-OOCYTE COMPLEXES FROM YOUNG
AND AGED MARES ......................................................................................... 108

Abstract ................................................................................................... 108
Introduction ............................................................................................. 109
Materials and Methods ............................................................................ 111
Results ..................................................................................................... 118
Discussion ............................................................................................... 120
Tables and Figures .................................................................................. 129
References ............................................................................................... 135

4. IMPACT OF TOTAL WESTERN DIET ON GAMETE
QUALITY IN SWINE ........................................................................................ 144

Abstract ................................................................................................... 144
Introduction ............................................................................................. 145
Materials and Methods ............................................................................ 149
Results ..................................................................................................... 157
Discussion ............................................................................................... 163
Tables and Figures .................................................................................. 172
References ............................................................................................... 196

5. SUMMARY ........................................................................................................ 207
References ............................................................................................... 215

APPENDICES ................................................................................................................. 216
APPENDIX A. The complete data set for all of the biological processes
found to be significantly enriched in IVM or IVV oocytes. ................... 205
APPENDIX B. License to publish chapter 3 ......................................... 264

CURRICULUM VITAE ................................................................................................. 265
ix

LIST OF TABLES

Table Page

2-1 Definition of gene names used in qPCR analysis .............................................. 84

2-2 Short read alignment summary for RNA sequencing data ................................. 86

2-3 List of the top 10 most significant genes for RNA-sequencing data found
to be more abundant in the IVM or IVV oocytes ............................................... 87

2-4 Gene ontology analysis of RNA sequencing data .............................................. 88

2-5 Gene set enrichment analysis results using DAVID/EASE functional
annotation tool .................................................................................................... 89

2-6 Genes tested for quantitative PCR and their functional gene categories ........... 90

Supplementary 2-1 Gene Names, corresponding accession numbers, and forward
and reverse primer sequences utilized for quantitative PCR ............................ 104

Supplementary 2-2 Detailed alignment statistics for all 31 lanes of data ................ 106

Supplementary 2-3 Summary of significant genes for RNA-sequencing data ........ 107

Supplementary 3-1 Gene names, corresponding accession numbers, and forward
and reverse primer sequences utilized for quantitative PCR ............................ 142

4-1 Porcine control diet guaranteed analysis .......................................................... 172

4-2 Porcine sTWD formulation .............................................................................. 173

4-3 Porcine diet compositions ................................................................................ 175

4-4 Statistical analysis of food intake and weight gain data ................................... 176

4-5 Mineral analysis for follicular fluid samples collected from CON and
sTWD pigs ........................................................................................................ 177

4-6 Genes tested for quantitative PCR and their functional gene categories ......... 178

Supplementary 4-1 Gene names, corresponding accession numbers, and forward and
reverse primer sequences utilized for quantitative PCR .................................. 201