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Regulation of the Preimplantation Bovine Embryo by Colony Stimulating Factor 2

Permanent Link: http://ufdc.ufl.edu/UFE0042116/00001

Material Information

Title: Regulation of the Preimplantation Bovine Embryo by Colony Stimulating Factor 2
Physical Description: 1 online resource (173 p.)
Language: english
Creator: Loureiro, Barbara
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: bovine, csf2, embryo, ifnt, ifv, microarray
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: REGULATION OF THE PREIMPLANTATION BOVINE EMBRYO BY COLONY STIMULATING FACTOR 2 Colony-stimulating factor 2 (CSF2) is a multifunctional cytokine originally recognized as a hematopoietic factor but now known to be expressed in several reproductive tract tissues including the bovine oviduct and endometrium. Colony-stimulating factor 2 may be an important intracellular regulator of endometrial, oviductal and embryonic functions during early pregnancy in the cow and other species. Addition of CSF2 to culture medium improved the proportion of in vitro produced embryos developing to the blastocyst stage in cow, mouse, human and pig and increased post-transfer embryonic survival in mice. A series of experiments was conducted to understand the role of CSF2 in preimplantation embryonic development including its effects on blastocyst formation, the embryonic transcriptome, and embryonic survival. The first experiment was conducted to test whether addition of CSF2 to culture medium could enhance development and post-transfer survival of in vitro produced bovine embryos. Treatment of embryos with CSF2 at Day 1 after insemination increased the percentageage of embryos that became transferable morulae or blastocysts at Day 7 but had no significant effect on pregnancy rate at Day 30-35 or calving rate. When CSF2 was added at Day 5 after insemination, there was increase in the percentageage of embryos that became transferable morulae or blastocysts at Day 7, an increase in the percentageage of recipient cows pregnant at Day 30-35, an increase in calving rate and a decrease in pregnancy loss after Days 30-35. Furthermore, blastocysts formed after treatment with CSF2 had an increase in the number of cells in the inner cell mass. The second experiment was conducted to analyze the transcriptome of CSF2 treated embryos to identify genes involved in mediating CSF2 effects on development to the blastoctcyst stage and survival after transfer. The Agilent bovine microarray platform was used. Embryos were treated with CSF2 at Day 5 and selected at Day 6 after insemination. A total of 160 genes were differentially expressed, with 67 being higher in CSF2-treated embryos and 93 being lower. Analysis identified 13 biological process ontologies that were grouped into three major groups. The first group included genes functionally involved in developmental processes and differentiation. Actions of CSF2 would tend to inhibit genes involved in neurogenesis and stimulate genes involved in mesoderm or muscle formation. The second group were genes involved in cell communication, with the most characteristic effect caused by CSF2 being inhibition of ?-catenin dependent WNT signaling. The third group were genes involved in apoptosis signaling, which was inhibited by CSF2. The antiapoptotic actions of CSF2 were confirmed in another experiment in which CSF2 decreased the percentageage of blastomeres in Day 6 embryos becoming apoptotic after heat-shock. The third study evaluated possible mechanisms by which CSF2 acts during Day 5 to 7 of development to improve embryonic and fetal survival. Embryos were treated with CSF2 or served as controls and were transferred to recipient cows at Day 7 after ovulation. Embryos were recovered at Day 15 and embryonic disc and trophoblast analyzed for gene expression by microarray analysis. Results suggest that higher pregnancy rates at Day 30-35 represent increased embryonic survival before Day 15 and a greater capacity of the embryo to elongate and secrete interferon tau (IFNT) at Day 15. This conclusion is based on greater recovery of embryos from cows receiving CSF2-treated embryos at Day 15 (P < 0.07), a tendency for CSF2 treated embryos to be longer than control embryos, and greater antiviral activity (a measure of IFNT bioactivity) in uterine flushings of cows receiving CSF2 treated embryos (P < 0.07, when considering those cows with detectable antiviral activity). Analysis of gene expression in filamentous embryos indicated no difference in transcription among this subset of embryos that survived to Day 15 and elongated successfully. Therefore, the reduction in embryonic and fetal loss after Day 30-35 caused by CSF2 is probably not a direct reflection of altered gene expression at Day 15. Taken together these results indicate that CSF2 can regulate embryonic development, increase embryonic survival after transfer and decrease pregnancy loss. The ability of CSF2 to improve development is probably due to a set of effects that include an increase in the number of cells in the inner cell mass, a decrease in cell death and differentially regulation of the embryo transcriptome. Furthermore, before implantation CSF2 embryos tend to be longer and secret more IFNT. The increased pregnancy rates observed after Day 35 of pregnancy may be a combined result of the factors mentioned above. Moreover, CSF2 treatment can be inflicting epigenetic changes that persist latter in development or even morphological changes that are consequence of the differentially gene expression early before implantation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Barbara Loureiro.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Hansen, Peter J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042116:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042116/00001

Material Information

Title: Regulation of the Preimplantation Bovine Embryo by Colony Stimulating Factor 2
Physical Description: 1 online resource (173 p.)
Language: english
Creator: Loureiro, Barbara
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: bovine, csf2, embryo, ifnt, ifv, microarray
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: REGULATION OF THE PREIMPLANTATION BOVINE EMBRYO BY COLONY STIMULATING FACTOR 2 Colony-stimulating factor 2 (CSF2) is a multifunctional cytokine originally recognized as a hematopoietic factor but now known to be expressed in several reproductive tract tissues including the bovine oviduct and endometrium. Colony-stimulating factor 2 may be an important intracellular regulator of endometrial, oviductal and embryonic functions during early pregnancy in the cow and other species. Addition of CSF2 to culture medium improved the proportion of in vitro produced embryos developing to the blastocyst stage in cow, mouse, human and pig and increased post-transfer embryonic survival in mice. A series of experiments was conducted to understand the role of CSF2 in preimplantation embryonic development including its effects on blastocyst formation, the embryonic transcriptome, and embryonic survival. The first experiment was conducted to test whether addition of CSF2 to culture medium could enhance development and post-transfer survival of in vitro produced bovine embryos. Treatment of embryos with CSF2 at Day 1 after insemination increased the percentageage of embryos that became transferable morulae or blastocysts at Day 7 but had no significant effect on pregnancy rate at Day 30-35 or calving rate. When CSF2 was added at Day 5 after insemination, there was increase in the percentageage of embryos that became transferable morulae or blastocysts at Day 7, an increase in the percentageage of recipient cows pregnant at Day 30-35, an increase in calving rate and a decrease in pregnancy loss after Days 30-35. Furthermore, blastocysts formed after treatment with CSF2 had an increase in the number of cells in the inner cell mass. The second experiment was conducted to analyze the transcriptome of CSF2 treated embryos to identify genes involved in mediating CSF2 effects on development to the blastoctcyst stage and survival after transfer. The Agilent bovine microarray platform was used. Embryos were treated with CSF2 at Day 5 and selected at Day 6 after insemination. A total of 160 genes were differentially expressed, with 67 being higher in CSF2-treated embryos and 93 being lower. Analysis identified 13 biological process ontologies that were grouped into three major groups. The first group included genes functionally involved in developmental processes and differentiation. Actions of CSF2 would tend to inhibit genes involved in neurogenesis and stimulate genes involved in mesoderm or muscle formation. The second group were genes involved in cell communication, with the most characteristic effect caused by CSF2 being inhibition of ?-catenin dependent WNT signaling. The third group were genes involved in apoptosis signaling, which was inhibited by CSF2. The antiapoptotic actions of CSF2 were confirmed in another experiment in which CSF2 decreased the percentageage of blastomeres in Day 6 embryos becoming apoptotic after heat-shock. The third study evaluated possible mechanisms by which CSF2 acts during Day 5 to 7 of development to improve embryonic and fetal survival. Embryos were treated with CSF2 or served as controls and were transferred to recipient cows at Day 7 after ovulation. Embryos were recovered at Day 15 and embryonic disc and trophoblast analyzed for gene expression by microarray analysis. Results suggest that higher pregnancy rates at Day 30-35 represent increased embryonic survival before Day 15 and a greater capacity of the embryo to elongate and secrete interferon tau (IFNT) at Day 15. This conclusion is based on greater recovery of embryos from cows receiving CSF2-treated embryos at Day 15 (P < 0.07), a tendency for CSF2 treated embryos to be longer than control embryos, and greater antiviral activity (a measure of IFNT bioactivity) in uterine flushings of cows receiving CSF2 treated embryos (P < 0.07, when considering those cows with detectable antiviral activity). Analysis of gene expression in filamentous embryos indicated no difference in transcription among this subset of embryos that survived to Day 15 and elongated successfully. Therefore, the reduction in embryonic and fetal loss after Day 30-35 caused by CSF2 is probably not a direct reflection of altered gene expression at Day 15. Taken together these results indicate that CSF2 can regulate embryonic development, increase embryonic survival after transfer and decrease pregnancy loss. The ability of CSF2 to improve development is probably due to a set of effects that include an increase in the number of cells in the inner cell mass, a decrease in cell death and differentially regulation of the embryo transcriptome. Furthermore, before implantation CSF2 embryos tend to be longer and secret more IFNT. The increased pregnancy rates observed after Day 35 of pregnancy may be a combined result of the factors mentioned above. Moreover, CSF2 treatment can be inflicting epigenetic changes that persist latter in development or even morphological changes that are consequence of the differentially gene expression early before implantation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Barbara Loureiro.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Hansen, Peter J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042116:00001


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REGULATION OF THE PREIMPLANTATION BOVINE EMBRYO BY COLONY
STIMULATING FACTOR 2














By

BARBARA LOUREIRO


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

2010

































2010 Barbara Loureiro





















To my husband, parents, family and friends









ACKNOWLEDGMENTS

First I thank my advisor, Dr. Peter J. Hansen, for the opportunity to join his group. I

truly appreciate his patience and support. He always challenged me to do my best and

above all he believed I could do it. His love for science and respect for his students is

something that I will take for the rest of my life. Also, I thank my committee members Dr.

Nasser Chegini, Dr. Jose Eduardo Portela Santos and Dr. Alan Ealy for their insight and

knowledge. Moreover, I am grateful for their accessibility and willingness to help, as well

as their encouragement and support during the completion of this dissertation.

I would like to thank my old lab mates Dr. Dean Jousan, Dr. Luiz Augusto de

Castro e Paula, Dr. Jeremy Block, Dr. Maria Beatriz Padua, Dr. Katherine Elizabeth

Hendricks, Dr. Lilian Oliveira, Moises Franco, Adriane Bell (in memorial) and Amber

Brad. I am grateful to them for helping me adapt to Gainesville, with the techniques in

the lab and foremost for their friendship. I also thank my present lab mates Aline and

Luciano Bonilla, Justin Fear and Sarah Fields for their help and assistance. Thanks are

extended to Dr. James Moss and Dr. Silvia Carambula for the great scientific

discussions and not so scientific conversations.

I thank the management and personnel at Central Packing Co. in Center Hill, FL

for providing the ovaries used in most of the experiments of this dissertation and William

Rembert for his assistance in collecting the ovaries and for always being so friendly.

Special thanks go to the management and personnel at North Florida Holsteins (Bell,

FL), the University of Florida Dairy Research Unit (Hague, FL) and Brooksco Dairy

(Quitman, GA).









I am also very grateful to the faculty, staff and students of the Department of

Animal Sciences and the Animal Molecular and Cell Biology Program for all of their

support and friendship.

I thank CAPES and Fulbright for the financial support, assistance with documents

and constant help. Specially the staff from Brazil Silvio dos Santos Salles, Sandra

Lopes, Giselle Melo and Glayna Braga.

My very special thanks go to my friends in Brazil Lemia, Aline, Luciana, Eruska,

Erissa and Virginia for cheering for me since the beginning of this journey. Also, I could

not forget the friends that supported me in every aspect Mr. Helio Alencar and family,

Mr. Reginaldo Barros (in memorial) and family and all friends from Recife.

Finally, I want to thank my husband Mauricio, for his love, support, help with the

experiments and endless patience at these final moments. I also thank my mother and

father in law, Odilon and Penha, my sister in law Tatiana and her family, and my brother

in law Willian for their involvement and support.

This accomplishment would not have happened without the encouragement,

support and love of my parents Maria Jose and Carlos Alberto Barbosa Loureiro, my

sister Emili, my grandmother Margarida, my uncle Jose Maria, my aunties Cau, Bam

and Te and my cousins Giu, Gabri and Lidi. Even from far way, they lived every moment

of this journey with me and I am forever grateful to them.









TABLE OF CONTENTS

page

A C KNOW LEDG M ENTS ......... ............... ............................................. ............... 4

LIST O F TABLES .............. ............................................................................. 9

LIST OF FIGURES.................................. ......... 10

LIST OF ABBREVIATIONS .................. ......................... 11

CHAPTER

1 LITERATURE REVIEW .................. .................. ......... 16

Introduction ......................................................................... .. ... ... ........ 16
Embryonic Development in the Bovine .......... ......... ............... 18
Time Course of Early Development............................................ 18
Changes in Gene Expression During Cleavage Stages ................. 19
Changes in Metabolism of the Early Embryo...................... ............ 20
Developmental Changes in Apoptosis............................... 22
Epigenetic Modifications....................................... ......... 24
Compaction and Blastocyst Formation .................................. ...... ....... 25
Hatching and Elongation ........ ........................................................ .. ...... 29
Maintenance of the Corpus Luteum ........... ................................. 30
A ttachm ent to the Endom etrium .................................. ..................... ........ 31
Placentomes............................. ........................ 32
Alterations in Embryonic Development In vitro ............ ........... ................. 32
Growth Factors and Cytokines as Uterine Regulators of Embryonic
Develop ent ...................................................................... .. ....... ....................... 35
Insulin-like Growth Factor 1 ............. ...................... .......... 37
Interleukin 1 ...................................................... ......... ............ 38
Fibroblast G row th Factor.................................................. .................... 39
Tum or Necrosis Factor.................................................. .................... 40
C olony Stim ulating Factor 2 ........................ ........ ........ .. .. .......................... 41
Biology and Signaling ............................. .......... .................... 41
CSF2 Secretion in the Uterine Tract.................. ... ............... ....... 43
Actions of CSF2 on Embryo Development and Survival .............................. 44
CSF2 and Interferon-tau Secretion............................. ....... 46
G oals of the C current Investigation ................................................... ............... 46

2 COLONY STIMULATING FACTOR IMPROVES DEVELOPMENT AND POST-
TRANSFER SURVIVAL OF BOVINE EMBRYOS PRODUCED IN VITRO............. 49

In tro d u c tio n ......... ............................................. ............... 4 9
M materials and M ethods....... ......... ........................ ................ ............... 51
M a te ria ls ........................ ............................................................... 5 1


6









Effects of CSF2 on Embryo Development and Blastocyst Properties ............. 52
Production of em bryos ......................... ..... ......... ................... ............ 52
Interactions between oxygen concentration and presence of CSF2 ......... 53
Cell number and differentiation of blastocysts.............................. 54
Apoptotic blastomeres ............ .... ....... ......... .................. 55
Effects of CSF2 and IGF1 on Pregnancy and Calving Success after
Transfer to Recipients ......................... .. ......... ...... .................. 55
Production of Holstein embryos using X-sorted semen ........................... 55
A n im a ls ............... ......................................... ......... ... ...... 5 7
Synchronization and timed embryo transfer.................................. .......... 57
Effect of CSF2 and IGF1 added at Day 1 of culture on development and
post-transfer survival of bovine embryos ................................................. 58
Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of
culture on development and post-transfer survival of bovine embryos ... 58
S tatistica l A na lysis ........................................................................ ......... 5 9
Results ......... ...................... .......... ........... ....... ......... 60
Effects of CSF2 on Embryo Development..................................... ................ 60
Effects of CSF2 on Blastocyst Total Cell Number, Cell Differentiation and
A po pto sis ............... ............ ... .................... ..... ........ ............ 6 1
Effects of CSF2 and IGF1 on Pregnancy and Calving Success after
Transfer to Recipients ...................... ........................... 61
D is c u s s io n .............. ..... ............ ................. ............................................. 6 2

3 COLONY STIMULATING FACTOR 2 CAUSES CHANGES IN THE
TRANSCRIPTOME OF THE BOVINE PREIMPLANTATION EMBRYO
INCLUDING ALTERATIONS IN EXPRESSION OF DEVELOPMENTAL AND
A PO PT O S IS G E N E S .............................................. ................... 72

Introduction .................................... ................................. ......... 72
M materials and M methods ............................................ ............ ....... 73
In vitro Production of Embryos ........... ..................................... 73
RNA Purification and Processing ............ ........... ................ ............. 74
Microarray Hybridization .............. ...... ........ ...... .............. 75
A analysis of M icroarray Data................................ .................................... 76
Quantitative Real Time PCR ............... .......... .... ................... 77
R regulation of A poptosis by C S F2 ................................................ .. .................. 78
R results .............. .......................... ................... ................ ................... 79
T ranscriptom al P profile ................................. ..................... ..... ............ 79
Biological Process Ontologies Affected by CSF2 ................ ... ............... 80
Genes Involved in Cellular Development and Differentiation........................... 81
Genes Involved in Signal Transduction and Cell Communication .................... 82
G enes Involved in W NT Signaling............................................ ..................... 82
Genes Involved in Apoptosis Signaling Pathway............................... 83
Q uantitative R eal T im e PC R ...................... ................................. .................. 83
Actions of CSF2 to Block Heat-Shock Induced Apoptosis............................. 84
D is c u s s io n ............. ......... .. .. ......... .. .. ......... .................................... 8 4









4 CONSEQUENCES OF EMBRYONIC EXPOSURE TO CSF2 FROM DAY 5 TO
7 AFTER INSEMINATION ON TROPHOBLAST ELONGATION, INTERFERON-
TAU SECRETION AND GENE EXPRESSION IN THE EMBRYONIC DISC AND
T R O P H EC TO D E R M ......... ......... ......... .......... ................ .............. 103

Introduction ............... .................... ........ ..... ................ ......... 103
M materials and M ethods........................................ .......... 104
In vitro Production of Embryos ........... .. ......................... ...... .. 104
Transfer Into Recipients ........... ............................... ............... 105
Embryo Recovery and Evaluation .......... .............. ........ .... ........ ... 106
A ntiv ira l A ssa y ............. .... .... ... ......................... ............................... .... 10 7
Analysis of the Transcriptome of Trophectoderm and Embryonic Disc.......... 108
Microarray Hybridization .............. .................. .. ............. 109
Analysis of Microarray Data.................................. ............... 109
Quantitative Real Time PCR ................................ .................... 110
Statistical Analysis ...................................... .......... 111
Results ......................... ........ ............. ......... ........... 112
Embryo Survival After Transfer ...... ..................... ............... 112
Embryonic Growth and Development.................................. ........ ........ 113
Antiviral Activity in Uterine Flushings.......................................................... 113
Changes in the Transcriptome of Embryonic Disc and Trophectoderm ........ 113
Characteristics of Genes Differentially Expressed Between Embryonic Disc
and Trophoblast ......................... .......... .... ................ ....... 114
Identification of Likely Candidate Genes for Use as Embryonic Disc Markers 115
Validation of M icroarray Results Using qPCR ............ ............................... 116
Discussion ...... ........ ............................... 117

G ENERAL D ISC USSIO N ................................... ...... .................... .......... 135

LIST OF REFERENCES ......... ........ ...... ................. ........................ 144

BIOGRAPHICAL SKETCH ......................... .. ............ .................... ............... 172









LIST OF TABLES


Table page

2-1 Effect of CSF2 on total cell number, inner cell mass (ICM), trophectoderm
(TE) and ICM/TE ratio of blastocysts at Day 7 after insemination ...................... 68

2-2 Effect of CSF2 on total cell number and TUNEL-positive blastomeres in
blastocysts at Day 7 after insemination ........... ....... .. ..... .... ........... 68

2-3 Effect of CSF2 and IGF1 added at Day 1 of culture on embryonic
development at Day 7, pregnancy risk at Day 30-35 (based on
ultrasonography), calving rate and pregnancy loss among recipient cattle that
received embryos that were cultured in 5% 02 ......................... ...................... 69

2-4 Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of culture
on embryonic development at Day 7, pregnancy risk at Day 30-35 (based on
ultrasonography), calving rate and pregnancy loss among recipient cattle that
received embryos that were cultured in 5% 02 ......................... ...................... 70

3-1 Primers and Probes used on qPCR........ ........................................ 92

3-2 Gene ontologies in the biological process category that were regulated by
CSF2. .................. ........ .. ..................... ......... ................. 95

3-3 Differentially-regulated genes involved in W NT signaling ................................ 98

3-4 Differentially-regulated genes involved in apoptosis................................. .... 99

4-1 Primers used for qPCR ......... .......... ........ ................................. 122

4-2 Estimates of effect of CSF2 on embryonic survival at Day 15 after expected
ovulation. .............. ......... .............................................. 123

4-3 Canonical pathways containing a significant number of genes differentially
expressed between embryonic disc and trophoblast. ........................... 124

4-4 Genes with the greatest fold change for embryonic Disc (ED) compared with
trophoblast (T r) ......... ...................................... ............... .. ......... 127

4-5 The 15 most abundant genes overexpressed in embryonic disc or
trophoblast............... .... ............................................................. 129

4-6 Differences in expression of selected genes between embryonic disk and
trophoblast as determined by microarray analysis and qPCR ....................... 131









LIST OF FIGURES


Figure page

1-1 Mechanisms by which CSF2 regulates cellular survival, differentiation,
functions and activation. ................. ...... ............................. 48

2-1 Percentageage of oocytes that developed to the blastocyst stage at Day 7
(Panel A) and 8 after insemination (Panel B). ............... ............. ............... 71

3-1 Genes expressed in control and CSF2-treated embryos at Day 6 of
develop ent ............. ...... ........... ............................... .. 100

3-2 Validation of microarray results using quantitative PCR................................. 101

3-3 Regulation of heat-shock induced apoptosis in Day 6 bovine embryos by
CSF. .................. ........ .. ..................... ......... ............. 102

4-1 Separation of a Day 15 concepts into embryonic disc and trophoblast. ........ 132

4-2 Individual values of antiviral activity in uterine flushings (top) and length of
recovered embryos (bottom)............................... ............... 133

4-3 Hierarchical Cluster of the transcriptomes of samples of embryonic disc (ED)
and trophoblast (Tr) for control and CSF2 treated embryos.............. ............ 134

5-1 Summary of effects of CSF2 on embryo development and post- transfer
survival. .............. ..... ........ ................................................... 143









LIST OF ABBREVIATIONS


AQP Aquaporins

ATP Adenosine triphosphate

3c Beta common

BNC Binucleated cell

CCCP Carbonyl cyanide 3-chloro-phenylhydrazone

CpG Cytosine-guanine dinucleotide

DNA Deoxyribonucleic acid

EAG Embryonic genome activation

ED Embryonic disc

ES Embryonic stem

FSH Follicle stimulating hormone

GnRH Gonadotropin-releasing hormone

GTP Guanosine triphosphate

H hour

ICM Inner cell mass

IFNT Interferon tau

IVP In vitro produced

JAK Janus kinase

KSOM Potassium simplex optimized medium

Min minute

mRNA Messenger RNA

mtDNA Mitochondrial DNA

NADPH Nicotinamide adenine dinucleotide phosphate

NKT Natural Killer T









PGF Prostaglandin F2a

P13K Phosphatidylinositol-3 kinase

PKC Protein kinase C

RNA Ribonucleic acid

RNAse Ribonuclease

STAT Signal transduction and activation of transcription

Sec Second

SOF Synthetic oviduct fluid

TE Trophectoderm

Tr Trophoblast

TUNEL Terminal deoxynucleotidyl transferase mediated dUTP

Vs Versus









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

REGULATION OF THE PREIMPLANTATION BOVINE EMBRYO BY COLONY
STIMULATING FACTOR 2

By

Barbara Loureiro

August 2010

Chair: Peter J. Hansen
Major: Animal Molecular and Cell Biology

Colony-stimulating factor 2 (CSF2) is a multifunctional cytokine originally

recognized as a hematopoietic factor but now known to be expressed in several

reproductive tract tissues including the bovine oviduct and endometrium. Colony-

stimulating factor 2 may be an important intracellular regulator of endometrial, oviductal

and embryonic functions during early pregnancy in the cow and other species. Addition

of CSF2 to culture medium improved the proportion of in vitro produced embryos

developing to the blastocyst stage in cow, mouse, human and pig and increased post-

transfer embryonic survival in mice. A series of experiments was conducted to

understand the role of CSF2 in preimplantation embryonic development including its

effects on blastocyst formation, the embryonic transcriptome, and embryonic survival.

The first experiment was conducted to test whether addition of CSF2 to culture

medium could enhance development and post-transfer survival of in vitro produced

bovine embryos. Treatment of embryos with CSF2 at Day 1 after insemination

increased the percentageage of embryos that became transferable morulae or

blastocysts at Day 7 but had no significant effect on pregnancy rate at Day 30-35 or

calving rate. When CSF2 was added at Day 5 after insemination, there was increase in









the percentageage of embryos that became transferable morulae or blastocysts at Day

7, an increase in the percentageage of recipient cows pregnant at Day 30-35, an

increase in calving rate and a decrease in pregnancy loss after Days 30-35.

Furthermore, blastocysts formed after treatment with CSF2 had an increase in the

number of cells in the inner cell mass.

The second experiment was conducted to analyze the transcriptome of CSF2

treated embryos to identify genes involved in mediating CSF2 effects on development to

the blastoctcyst stage and survival after transfer. The Agilent bovine microarray platform

was used. Embryos were treated with CSF2 at Day 5 and selected at Day 6 after

insemination. A total of 160 genes were differentially expressed, with 67 being higher in

CSF2-treated embryos and 93 being lower. Analysis identified 13 biological process

ontologies that were grouped into three major groups. The first group included genes

functionally involved in developmental processes and differentiation. Actions of CSF2

would tend to inhibit genes involved in neurogenesis and stimulate genes involved in

mesoderm or muscle formation. The second group were genes involved in cell

communication, with the most characteristic effect caused by CSF2 being inhibition of P-

catenin dependent WNT signaling. The third group were genes involved in apoptosis

signaling, which was inhibited by CSF2. The antiapoptotic actions of CSF2 were

confirmed in another experiment in which CSF2 decreased the percentageage of

blastomeres in Day 6 embryos becoming apoptotic after heat-shock.

The third study evaluated possible mechanisms by which CSF2 acts during Day 5

to 7 of development to improve embryonic and fetal survival. Embryos were treated

with CSF2 or served as controls and were transferred to recipient cows at Day 7 after









ovulation. Embryos were recovered at Day 15 and embryonic disc and trophoblast

analyzed for gene expression by microarray analysis. Results suggest that higher

pregnancy rates at Day 30-35 represent increased embryonic survival before Day 15

and a greater capacity of the embryo to elongate and secrete interferon tau (IFNT) at

Day 15. This conclusion is based on greater recovery of embryos from cows receiving

CSF2-treated embryos at Day 15 (P<0.07), a tendency for CSF2 treated embryos to be

longer than control embryos, and greater antiviral activity (a measure of IFNT

bioactivity) in uterine flushings of cows receiving CSF2 treated embryos (P<0.07, when

considering those cows with detectable antiviral activity). Analysis of gene expression

in filamentous embryos indicated no difference in transcription among this subset of

embryos that survived to Day 15 and elongated successfully. Therefore, the reduction

in embryonic and fetal loss after Day 30-35 caused by CSF2 is probably not a direct

reflection of altered gene expression at Day 15.

Taken together these results indicate that CSF2 can regulate embryonic

development, increase embryonic survival after transfer and decrease pregnancy loss.

The ability of CSF2 to improve development is probably due to a set of effects that

include an increase in the number of cells in the inner cell mass, a decrease in cell

death and differentially regulation of the embryo transcriptome. Furthermore, before

implantation CSF2 embryos tend to be longer and secret more IFNT. The increased

pregnancy rates observed after Day 35 of pregnancy may be a combined result of the

factors mentioned above. Moreover, CSF2 treatment can be inflicting epigenetic

changes that persist latter in development or even morphological changes that are

consequence of the differentially gene expression early before implantation.









CHAPTER 1
LITERATURE REVIEW

Introduction

The maternal microenvironment of the oviduct and uterus is a major determinant

of the health and well being of the newly formed offspring during embryonic, fetal,

neonatal and adult life. During development, the needs of the embryo constantly

changes and, to cope with those changing requirements, the reproductive tract

undergoes specific physiological and biochemical modifications throughout gestation

(Buhi 2002; Spencer et al., 2007; Hugentobler et al., 2010).

The importance of the uterine environment for pregnancy has been shown in

several experiments. By comparing the probability that twin embryos would survive

pregnancy in embryo transfer studies, McMillam (1998) estimated that only about 50-

70% of recipients are capable of maintaining a transferred embryo. In other embryo

transfer experiments with cows, pregnancy rates were affected by a variety of

physiological states of the recipient animal including heat stress (Chebel et al. 2008),

circulating concentrations of urea (Tillard et al. 2007), treatment with somatotropin

(Moreira et al. 2001), milk yield (Yaniz et al. 2008), and asynchrony between recipient

and embryo (Kubisch et al. 2004). Evidence that the uterine environment can affect

adult life comes from many species including the sheep, in which a reduction in vitamin

B12, folate and methionine content of the maternal diet around the time of conception

caused high blood pressure, increased adiposity, insulin resistance and altered immune

function in adult offspring (Sinclair et al. 2007). Males suffered greater effects than

females. Long term effects in adult sheep are probably manifestation of epigenetic

modifications of the genome.









The importance of the uterine environment for embryonic development means that

in vitro systems for embryo production can be compromised unless critical features of

the reproductive tract environment are replicated in vitro. In beef and dairy production

systems, the in vitro produced (IVP) embryo is important for increasing genetic selection

and as a required technical procedure for production of transgenic animals (Hansen and

Block 2004). Embryo transfer can also be used to improve fertility in heat-stressed

females (Block et al. 2003; Block and Hansen 2007). Embryo transfer is effective in this

regard because embryos are transferred to the recipient at Day 7 of pregnancy when

the embryo is resistant to heat stress (Ealy et al. 1993; Ealy et al. 1995).

Early embryonic development is not absolutely dependent upon regulatory

molecules present in the reproductive tract because embryos produced in vitro in

medium without growth factors can give rise to live calves after transfer to recipients

(Block and Hansen 2007). However, culture conditions are suboptimal and result in

alterations at the morphological (Fischer-Brown et al. 2002; Fischer-Brown et al. 2004),

ultrastructural (Rizos et al. 2002a), physiological (Ushijima et al. 1999) and

transcriptional levels (Bertolini et al. 2002; Sagirkaya et al. 2006; Smith et al. 2009)

compared with embryos produced in vivo. Using microarray analysis, 200 genes were

found to be differentially expressed between embryos produced in vivo and in vitro

(Smith et al. 2009). In addition, several abnormalities have been reported in calves

resulting from IVP embryos (Farin and Farin 1995; Lazzari et al. 2002; Miles et al.

2005). Thus, one or more components of the maternal environment are necessary for

optimal embryonic development and birth of a healthy calf.









There are strong lines of evidence to implicate colony stimulating factor 2 (CSF2),

otherwise called granulocyte-macrophage colony-stimulating factor, as a physiologically

important regulator of early embryonic development. This cytokine is expressed in

several reproductive tract tissues including the oviduct and endometrium of the cow (de

Moraes et al. 1999) and human (Zhao and Chegini 1994; Chegini et al. 1999). Addition

of CSF2 to culture medium improved the proportion of in vitro embryos developing to

the blastocyst stage in cow (de Moraes and Hansen 1997a), mouse (Robertson et al.

2001), human (Sjoblom et al. 1999) and pig (Cui et al. 2004) and increased post-

transfer embryonic survival in mice (Sjoblom et al. 2005).

The literature review here presented will focus on the events that take place during

early embryonic development in the cow, unless stated otherwise, and the effects of

CSF2 on preimplantation embryonic development.

Embryonic Development in the Bovine

Time Course of Early Development

After ovulation, the bovine oocyte is picked-up by the cilia-covered fimbria and

guided through the infundibulum and ampulla of the oviduct (Oxenreider and Day 1965).

It is in the ampullary-isthmic junction of the oviduct that the oocyte is fertilized (Bazer et

al. 2009). Fusion of the two gametes and release of the second polar body represents

the completion of meiosis (Marteil et al. 2009). The first cleavage of the zygote occurs

around 23-31 hours after insemination (Maddox-Hyttel et al. 1988). The developing

embryo leaves the oviduct and moves into the uterus approximately at Day 5 of

pregnancy when it is about 16 cells (Betteridge et al. 1988).

Until hatching at Day 7-8, the embryo is surrounded by a protein coat called the

zona pellucida (composed of three glycoproteins; ZP1-ZP3) that is important for









prevention of polyspermy, to keep the blastomeres together and to conserve the

microenviroment of the peritvitelline space (Litscher et al. 2009; Van Soom et al. 2010).

After hatching, the blastocyst is transformed to an ovoid form until elongation of the

trophoblast is initiated between Days 12 and 14. Also around this period, gastrulation

and specification of the germ layers occur in the embryonic disc (ED). By Day 24 the

concepts can fill the entire length of both uterine horns (Blomberg et al. 2008).

Implantation starts on Day 20, when the trophectoderm (TE) adheres to the endometrial

luminal epithelium of the mother (Blomberg et al. 2008; Bazer et al. 2009).

Changes in Gene Expression During Cleavage Stages

During early development, the embryonic genome is inactive and the embryo

relies on maternal messenger ribonucleic acid (mRNA) for protein synthesis (Thelie et

al. 2009). The recruitment mechanisms by which dormant RNA is either targeted for

translation or decay are still largely uncharacterized. The current model involves

lengthening of the poly(A) tail, which triggers binding of the poly(A) binding protein and

binding of translation initiation factors (Memili and First 2000; Groisman et al. 2002).

RNA concentration is highest in the germinal vesicle stage oocyte and from then until

the 8-cell stage, RNA is gradually depleted (Gilbert et al. 2009; Vallee et al. 2009).

Evidence in the mouse suggests that this decline is important for activation of the

embryonic genome (Li et al. 2010). Depletion of maternal argonaute 2 (Ago2), which

encodes a catalytic RNA hydrolase (RNase), disrupts gene expression and the 2 cell

embryo fails to become a blastocyst (Li et al. 2010). In the bovine, embryonic genome

activation (EGA) occurs at the 8 to 16-cell stage (Memili and First 2000) through an

unknown mechanism.









Changes in Metabolism of the Early Embryo

The type of substrate metabolized by the embryo changes with development. At

early stages, the fuel for ATP formation by the mitochondria is provided by pyruvate,

while the uptake of glucose is low. As the embryo develops to the compact morula and

blastocyst stage, ATP synthesis increases and glucose starts to contribute to the citric

acid cycle through conversion to lactate and then pyruvate (Thompson 2000). Glucose

can also generate ribose required for nucleic acid synthesis, and nicotinamide adenine

dinucleotide phosphate (Thompson 2000). Furthermore, glucose, pyruvate and lactate

production and/or consumption can be different between the two cell types of the Day 8

blastocyst. While inner cell mass (ICM) cells consume more glucose than pyruvate, the

converse is true for TE cells (Gopichandran and Leese 2003). Lactate production is

higher for TE than for ICM cells (Gopichandran and Leese 2003).

The number and activity of mitochondria also change as the embryo develops. A

primordial oocyte contains as few as 10 mitochondrial DNA (mtDNA) copies whereas a

fully grown oocyte can contain more than 100.000 copies of mtDNA, with one or two

copies of mtDNA per organelle (Ferreira et al. 2009; Chiaratti et al. 2010). At this stage

the mitochondria is immature and does not present any activity, therefore the energy is

supplied through the granulosa cells (Tarazona et al. 2006). Initially present near the

periphery of the cell, oocyte mitochondria become more dispersed at the germinal

vesicle breakdown stage, presumably to better serve the cell (Ferreira et al. 2009;

Marteil et al. 2009). At this point the levels of activity have highly increased and the

mitochondria are capable of generating the necessary ATP (Tarazona et al. 2006). The

bovine embryo does not gain the capacity for replenishing the mtDNA until the morula

and blastocyst stages when there is increased expression of nuclear respiratory factor 1









(NRF1) and transcription factor A, mitochondria (TFAM) that are regulators of mtDNA

replication and transcription (Chiaratti et al. 2010). Embryos with non-competent

mitochondria stop development before the EGA (Tarazona et al. 2006).

High concentrations of glucose are toxic to bovine embryos, especially female

embryos undergoing the transition from the morula to blastocyst stage (Gutierrez-Adan

et al. 2000). This fact along, with the possibility that male embryos develop faster in

culture than female embryos (Gutierrez-Adan et al. 2000; Kimura et al. 2005), could

explain the skewing of the sex ratio towards males often seen in calves from IVP

embryos (Block and Hansen 2007; Camargo et al. 2010).

The gender difference in growth rate and sensitivity to glucose could be due to

differential gene expression either because of differences in activity of sex or autosomal

chromosomes. Both X-chromosomes are active in the cleavage-stage female embryo

(Mak et al. 2004). Unbalanced expression of X-linked genes can increase the activity of

enzymes involved in energy metabolism and detoxification of oxygen radicals (Perez-

Crespo et al. 2005). For example, the gene for glucose 6-phosphate dehydrogenase

(G6PDH), an enzyme that controls the entry of glucose into the pentose-phospate

pathway, is located on the X-chromosome. With both X-chromosomes active in the

female embryo there is more pentose-phospate pathway activity which leads to poor

tolerated imbalance in carbohydrate metabolism (Gutierrez-Adan et al. 2000).

Hypoxanthine phosphoribosyl transferase 1, an enzyme involved in controlling the

amount of oxygen radicals, is also on the X-chromosome (Goldammer et al. 2003). Free

radical actions involve not only cellular damage but also cellular growth (Rieger 1992).









The retarded development in female embryos could be caused by a decrease in oxygen

radical levels due to increased activity of the enzyme.

By the time the embryo reaches the blastocyst stage, 88% of the genes (193

genes) in the X-chromosome are upregulated in the female embryo comparing to the

male embryo. However, only 10% of these genes had a 2 fold-increase and in fact, 70%

of the genes had a fold-increase lower than 1.6 comparing to the male embryo

(Bermejo-Alvarez et al. 2010). These suggest that at the blastocyst stage the X-

chromosome is starting to be inactivated.

Glutathione is the most important cellular antioxidant in the cytosol. There is a

large decline in its concentration after fertilization because it is consumed as part of the

chromatin decondensation process (Lim et al. 1996; Luberda 2005). However, de novo

synthesis of glutathione increases at the 16 cell stage (around EGA), and reaches its

maximum peak at the hatched blastocyst stage (Lim et al. 1996).

Developmental Changes in Apoptosis

Apoptosis is a developmentally regulated process that plays an important role in

the survival of the preimplantation embryo. Although the maturing oocyte can undergo

apoptosis (Roth and Hansen 2004a; Roth and Hansen 2004b; Roth and Hansen 2005),

the capacity for apoptosis is lost at the 2-cell stage and does not become reacquired

until sometime between the 8 and 16-cell stages (Paula-Lopes and Hansen 2002a;

Gjorret et al. 2005). As the embryo undergoes further development, there is little

change in the degree of apoptosis (Loureiro et al. 2007). The lack of apoptosis at the

two cell stage caused is in part by a block in activation of caspases (Brad et al. 2007; de

Castro e Paula and Hansen 2008a), which in turn reflects increased resistance of the

mitochondria to depolarization (Brad et al. 2007). A second block to the pathway exists









at the level of the nucleus since addition of carbonyl cyanide 3-chloro-phenylhydrazone,

a mitochondria depolarization agent, did not cause increase in DNA fragmentation even

though group II caspases were activated (Brad et al. 2007; Carambula et al. 2009).

The sensitivity of embryonic DNA to fragmentation coincides with its levels of

methylation. DNA is highly methylated at the 2-cell stage and then becomes

progressively more demethylated as development progresses until by the 8-16 cell

stage the embryo has low methylation and transcription is activated (Dean et al. 2001;

Park et al. 2007). The interaction between apoptosis and methylation was shown when

sensitivity of 2-cell embryos to carbonyl cyanide 3-chloro-phenylhydrazone could be

induced with 5-aza-20-deoxycytidine, a DNA methylation inhibitor (Carambula et al.

2009).

In the bovine embryo, the consequences of apoptosis are dependent upon stage

of development. Induction of apoptosis is a major cause for the reduced oocyte

competence for fertilization and development caused by heat shock (Roth and Hansen

2004b). Cell stressors like arsenic (Krininger et al. 2002), heat shock (Loureiro et al.

2007) and ceramide (de Castro e Paula and Hansen 2008b) induce apoptosis through

the mitochondrial pathway that causes mitochondria depolarization and activation of

caspases. It is likely that massive activation of apoptosis by these stresses

compromises development. However, when the stress is less severe so that the

increase in apoptosis is limited, the apoptotic process itself may be beneficial to embryo

survival. At Day 4 of development, inhibition of apoptosis with a caspase inhibitor

exacerbated deleterious effects of heat shock on development to the blastocyst stage

(Paula-Lopes and Hansen 2002b).









Epigenetic Modifications

Cytosine methylation at the cytosine-guanine dinucleotide (CpG) islands in the

genomic DNA is necessary for regulation of chromatin configuration and normal gene

expression (Geiman and Muegge 2010). In general, hypermethylated DNA is

transcriptionally inactive whereas hypomethylated DNA is highly transcribed (Corry et

al. 2009). Conservation of methylation in only one parental allele is a form of epigenetic

regulation known as imprinting (Wilkins 2006; Tveden-Nyborg et al. 2008). This

monoallelic expression can be tissue specific. One example of tissue specific imprinting

is the X-chromosome, in which the paternally derived copy is inactivated in

extraembryonic tissue while its expression is random in fetal and adult somatic tissues

(Wilkins 2006).

The degree of methylation change with development. In the bovine zygote, the

male pronucleus is partially demethylated at 20 hours after fertilization so that paternal

levels of reactivity to antibody to 5-methyl cytosine is 51% of that of maternal DNA (Park

et al. 2007). Methylation of the paternal chromosomes increases to levels similar to the

female by 28 hours after fertilization when embryos are at about the 2-cell stage (Park

et al. 2007). Embryonic genome activation occurs at the 8 to16-cell stage (Gilbert et al.

2009). Up to this stage, embryos are highly methylated (Dean et al. 2001) and

coincident with embryonic genome activation, methylation declines (Dean et al. 2001).

De novo methylation after the 16-cell stage is not identical for all the nuclei. By the

blastocyst stage, the ICM contains highly methylated nuclei and the TE lower amounts

of methylated DNA (Dean et al. 2001). The degree of methylation influences cell

potential for differentiation (Western et al. 2010).









Compaction and Blastocyst Formation

Around the 32-cell stage, at Day 5 of pregnancy, the bovine embryo forms a solid

mass known as the morula that then undergoes a process called compaction

(Betteridge et al. 1988). Prior to compaction, the blastomeres are spherical and lack

specialized intercellular junctions. During compaction, cells become flattened against

one another, thus maximizing intercellular contact and obscuring intercellular

boundaries (Sheth et al. 2000; Johnson and McConnell 2004). This process is mediated

primarily by activation of e-cadherin, a Ca2+ dependent adhesion molecule. For full

adhesive function and cytoskeletal anchorage, e-cadherin forms a core adhesion

complex with a protein known as catenin (Niessen and Gottardi 2008). This type of

junction is responsible for generating contact dependent growth and polarity signals with

apical and basolateral domains in all blastomeres (Johnson and McConnell 2004).

Blastomere initiation of polarity seems to be mediated by the Par complex protein (Par-3

and -6), atypical protein kinase C (aPKC) and caudal cell division cycle homolog (S.

cerevisiae; cdc42) which are localized on the embryo's apical domain (Eckert and

Fleming 2008). Inhibition of aPKC and Par-3 causes failure of asymmetric cleavage

latter in development (Plusa et al. 2005).

Another important junctional complex for epithelial differentiation is the tight

junction. Formed by claudin, ocludin and zona ocludins proteins (ZO-1 and -2), this

junction tightly connects opposing cell membranes, creating a barrier that is virtually

impermeable to fluid diffusion through the intercellular space (Tsukita et al. 2001). In

mouse embryos, after compaction ZO-1 binds to rab-guanosine triphosphate hydrolase

(GTPase) and Par-3-6/aPKC to the e-cahdehirin-catenin complex. When cells start to

differentiate, the peripheral membrane proteins cingulin and ZO-2 assemble to the









complex which in turn results in loss of Par-3-6/aPKC. Finally, during blastocoel

formation, ZO-1a+ and the transmembrane proteins occludins and claudins join the

complex. At this point the embryo generates an impermeable seal between TE cells and

the blastocoel cavity (Sheth et al. 2000).

Cavitation, in which the fluid-filled blastocoel is formed, involves polarized

transport of ions and water (Watson et al. 2004). Vectorial transport of Na+ and Cl- ions

through the TE into the blastocoel generates an osmotic gradient that drives fluid across

the epithelium (Kawagishi et al. 2004). In the mouse embryo, it is a carrier-mediated

process that involves several types of ion transporters, including a Na+ channel, Na+/H+

exchangers and ATPase Na+/K+ transporting, alpha 1 (Atplal). The Atplal are

localized in the basolateral membrane region of the TE (Madan et al. 2007). Blastocoel

expansion is significantly retarded in the absence of extraembryonic Na+, in the

presence of inhibitors of Na+ channels or silencing RNA (siRNA) for Atplal (Kidder

2002; Madan et al. 2007). Also present in the TE membrane are water channels known

as aquaporins (AQP). They allow rapid water flow across the membrane in the direction

of the osmotic gradient (Liu and Wintour 2005). Murine preimplantation embryos

express mRNA for multiple AQP throughout preimplantation development (Liu and

Wintour 2005). AQP-3 mRNA increases at the morula to blastocyst transition and AQP-

8 protein is first detected in the cell margins at the morula stage (Barcroft et al. 2003).

Both AQP-3 and -8 are found in the basolateral membrane of the TE while AQP-9

is predominantly observed in the apical membrane domain of the TE (Liu and Wintour

2005). AQP-8 is highly selective for water molecules while AQP-3 and -9 are less

selective, allowing the passage of small solutes (van Os et al. 2000).









Following compaction and cavitation, the bovine blastocyst at Days 6-7 starts to

differentiate into ICM cells, which retain a pluripotent phenotype (Pant and Keefer 2009)

and TE cells which will become the outer layer of the placenta (Hamilton 1946;

Betteridge et al. 1988). TE cells are the first to differentiate and have the characteristics

of an epithelium (Marikawa and Alarc6n 2009). The ICM cells go through a second

lineage segregation becoming the epiblast, which will give rise to the fetus itself, and

primitive endoderm that becomes the parietal and visceral endoderm, which latter

contributes do the yolk sac (Blomberg et al. 2008). Around the third week of

development gastrulation, neurulation and formation of the somites will take place in the

epiblast (Maddox-Hyttel et al. 2003).

In the bovine, it is not known how the blastomere decides to become a TE cell or

ICM cell. There are two theories to explain TE and ICM differentiation in the mouse

embryo. According to the inside-out theory, position determines cell fate. At the morula

stage, cell to cell contact increases and some cells become enclosed by surrounding

cells while other cells in the outside layer are in contact with the external environment

for part of their surface. Asymmetric cell contact induces epithelial differentiation into TE

whilst symmetric contact of the enclosed ICM inhibits differentiation (Eckert and Fleming

2008; Marikawa and Alarc6n 2009). An alternative theory suggests that the cell decides

its fate and the location in which it will reside, rather than the location deciding what fate

the cell will have (Yamanaka et al. 2006). Several studies have corroborated the inside-

outside model. For example, imunosurgically-isolated ICM have the potential to form

TE in vivo (Rossant and Lis 1979) and tight junction and a blastocoel in vitro (Eckert et

al. 2005).









A few genes have been identified as being responsible for formation of the ICM

and TE in human and mouse. The first transcription factor to appear in the late morula

ICM is Sex determining region Y-box2 (Sox2) (Guo et al. 2010). It is a key factor for

maintenance of pluripotency and for reprogramming of differentiated cells into induced

stem cells (Takahashi and Yamanaka 2006). Pou domain class 5 transcription factor-1

(Pou5fl or Oct4) is a transcription factor necessary for the maintenance of the

pluripotency in the ICM and it prevents ICM transformation to TE (Zernicka-Goetz et al.

2009). Another stem cell marker present in the ICM is Nanog homeobox (Nanog)

(Marikawa and Alarc6n 2009). Caudal type homeobox 2 (Cdx2) is specifically

expressed in TE and its presence is necessary to repress Pou5fl expression in TE.

Loss of Cdx2 results in failure to downregulate Pou5fl and Nanog in outer cells of the

blastocyst and subsequent death of those cells (Strumpf et al. 2005). Nodal is found to

be expressed in the ICM and primitive endoderm of the blastocyst and also latter in the

epiblast and visceral epithelium (Mesnard et al. 2006).

In the bovine, the mechanism of early segregation and differentiation is different in

some respects from the mouse. POU5F1 is expressed in both ICM and TE of in vitro

and in vivo produced blastocysts until Day 10 of development (Eijk et al. 1999; Kirchhof

et al. 2000; Mesnard et al. 2006) whereas CDX2 has a weak expression (Degrelle et al.

2005). NANOG mRNA and protein are also found in both ICM and TE of bovine

blastocysts and elongated embryos, but with greater expression in the ICM and ED

(Degrelle et al. 2005; Muioz et al. 2008).

A microarray experiment that analyzed the transcriptome of ICM and TE of human

blastocysts has identified new marker genes to complement the existing markers for









ICM/TE such as POU5F1 and CDX2. Pathway analysis of the microarray data identified

signaling pathways related to integrin mediated cell adhesion and overexpression of

several NA+/K+-ATPases in TE (Adjaye et al. 2005), reflecting their role in controlling

permeabilization and fluid transport across the epithelium. Keratin 18 (KRT18), a

cytoeskeletal protein, was predominantly expressed in the TE cells and

imunocytochemistry detected its expression only in the cell to cell contacts of the TE

(Goossens et al. 2007). Pathways involved in cell cycle were also differentially

expressed. Genes that activate the WNT signaling pathway were shown to be

overexpressed in the ICM while genes encoding Casein kinase 1 alpha (CSNKIA) and

disheveled activator of morphogenesis 1 (DAAM1), which are agonists of the WNT

pathway, were both over expressed in the TE (Adjaye et al. 2005).

Hatching and Elongation

The blastocyst becomes fully expanded when it reaches about 160 tol80-cells

(Van Soom et al. 1997). Thereafter, the blastocyst hatches from the zona pellucida by a

combination of cell growth and volume increase in the blastocoel (Van Soom et al.

1997; Houghton et al. 2003). The hatching process in the bovine embryo is apparently a

mechanical process more than an enzymatic one (Flechon and Renard 1978; Massip

and Mulnard 1980; Massip et al. 1982).

Once the blastocyst has hatched, the ICM forms a protuberance called the ED,

which is still covered with TE (Rauber's layer) until about Day 12. By Day 12, the layer

of TE cells covering the ED is degraded via apoptosis and the disc cells are exposed to

the maternal milieu (Williams and Biggers 1990; Guillomot et al. 2004).

Elongation begins between Days 12 and 14 (Betteridge et al. 1988; Vejlsted et al.

2006) and is concomitant with gastrulation (Hue et al. 2001). The development of the









trophoblast provides a large placental surface area that is better able to initiate the

maternal-conceptus cross-talk and exchange essential nutrients for survival of the

concepts (Spencer and Bazer 2004). As part of the process of elongation, the

concepts undergoes shape changes going from spherical to ovoid, then tubular, and

finally to the elongated stage. As a result, the concepts increases in size more than

1000-fold by Day 24 of gestation so that it can extend the entire length of both uterine

horns (Maddox-Hyttel et al. 2003). Elongation is accomplished by an increase in cell

number accompanied by an increase in protein synthesis (Thomson 1998; Degrelle et

al. 2005). Elongation of bovine embryos appears to be in part determined by uterine

signals, given that extended in vitro culture beyond the blastocyst stages results in

formation of TE outgrowths and attachment of the embryo to the bottom of the culture

dish rather than elongation (Alexopoulos et al. 2005). However, in vitro produced

blastocysts elongate when transferred to recipients (Block et al. 2007). One candidate

as a uterine elongation factor is insulin-like growth factor binding protein 1 (IGFBP1). In

the bovine endometrium, IGFBP1 mRNA increases in amount by Day 16 of pregnancy

and is significantly different in pregnant versus non pregnant animals (Simmons et al.

2009). In vitro, IGFBP1 stimulates migration and mediated attachment of ovine TE cells

while had no effect on cell proliferation (Simmons et al. 2009).

Maintenance of the Corpus Luteum

One of the roles of the elongated concepts is to produce the pregnancy

recognition signal, interferon-tau (IFNT), which blocks luteal regression caused by

uterine prostaglandin F2a (PGF2a) release and allowed for continued secretion of

progesterone (Thatcher et al. 2001; Spencer et al. 2007). IFNT is secreted by the

mononuclear cells of the primitive extra-embryonic trophoblast a few Days prior to when









the concepts attaches to the uterine wall (Helmer et al. 1987). The peak production of

IFNT occurs on Days 16 to 17 of pregnancy (Thatcher etal. 2001). The way IFNT

prevents development of the luteolytic mechanism has been well documented in sheep.

It inhibits transcription of the gene for the estrogen receptor a in the luminal and

superficial ductal glandular epithelia (Spencer and Bazer 2004). This action prevents

the induction of oxytocin-receptor transcription by estrogen and, therefore, oxytocin

induced luteolytic pulses of PGF2a (Asselin et al. 1997; Spencer et al. 1998; Pru et al.

2001; Spencer and Bazer 2004). In the cow the mechanism must be a little different,

since there is no change in estrogen receptor mRNA in the endometrium from Day 16

pregnant cows and cyclic cows but there is a decrease in oxytocin receptor mRNA

(Robinson et al. 1999). Therefore, in the cow, pregnancy may alter the oxytocin receptor

regardless of the estrogen one. Between Days 8 and 17 of pregnancy is when the

concepts ordinarily inhibits pulsatile PGF2a secretion but is also when at least 40% of

total embryonic losses occur (Thatcher et al. 2001).

Attachment to the Endometrium

The ruminant concepts does not actually implant in the uterus. Rather

placentation occurs because of apposition and interdigitation with limited invasion of

trophoblast cells into the endometrial epithelium. Placentation starts at about Day 20 of

pregnancy (Chavatte-Palmer and Guillomot 2007). Cell contact is initiated in the region

of the ED and extends towards the ends of the concepts (Assis Neto et al. 2009a). In

sheep and cow embryos, the trophoblast attaches to the caruncular epithelium mainly

and to the intercaruncular mucosa to a lesser extent. In these areas, transitory villi grow

on the trophoblast and invade the uterine glands. This process ensures an anchorage of









the concepts in the uterine cavity and localized absorption sites of the glandular

secretions (Chavatte-Palmer and Guillomot 2007).

The limited invasion of the maternal tissue is caused by trophoblast cells that

migrate into the endometrial epithelium and fuse with uterine epithelial cells to form

binucleated cells (BNC). The BNC represent 20% of the trophoblast and produce

placental lactogen and a group of native aspartyl proteinases called pregnancy

associated glycoproteins that are delivered to the maternal compartment (Szenci et al.

1998; Klisch et al. 1999). Fusion of the BNC with uterine epithelial cells forms syncytial

plaques (Schlafke and Enders 1975). Due to the presence of uterine syncytium, the

ruminant placenta is classified as synepitheliochorial.

Placentomes

The definitive placenta is differentiated into two regions cotyledons and

intercotyledonary tissue. Placentomes are formed by interdigitation of cotyledons with

corresponding structures on the endometrium called caruncles. Their primary role is

nutrient and gas exchange between the fetus and the mother (Schlafer et al. 2000;

Enders and Carter 2004). Cotyledons can be observed macroscopically after Day 37 of

gestation and 80 to 120 cotyledons eventually form (Schlafer et al. 2000; Assis Neto et

al. 2009). By the second trimester of gestation, the number of viable luteal cells

decrease and the cotyledons are responsible for producing the progesterone necessary

for maintenance of pregnancy (Shemesh 1990; Izhar et al. 1992).

Alterations in Embryonic Development In vitro

Pregnancy risk in recipients of IVP embryos are generally not superior to

pregnancy risk following artificial insemination (Rodrigues et al. 2004; Sartori et al.

2006) and are less than following the transfer of in vivo derived embryos (Hasler 2000).









Furthermore, in vitro produced (IVP) embryos that survive the embryonic period are

more likely to be lost later on. While pregnancy loss after the first two months of

gestation for Al and superovulated embryos is generally around 5 to 14% (Santos et al.

2004; Demetrio et al. 2007; Jousan et al. 2007), pregnancy loss after Day 40 of

gestation for IVP embryos ranged from 12 to 24% (Hasler 2000; Block et al. 2003;

Demetrio et al. 2007).

A major problem with IVP embryos compared with embryos produced in vivo is

poor survival to cryopreservation (Enright et al. 2000; Rizos et al. 2002). The high

sensitivity of IVP embryos to chilling and freezing can be related to a higher

accumulation of cytoplasmic lipid droplets (Ushijima et al. 1999). In addition, blastocysts

cultured in synthetic oviduct fluid medium (SOF) had lower expression of connexin 43, a

gap junction protein that is essential for the transport of cryoprotectants and fluids

during freezing and thawing, when compared with embryos developed in coculture or in

vivo (Rizos et al. 2002a).

The period of embryo development, rather than the period of maturation or

fertilization, is the most critical one for perturbations resulting in reduced capacity of the

blastocyst for cryopreservation. Survival rates after cryopreservation of blastocysts that

were produced by maturation and fertilization in vivo but cultured in vitro were 0%

compared with 70% for blastocysts that were matured, fertilized and cultured in vivo

(Rizos et al. 2002a). A similar conclusion that the period of embryonic development is

crucial for high competence for cryosurvival was acquired when cryopreservation rates

of embryos produced by in vitro maturation, fertilization and embryo culture (0%) were

compared with cryopreservation rates of embryos that were matured and fertilized in









vitro but cultured in the ewe oviduct (63%) (Enright et al. 2000; Rizos et al. 2010).

Transcript abundance of 5 genes, elongation factor 1 gamma (EEFIG),

guaninenucleotide binding protein (GNB3), forkhead transcription factor (FOXP3),

represssor of estrogen receptor activity and high mobility group protein 2 (ESR1) were

significantly lower for blastocysts cultured in SOF than for blastocysts produced in vivo

or produced in vitro and allowed to develop in the ewe oviduct (Corcoran et al. 2007).

Abnormalities for calves resulting from IVP embryos have included increased calf

birth weight (Lazzari et al. 2002), altered organ development (Farin and Farin 1995) and

alterations in placentome number and placental structure (Miles et al. 2005). Those

abnormalities can result in an increase in the cases of dystocia and cesarean section as

well as perinatal mortality (van Wagtendonk-de Leeuw et al. 2000).

The low pregnancy risk and increased fetal loss that are characteristic of transfers

with IVP embryos are probably connected to a variety of cellular and molecular

deviations during early embryonic development. Morphological evaluation of Day 14

embryos revealed that IVP embryos have high incidence of no detectable ED (Bertolini

et al. 2002; Fischer-Brown et al. 2002; Fischer-Brown et al. 2004). In vitro culture of

bovine embryos in the presence of high concentrations of serum or bovine serum

albumin resulted in increased number of cells in Day 7 blastocysts, size of blastocysts

on Day 12, and the relative abundance of the transcripts for heat shock protein 70.1

(HSP70.1), copper/zinc-superoxide dismutase, glucose transporters-3 and -4 (SLC2A3

and SLC2A4), fiblroblast growth factor 2 (basic) (FGF2), and insulin like growth factor 1

receptor (IGFIR) when compared with in vivo derived embryos (Lazzari et al. 2002).

Other studies show IVP embryos with alterations in the level of expression of X-linked









genes, increased chomosomally-abnormal cells (King 2008) and differential expression

of IGF family genes (Bertolini et al. 2002; Sagirkaya et al. 2006; Moore et al. 2007). The

expression of BCL2-associated X protein (BAX), an apoptosis related gene, was higher

in blastocysts produced in vitro using SOF than for those developed in coculture or in

vivo (Rizos et al. 2002a). Oxidative stress genes are also differentially expressed, with

mitochondrial manganese-superoxide dismutase, an important antioxidant defense in

cells exposed to oxygen, strongly expressed in blastocyts developed in vivo when

compared with IVP embryos while sarcosine oxidase, an oxidative enzyme, highly

expressed on IVP embryos when compared with its in vivo counterparts (Rizos et al.

2002a).

Microarray techniques have been used to investigate differences in the

transcriptome between IVP and in vivo embryos (Smith et al. 2009). In vivo produced

embryos have significant overexpression of genes in the category 'response to

stimulus', unfold protein and carboxy-lyase activity while genes in the G-protein coupled

receptor signaling pathway were significantly lower for in vivo embryos. However, none

of the genes differentially regulated in this study matched the imprinted genes thought

to be responsible for large offspring syndrome. Transcripts for enzymes involved in the

de novo methylation process were downregulate in the IVP embryos compared with the

in vivo derived embryos; this could lead to aberrant methylation and subsequent fetal

abnormalities (Smith et al. 2009).

Growth Factors and Cytokines as Uterine Regulators of Embryonic Development

Among the molecules secreted by the reproductive tract that can regulate

embryonic development are various growth factors and cytokines. Originally described

as protein molecules that promote cell proliferation and inhibit apoptosis, growth factors









are now known to play roles in endocrine, paracrine, and autocrine regulation of a wide

variety of cell functions. Historically, cytokines are associated with hematopoietic and

immune cells; however, they are now known to be secreted by a wide variety of cells

and tissues including endocrinologically responsive tissues within the reproductive tract.

While some cytokines can be growth factors, like CSF1 and CSF2, others have an

inhibitory effect on cell growth, and they can target cells to undergo apoptosis and cell

death. The embryo itself expresses receptors for an array of growth factors and

cytokines. In bovine embryos, receptors for platelet derived growth factor a (PDGFR-a)

and IGFIR and IGF2R are found at the oocyte stage and throughout embryo

development (Yoshida et al. 1998; Wang et al. 2008a). Messenger RNAs for FGF2

receptor (FGF2R) are present in all stages of oocyte maturation and embryonic

development up to the 2-cell stage, and again at the blastocyst stage (Yoshida et al.

1998). Transcripts for FGFRlc, FGFR2b, FGFR3c and FGFR4 are found on in vitro

produced blastocysts and the in vivo elongated Day 17 concepts (Cooke et al. 2009).

Epidermal growth factor receptor (EGFR) mRNA and protein have been shown to be

present in spherical embryos on Day 13 and elongated embryos on Day 16 (Kliem et al.

1998). Expression of mRNA and protein of the CSF2 receptor alpha subunit (CSF2R-a)

has only been shown in mouse embryos; it is present from the first cleavage through the

blastocyst stage (Sjoblom et al. 2002).

The list of cytokines and growth factors present in the uterus is large and their

physiological roles are still being resolved. The purpose of this section of the literature

review is to provide examples of specific growth factors and cytokines implicated in









regulation of embryonic development in the cow and to illustrate the specific roles they

may play.

Insulin-like Growth Factor 1

The main source of insulin like growth factor 1 (IGF1) is the liver, which secretes

IGF1 in response to growth hormone (Scharf et al. 1996). However, it is not assured

that changes in circulating IGF1 cause changes in uterine IGF1 concentrations since

IGF1 concentrations in uterine fluid did not change in parallel with plasma

concentrations of IGF1(Bilby et al. 2004). The uterus, oviduct and the embryo also

produce IGF1 (Velazquez et al. 2008) although their contribution to the total IGF1 pool

in uterine and oviductal fluid is unknown.

Addition of IGF1 to culture medium increases the proportion of IVP bovine

embryos that develop to the blastocyst stage (Block et al. 2003; Lima et al. 2006). In

addition, IGF1 increased blastocyst total cell number (Makarevich and Markkula 2002;

Sirisathien et al. 2003) and altered the relative abundance of developmentally important

mRNA transcripts at the blastocyst stage including increases in IGFBP1-2-3-5 (Prelle et

al. 2001; Block et al. 2007), desmocollin 2, ATPIA1 (Block etal. 2007) and SLC2 genes

(Oropeza et al. 2004) and decreases in expression of the gene for heat shock protein

70 (HSP70) (Block et al. 2007), and IGFIR (Enright et al. 2000; Prelle et al. 2001; Block

et al. 2007).

In addition, IGF1 protected embryos from heat shock, allowing increased

development and reduced apoptosis (Jousan and Hansen 2004; Jousan et al. 2007;

Jousan et al. 2008), and protected bovine embryos from the anti-developmental actions

of the prooxidant menadione (Moss et al. 2009). Furthermore, IGF1 treated embryos

showed increased pregnancy rates after they were transferred to recipients exposed to









heat stress (Block et al. 2003; Block and Hansen 2007). Thus, IGF1 may function in

pregnancy to increase embryonic development and protect embryos from specific

stresses capable of disrupting development.

Interleukin 1

Interleukinl, beta (IL1B) is a polypeptide found in uterine flushes at least from

Days 11, 14 and 17 of cyclic cows (Davidson et al. 1995). There is evidence that the

source of this ILB1 is the luminal and glandular epithelium and stroma of the

endometrium (Paula-Lopes et al. 1999). On the other hand, ILB1 could not be detected

in pregnant cows on Days 25 and 30 of gestation (Davidson et al. 1995). When

endometrium collected from pregnant cows was cultured with ILB1 it increased the

secretion of PGE2 and PGF2a from epithelial cells (Betts and Hansen 1992; Davidson et

al. 1995) and from stromal cells (Davidson et al. 1995). ILB1 treatment also decreased

DNA synthesis in stromal cells but had no effect on epithelial cells (Davidson et al.

1995).

The presence of ILB1 in the uterus indicates that this cytokine might have a role in

early embryonic development in cattle. Addition of ILB1 to cultured bovine embryos

increased the percentageage of embryos becoming a blastocyst but only if ILB1 was

added at the first Day of culture and if the embryos were cultured in high density (25-30

embryos/drop) (Paula-Lopes et al. 1998). The fact that effects are seen at high embryo

density may mean that ILB1 acts to stimulate some embryo-derived growth factor that in

turn increases embryonic development.

The importance of ILB1 for pregnancy was demonstrated in mouse (Sim6n et al.

1998). Injections of ILB1 receptor antagonist around the preimplantation period

significantly decreased the number of implantation sites in this species. When flushed at









Day 8, 10 times more blastocysts were found in the uterine flush of the ILB1 receptor

antagonist-treated mice. Even though all the blastocysts appeared to be

morphologically normal, they were delayed in development comparing to the non

injected group, were still surrounded by the zona pellucida and were not able to implant

(Sim6n et al. 1998).

Fibroblast Growth Factor

Fibroblast growth factors (FGFs) are represented by more than 22 FGF genes and

4 FGFR genes with different temporal and spatial patterns of expression during

development (Itoh and Ornitz 2004; Itoh and Ornitz 2008; Gotoh 2009). Specifics FGFs,

i.e. FGF2, are required for self-renewal and maintenance of pluripotency activity in

mouse and human embryos and embryonic stem (ES) cells (Gotoh 2009). Interestingly,

FGF4 is necessary for cellular differentiation in mouse and human ES cells (Kunath et

al. 2007) but is needed to maintain multipotency and self renewal in trophoblast stem

cells (Guzman-Ayala et al. 2004).

In the cow, FGF2 has been identified within the endometrium and in the uterine

lumen at Days 17-18 of the estrous cycle in pregnant and non-pregnant females

(Michael et al. 2006). Moreover, in the ewe flush, FGF2 concentrations in uterine

flushings increase around Days 12-13 after estrus (Oc6n-Grove et al. 2008).

In bovine embryos, the best characterized effect of FGF2 is on TE growth and

IFNT secretion. Supplementation of a TE bovine cell line (CT-1) with FGF2 increases

cell proliferation and IFNT secretion (Michael et al. 2006). In addition, treatment of IVP

blastocysts with FGF2 increased the expression of IFNTmRNA but had no effect on

blastocyst cell number. Other FGFs, i.e. FGF1 and FGF10, have also increased the









steady state amounts of mRNA for IFNT in CT-1 cells as well as IFNT biological activity

(Cooke et al. 2009).

Tumor Necrosis Factor

Tumor necrosis factor (TNF) is a multifunctional cytokine that first identified as a

regulator of immunological and inflammatory responses in several tissues, including the

reproductive tract (Hunt et al. 1996). It is produced by macrophages and oviductal

epithelial cells in the mouse (Hunt 1993), human (Morales et al. 2006) and bovine

(Wijayagunawardane and Miyamoto 2004) and by blastocysts in mouse and human

(Hunt et al. 1996). In the bovine endometrium, TNF can increase PGF2a secretion and

lead to luteolysis (Murakami 2001; Skarzynski et al. 2005; Siemieniuch et al. 2009).

Exposure of mouse blastocysts to TNF decreased cellular proliferation and

increased the percentageage of blastomeres that were apoptotic (Pampfer et al. 1997).

Furthermore, there was a decrease in the number of cells in the ICM and an increase in

reabsorption rate when these blastocysts were transferred to recipients (Wuu et al.

1999). In bovine, TNF did not affect development of embryos to the blastocyst stage

(Soto et al. 2003) but it did increased the percentageage of apoptotic blastomeres in

embryos exposed to the cytokine at Days 4, 5 and 6 after insemination (Loureiro et al.

2007).

It is also possible that TNF is associated with mastitis, as elevated concentrations

of TNF are found in the animals after infections in the mammary gland or infusion of

lipopolysaccharide (Hansen et al. 2004). Cows that present mastitis have a reduction in

fertility as there is an increase in the Days to first service, Days open and service per

conception (Barker et al. 1998; Schrick et al. 2001). In mice, embryonic losses









associated with diabetes have been related to excessive production of TNF in the

uterus (Pampfer 2001).

Colony Stimulating Factor 2

Biology and Signaling

CSF2 is a polypeptide growth factor of 124 amino acids and with a molecular

weight of 14,138 (Metcalf 1985). Most adult organs synthesize detectable amounts of

CSF2 (Burgess and Metcalf 1980); however increased secretion requires stimulation of

cytokines, antigens, microbial products or inflammatory agents (Conti and Gessani

2008). In the human serum, concentrations of CSF2 range from 20 to 100 pg/ml (Conti

and Gessani 2008). The main role of CSF2 is to promote survival and activation of

neutrophils, eosinophils and macrophages, as well as dendritic cell maturation and

differentiation of alveolar macrophages and invariant natural killer T cells (iNKT)

(Barreda et al. 2004; Conti and Gessani 2008; Hercus et al. 2009). It is thought to

promote the necessary communication between the hematopoietic cells and local

tissues in the event of inflammation (Hercus et al. 2009) as well as to enhance

proinflamatory cytokine production (Brissette et al. 1995) (Figure 1-1).

CSF2 deficient mice present deficient alveolar macrophage maturation, which

leads to the development of abnormal lungs (Stanley et al. 1994), and compromised

iNKT cellular differentiation (Bezbradica et al. 2006). Administration of exogenous CSF2

corrected these defects (Bezbradica et al. 2006). Furtheremore, CSF2 injection in mice

caused an increase in the levels of circulatory neutrophils and cycling peritoneal

macrophages (Hamilton 2002) and administration of CSF2 specific antibody caused a

decrease in inflammation in the skin (Schon et al. 2000). Other inflammatory reactions









are promoted through activation of adhesion events and increased cell survival through

inhibition of apoptosis (Hamilton 2002).

Many or all cells in the stem and progenitor compartments exhibit receptors for

CSF2 and are responsive to stimulation by this molecule (Metcalf et al. 1980). CSF2

receptors are expressed at very low levels (100-1000 per cell) and comprise a cytokine-

specific a subunit and a 3 common (3c) subunit that interacts with various receptor-

associated proteins important for the signaling downstream of the receptor (Quelle et al.

1994; Guthridge et al. 1998; Carr et al. 2001; Mirza et al. 2010). Each a subunit binds

the cytokine with low affinity but the presence of 3c converts this to a high affinity

reaction causing dimerization of both subunits and receptor activation (Carr et al. 2001;

Mirza et al. 2010). Upon binding to CSF2, the CSF2 receptor complex is a high-order

dodecamer composed of two hexamers with a stoichiometry of 2 CSF2, 2 a subunits

and 2 3c subunits (Hansen et al. 2008) (Figure 1-1).

CSF2 and CSF2 receptors can be rapidly consumed by internalization of the

complex followed by ligand endocytosis, lysosomal degradation and direct proteosomal

degradation of the 3c cytoplasmic domain. Furthermore, the a subunit mRNA is

downregulated in response to the 3c stimulation (Barreda et al. 2004).

One prominent interaction partner of CSF2 is Janus kinase 2 (JAK2), a tyrosine

kinase that binds to the 3c subunit for subsequent transphosphorylation and activation.

Activated JAK2 phosphorylates tyrosine residues of the 3 subunit and generates binding

sites for Src-homology 2 domains of other proteins such as members of the signal

transducer and activator of transcription (STAT) family. JAK2 can also phosphorylate

STAT proteins themselves. In parallel, additional signaling pathways can be activated,









such as the Ras-Raf-extracellular signal-regulated kinase (Ras-Raf-ERK) pathway,

which is important for triggering the cell cycle and is activated by binding of the adapter

proteins She and Grb2 to the 3c (Degroot 1998; Barreda et al. 2004; Choi et al. 2007).

While activation of JAK2/STAT is mainly responsible for CSF2 induced cell proliferation,

phosphatidylinositol-3 kinase (P13K), another signaling pathway triggered by the 3c

subunit has a role in regulation of apoptosis and cell survival (Degroot 1998; Dhar-

Mascareno et al. 2005). One difference between these pathways in response to CSF2

is the concentration necessary for activation. For example, the apoptosis inhibition

reaction occurs at significantly lower concentrations (100 fold) than those required for

stimulation of cell proliferation (Barreda et al. 2004) (Figure 1-1).

Data indicate that the a subunit, but not the 3c protein, is present in blastomere

cell membranes of mouse and human preimplantation embryos (Robertson et al. 2001;

Sjoblom et al. 2002).

CSF2 Secretion in the Uterine Tract

CSF2 is expressed at the protein and mRNA level in endometrial epithelial cells of

humans and mice (Chegini et al. 1999; Robertson et al. 2001), in human fallopian tube

(Zhao and Chegini 1994) and in human first trimester decidua (Segerer et al. 2009). In

the cow, CSF2 has been localized in the oviductal epithelium (greater in ampulla) (de

Moraes et al. 1999), endometrium (mostly in the luminal epithelium and the apical

portions of the glands) (de Moraes et al. 1999; Emond et al. 2004) and in the

myometrium after Day 30 of pregnancy (Emond et al. 2004).

On Days 14-17 after estrus, concentrations of CSF2 in uterine flushing in cattle

tended to be higher in pregnant cows than cyclic cows (de Moraes et al. 1999). For









cyclic cows, concentrations of CSF2 tended to be lowest during estrus compared with

Days 7, 13-16 and 18 of the cycle (de Moraes et al. 1999).

This pattern in the cow is unlike the mouse where CSF2 concentrations in the

uterus peak during estrus (Robertson et al. 1996). Moreover, CSF2 release in the

mouse is stimulated by specific factors in seminal plasma, including transforming growth

factor (TGFB) (Tremellen et al. 1998). Expression declines at implantation under the

inhibitory influence of progesterone (Robertson et al. 1996), but bioactive CSF2 can be

detected in placental and decidual tissues for the duration of pregnancy (Crainie et al.

1990). In pregnant bitches, CSF2 mRNA was found at the onset of

implantation/placentation (Beceriklisoy et al. 2009).

Actions of CSF2 on Embryo Development and Survival

The use of knock-out models in mice have indicated that CSF2 is important for

normal embryonic development. Despite a regular number of implantation sites,

offspring from CSF2 null mice are 25% of normal size, have a 4.5 fold increase in

mortality rate during the first three weeks of life and a fetal growth retardation that

persists through adulthood (Seymour et al. 1997; Robertson et al. 1999). Total cell

number of blastocysts from CSF2-/- mice was reduced by 14-18% (Robertson et al.

2001). Cell number of embryos from CSF2 -/- mice was increased 24% when cultured

with exogenous CSF2 (Robertson et al. 2001). Mitogenic actions of CSF2 were also

demonstrated in a study in which mouse embryos were cultured in media supplemented

with CSF2. The cytokine increased total cell numbers and ICM cell numbers of CF1

mice blastocysts (more sensitive strain), but only in the absence of human serum

albumin (Karagenc et al. 2005). There was no effect of CSF2 on TE cell numbers.









Another line of evidence for beneficial effects of CSF2 on preimplantation

embryonic development is the improvement in the proportion of cultured embryos that

develop to the blastocyst stage in vitro in the cow (de Moraes and Hansen 1997a),

human (Sjoblom et al. 1999), mouse (Robertson et al. 2001), and pig (Cui et al. 2004).

Bovine embryos seem to be more responsive to CSF2 when it is added to culture at

Day 5 after insemination than when added within 24 hours of insemination. When added

at Day 5, CSF2 increased the proportion of oocytes becoming blastocysts (de Moraes

and Hansen 1997a). When added after insemination, CSF2 had no effect on the

proportion of embryos developing past the 8 cell stage (de Moraes and Hansen 1997a).

The late effect of CSF2 coincides with the time that the embryo enters the uterus

(Betteridge et al. 1988).

Murine embryos cultured in CSF2 have a higher rate of metabolic activity and

requirements. CSF2 elicited a 50% increase in the uptake of the non-metabolizable

glucose analogue, 3-O-methyl glucose, in mouse blastocysts (Robertson et al. 2001).

One possible reason for effects of CSF2 on the proportion of embryos becoming

blastocysts may be the antiapoptotic effects of the cytokine. Culture of mouse embryos

with CSF2 decreased the number of apoptotic blastomeres by 50% and increased the

number of viable ICM cells by 30% as a result of both antiapoptotic and proliferative

effects (Sjoblom et al. 2002). In another study, CSF2 protected one cell mouse embryos

from freezing damage and decreased the apoptotic index to zero (Desai et al. 2007).

The means by which CSF2 protects against apoptosis in embryos is not clear. In

neurons, polymorphonuclear neutrophils and HeLa cells, CSF2 can activate the PI3K-

Akt pathway which decreases apoptosis by induction of BCL-2 and BCL2L1 (previous









known as BcL-XL) (Antignani and Youle 2007; Schabitz et al. 2008). Using microarray

analysis, CSF2 was recently shown to decrease expression of stress response genes

and apoptosis genes in mouse blastocysts (Chin et al. 2009). In vivo, CSF2 null mutant

mice had elevated expression of heat shock protein 1 (Hsphl) (Chin et al. 2009).

Sjoblom et al. (2005) found that survival of mouse embryos after transfer into

recipient females was greater when embryos were cultured with CSF2 than when

embryos were cultured without the cytokine (90% vs 76%). Along with this finding, there

was evidence that CSF2 treatment during the preimplantation period affected placental

development after transfer. Compared with values for fetuses produced in vivo, fetal

placental weight ratio decreased by 8% for fetuses from CSF2 treated embryos versus

11 % for fetuses from embryos cultured in medium alone.

CSF2 and Interferon-tau Secretion

One way in which CSF2 could improve post-transfer embryonic survival in cattle

would be through increased secretion of IFNT by elongated conceptuses. Imakawa et

al. (1993 and 1997) and Rooke et al. (2005) demonstrated that CSF2 stimulates

production of IFNT by sheep trophoblast in vitro. Similar results were found when a cell

line derived from bovine trophoblast cells (CT-1 cells) was treated with porcine CSF2

(Michael et al. 2006). On the other hand, de Moraes et al. (1997b) found no beneficial

effect of bovine CSF2 on IFNT secretion by bovine blastocysts at Day 7-8 after

insemination.

Goals of the Current Investigation

The aim of this dissertation is to understand the role of CSF2 in preimplantation

embryonic development including its effects on blastocyst formation, the embryonic

transcriptome, and long term embryonic survival. Based on the literature review, it is









likely that CSF2 can act as a regulator of preimplantation embryonic development,

enhance embryo competence for survival, induce genes that promote proliferation,

inhibit apoptosis and promote critical functions necessary for post-transfer survival.

To accomplish our goal we pursued the following research objectives:

Research objective 1: to determine the effects of CSF2 on blastocyst yield and

capacity for survival after transfer into recipients, and evaluate properties of the

blastocyst formed after CSF2 treatment.

Research objective 2: to determine the transcriptome of CSF2 treated embryos.

Research objective 3: test whether treatment of embryos with CSF2 increases

post-transfer growth and IFNT secretion and alters the transcriptome of the ICM and

TE.

Results from the proposed studies will add information regarding the biology of

CSF2 during preimplantation embryonic development. The first objective establishes

that CSF2 is an important regulator of embryonic development and post-transfer

survival. The second objective seeks to identify genes and pathways through which

CSF2 regulates embryo development and survival. The third objectives evaluates

whether exposure to CSF2 during the preimplantation period leads to changes in gene

expression later in pregnancy that could enhance subsequent embryonic and fetal

development.










GM GM QGM GM-CSF
(I-1 _P i ,la._ Gautoan-ibody


SG196R
mutation


Cell survival BCL2A1 -*--


S -0 ? ,---* Surfac


tant catabolism


Cell adhesion CD11 Evil I Proliferation
Celladhesion4 11 MR A ROS
Pathogen / FR TLR4 I -12 GM-CSF,
recognition TLR2 TNF IL-18 M-C Microbial killing
Phagocytosis Macrophage
Proinflammatory IFNy activation
cytokine signaling /
Innate Adaptive
immunity immunity
Cuienml Opbonn in Immunoogy


Figure 1-1. Mechanisms by which CSF2 regulates cellular survival, differentiation,
functions and activation. CSF2 initiates signaling by first binding to the CSF2
receptor a, which then associates with homodimers of the affinity-enhancing
CSF2 receptor 3c. Jak2 is bound constitutively to the 3c chain and signals
through an intracytoplasmic 3c chain motif. At low concentrations, CSF2
activates P13K and Akt signaling resulting in cell survival without proliferation.
At high concentrations, CSF2 activates STAT or Shc-dependent pathways
stimulating cell survival, cellular activation and proliferation. Pulmonary CSF2
regulates the expression of numerous genes enabling multiple immune and
non-immune functions. CSF2 is also known as GM-CSF. Figure reproduced
with permission from Current Opinion in Immunology 2009, 21:514-521.


Cellsurvval nly









CHAPTER 2
COLONY STIMULATING FACTOR IMPROVES DEVELOPMENT AND POST-
TRANSFER SURVIVAL OF BOVINE EMBRYOS PRODUCED IN VITRO

Introduction

The embryo executes its developmental program in the female reproductive tract

in an environment largely dictated by the mother. An inadequate maternal environment

can lead to reduced embryonic survival (Hansen 2007; Leroy et al. 2008; Robinson et

al. 2008) and epigenetic changes that persist into adulthood (Sinclair et al. 2007). The

maternal environment affects embryonic development by providing to the embryo an

array of nutrients and regulatory molecules and by expression of cell adhesion

molecules that facilitate eventual attachment and placentation (Spencer et al. 2008).

Among the regulatory factors shown to affect preimplantation development in the cow,

for example, are insulin-like growth factor-1 (Block 2007), interleukin 1, beta (Paula-

Lopes et al. 1998), activin (Park et al. 2008) and granulocyte-macrophage colony

stimulating factor (CSF2) (de Moraes and Hansen 1997a).

There are strong lines of evidence to implicate CSF2 as a physiologically-

important regulator of early embryonic development. The cytokine is expressed in

luminal epithelium and other tissues of the oviduct and endometrium (Zhao and Chegini

1994; Chegini etal. 1999; de Moraes etal. 1999; Emond etal. 2004). In mice,

production of CSF2 fluctuates during the estrous cycle, peaking during estrus

(Robertson et al. 1996). Expression declines at implantation under the inhibitory

influence of progesterone (Robertson et al. 1996). Release of CSF2 into the uterine

lumen is stimulated by specific factors in seminal plasma, including TGFB (Tremellen et

al. 1998). In cattle, amounts of immunoreactive CSF2 in the endometrium of cyclic cows

tends to be low during estrus and high from Days 13 to 17 after estrus before declining









as estrus approaches (de Moraes et al. 1999; Emond et al. 2004). Immunoreactive

CSF2 in the endometrial luminal epithelium increases in response to the maternal

recognition of pregnancy signal interferon-tau (IFNT) so that amounts of

immunoreactive CSF2 are higher in pregnant cows at Day 16 and 18 after estrus

compared with cyclic cows (Emond et al. 2004).

Addition of CSF2 to culture medium improved the proportion of in vitro embryos

developing to the blastocyst stage in the cow (de Moraes and Hansen 1997a), human

(Sjoblom et al. 1999) and pig (Cui et al. 2004) and increased post-transfer embryonic

survival in mice (Sjoblom et al. 2005). Total cell numbers of blastocysts from CSF2-/-

mice were significantly reduced by 14-18% compared with wild-type controls (Robertson

et al. 2001). Despite a regular number of implantation sites, offspring from CSF2-/- mice

were 25% of normal size, exhibited fetal growth retardation that persists through

adulthood, and experienced increased mortality during the first three weeks of life

(Seymour et al. 1997; Robertson et al. 1999).

The bovine preimplantation embryo is a good model for studying maternal

regulation of embryonic development. Development to the blastocyst stage is not

absolutely dependent upon regulatory molecules present in the reproductive tract

because embryos that give rise to live calves after transfer to recipient can be produced

in culture in growth-factor free media (Block and Hansen 2007). However, embryos

produced by in vitro oocyte maturation, fertilization and culture have aberrant

biochemical and molecular properties compared with embryos produced in vivo (Lazzari

et al. 2002; Rizos et al. 2002). Deviation in embryonic function associated with

production in vitro is due in part to an inadequate environment during the









preimplantation period. This is so because the gene expression and cryotolerance of

bovine embryos produced by in vitro fertilization was enhanced when embryos were

cultured in vivo in the sheep oviduct after insemination (Rizos et al. 2002; Rizos et al.

2002).

Here we tested the possible role of CSF2 as one of the regulatory molecules that

mediate maternal effects during the preimplantation period on survival through the

embryonic and fetal periods. The approach was to determine whether addition of CSF2

to culture medium enhances the development and post-transfer survival of bovine

embryos produced in vitro. As a positive control, some embryos were treated with

insulin-like growth factor-1 (IGF1), which to date, is the only growth factor shown to

improve survival of bovine embryos transferred to recipients. This molecule protects

embryos from the effects of elevated temperature (Jousan and Hansen 2004; Jousan et

al. 2008) and pregnancy and calving rates of cows exposed to heat stress were

improved when embryos used for transfer were treated with IGF1 (Block et al. 2003;

Block and Hansen 2007). However, no improvement in post-transfer survival was seen

when cows were not heat-stressed (Block and Hansen 2007). The current studies were

conducted in both hot and cool seasons.

Materials and Methods

Materials

The media HEPES-Tyrodes Lactate (HEPES-TL), in vitro fertilization (IVF)-TL, and

Sperm-TL were purchased from Caisson (Sugar City, ID) and used to prepare HEPES-

Tyrodes albumin lactate pyruvate (TALP) and IVF-TALP as previously described (29).

Oocyte collection medium (OCM) was Tissue Culture Medium-199 (TCM-199) with

Hank's salts without phenol red (Hyclone, Logan UT) supplemented with 2% (v/v)









bovine steer serum (Pel-Freez, Rogers, AR) containing 2 U/ml heparin, 100 U/ml

penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocyte maturation medium

(OMM) was TCM-199 (Invitrogen, Carlsbad, CA) with Earle's salts supplemented with

10% (v/v) bovine steer serum, 2 [Lg/ml estradiol 17-P, 20 [Lg/ml bovine follicle stimulating

hormone (Folltropin-V; Belleville, ON), 22 [Lg/ml sodium pyruvate, 50 [Lg/ml gentamicin

sulfate, and 1 mM glutamine. Percoll was from Amersham Pharmacia Biotech

(Uppsala, Sweden).

Potassium simplex optimized medium (KSOM) that contained 1 mg/ml bovine

serum albumin (BSA) was obtained from Caisson. Essentially fatty-acid free (EFAF)

BSA was from Sigma (St. Louis, MO). On the Day of use, KSOM was modified for

bovine embryos to produce KSOM-BE2 as described elsewhere (Soto et al. 2003).

Prostaglandin F2a (PGF) was Lutalyse from Pfizer (New York, NY, USA) and

gonadotrophin releasing hormone (GnRH) was Cystorelin from Merial (Duluth, GA,

USA). Dithiothreitol (DTT) was from Sigma-Aldrich (St. Louis MO, USA).

Recombinant bovine CSF2 was donated by CIBA-GEIGY (Basle, Switzerland).

Recombinant human modified IGF1 (E3R) was obtained from Upstate Biotech (Lake

Placid, NY, USA).

Effects of CSF2 on Embryo Development and Blastocyst Properties

Production of embryos

Embryo production was performed as previously described (Soto et al. 2003).

Cumulus oocyte complexes (COCs) were obtained by slicing 2- to 10-mm follicles on

the surface of ovaries (a mixture of beef and dairy cattle) obtained from Central Beef

Packing Co. (Center Hill, FL). Those COCs with at least one complete layer of compact









cumulus cells were washed two times in OCM, placed in groups of 10 in 50-[Ll

microdrops of OMM overlaid with mineral oil and matured for 20-22 h at 38.50C in a

humidified atmosphere of 5% (v/v) C02 in humidified air.

Matured COCs were washed once in HEPES-TALP and transferred in groups of

50 to 4-well plates containing 425 [l of IVF-TALP and 20 [l of PHE (0.5 mM

penicillamine, 0.25 mM hypotaurine, and 25 pM epinephrine in 0.9% [w/v] NaCI) per

well and fertilized with ~1 x 106 Percoll-purified spermatozoa from a pool of frozen-

thawed semen from three bulls. After 8-10 h at 38.50C in a humidified atmosphere of

5% (v/v) C02 in humidified air, putative zygotes were removed from fertilization wells,

denuded of cumulus cells by vortex in HEPES-TALP, and placed in groups of 30 in 50-

il microdrops of KSOM-BE2. Putative zygotes were cultured at 38.50C in a humidified

atmosphere of 5% C02 or 5% C02, 5% 02, and 90% N2. Embryos received treatment at

Day 0 (i.e., immediately after insemination) or Day 5 after insemination according to the

specific experimental design.

Interactions between oxygen concentration and presence of CSF2

This experiment was designed to determine the effects of CSF2 on embryonic

development at culture conditions of high oxygen [5% (v/v) C02 in air] or low oxygen

(5% C02, 5% 02, and 90% N2, v/v) and when added at Day 0 or 5 after insemination.

After removal from fertilization drops, embryos were washed, placed in microdrops of

KSOM-BE2 medium and cultured in high 02or low 02. If treatment was Day 0 after

insemination, embryos were placed in a 50 pl KSOM-BE2 drop 10 ng/ml CSF2. When

treatment was added at Day 5, embryos were placed in a 45 pl KSOM-BE2 drop and 5

pl of KSOM-BE2 10 ng/ml CSF2 were added to the drop at Day 5 after insemination.









The concentration was chosen based on results from other experiments that addition of

CSF2 at this concentration would increase blastocyst yield (de Moraes and Hansen

1997a). Cleavage rate was assessed at Day 3 after insemination and the

percentageage of oocytes that became blastocysts was assessed at Days 7 and 8 after

insemination. The experiment was replicated 11 times using 2673 oocytes.

Cell number and differentiation of blastocysts

The experiment was designed to test whether CSF2 would increase cell number of

bovine blastocysts and alter cell allocation between the trophectoderm (TE) and inner

cell mass (ICM). After insemination, embryos were cultured in 45 pl microdrops of

KSOM-BE2 at 38.5C in a humidified atmosphere 5% CO2, 5% 02, and 90% N2. At Day

5 after insemination, 5 pl of CSF2 (to create a final concentration of 10 ng/ml) or 5 pl of

vehicle (KSOM-BE2) were added to the drops. Zona-intact blastocysts were removed

from culture at Day 7, washed two times in 50-tl microdrops of 10 mM KPO4, pH 7.4

containing 0.9% (w/v) NaCI and 1 mg/ml PVP (PBS-PVP) by transferring the embryos

from microdrop to microdrop. To label TE cells, embryos were placed in 100 pl PBS-

PVP containing 0.5% (v/v) Triton X-100 and 100 pg/ml propidium iodine for 30 s at

37C. Embryos were immediately washed in PBS-PVP. Embryos were then incubated

in 50 pl drops of PBS-PVP containing 4% (w/v) paraformaldehyde and 10 pg/ml

Hoechst 33258 for 15 min at room temperature to fix the embryos and stain ICM.

Embryos were washed in PBS-PVP and mounted on slides using Prolong Gold Antifade

(Invitrogen, Eugene, Or, USA) and coverslips placed on the slides. Labeling of

propidium iodine and Hoechst was observed using a Zeiss Axioplan 2 epifluorescence

microscope (Zeiss, Gottingen, Germany). Each embryo was analyzed for the number of









ICM (blue nuclei) and TE (pink nuclei), and total cell number (blue + pink nuclei) with a

DAPI filter using a 20x objective. Digital images were acquired using AxioVision

software (Zeiss) and a high-resolution black and white Zeiss AxioCam MRm digital

camera. The experiment was replicated 3 times using 18-36 embryos/group.

Apoptotic blastomeres

This experiment was designed to test the hypothesis that CSF2 decreases the

percentageage of blastomeres that are positive for the Terminal deoxynucleotidyl

transferase dUTP nick end labeling (TUNEL) reaction. Embryos were cultured and

treated with CSF2 as described above for analysis of TE and ICM. On Day 7 after

insemination, blastocysts, expanded blastocysts and hatching blastocysts were

removed from culture and washed two times in 50-[Ll microdrops of PBS-PVP. Embryos

were fixed in a 50-[Ll microdrop of 4% (w/v) paraformaldehyde in PBS for 15 min at

room temperature, washed twice in PBS-PVP, and stored in 600 [il of PBS-PVP at 40C

until analysis for TUNEL labeling as described previously (Jousan and Hansen 2004;

Jousan et al. 2008). The experiment was replicated 3 times using 31-58

embryos/group.

Effects of CSF2 and IGF1 on Pregnancy and Calving Success after Transfer to
Recipients

Production of Holstein embryos using X-sorted semen

Embryo production was performed as described above with slight modifications.

Holstein COCs were purchased from one of three suppliers (Evergen, Storrs, CT, USA;

Bomed, Madison, WI, USA; Trans Ova, Sioux City, IA, USA) and shipped overnight in a

portable incubator at 38.50C in maturation medium or collected from slaughter house

ovaries. After 20-24 hours, COCs were removed from maturation medium, washed one









time in Hepes-TALP and transferred in groups of 30 to fertilization drops covered with

mineral oil. Each drop contained 50 pl IVF-TALP and then 3 pl PHE and 20 pl sperm

purified by Percoll gradient were added to this. The final concentration of sperm was

1x106 per ml. For each replicate, two to four straws of X-sorted semen from one

Holstein bull (Sexing Technologies Inc., Navasota TX) were used depending on the

number of oocytes that had to be fertilized. In total 4 different bulls were used

throughout the experiment.

After 20-22 h at 38.5C and 5% (v/v) C02, presumptive zygotes were removed

from fertilization drops, vortexed in 30 pl Hepes-TALP for 5 min in a microcentrifuge

tube and washed two times to remove cumulus cells and associated spermatozoa.

Putative zygotes (25-30/drop) were placed in 45 pl (treatment at Day 5) or 50 pl

(treatment at Day 0) of KSOM-BE2 overlaid with mineral oil. Embryos were cultured at

38.5C in a humidified atmosphere of 5% C02, 5% 02, and 90% N2 (v/v). Treatments

were added at either Day 0 (i.e., immediately after insemination) or Day 5 after

insemination as described for each experiment.

The proportion of oocytes that cleaved was recorded at Day 3 after insemination.

Morula, blastocyst and expanded blastocyst stage embryos classified as Grade 1

(Robertson and Nelson 1998) were harvested on Day 7 (Farm 1 and 2) and Day 8

(Farm 3) and loaded into 0.25 ml straws in HEPES-TALP supplemented with 10% fetal

calf serum and 50 pM DTT. Straws containing selected embryos were then placed

horizontally into a portable incubator (Cryologic, Mulgrave, Vic, Australia) at 38.5C and

transported to the respective farm.









Animals

The experiment was conducted at 3 locations: Farm 1 (Brooksco Dairy, Quitman,

GA, USA; 30o47'5" N, 83033'39" W), Farm 2 (University of Florida Dairy Research Unit,

Hague, FL, USA; 29046'21"N, 82024'54"W) and Farm 3 (Alliance Dairy, Trenton, FL,

USA; 29034'35"N, 82051'17"W). At Farm 1, 271 primiparous and multiparous lactating

Holstein cows between 76 and 154 Days in milk (mean = 84) were used as recipients

from June 29 December, 12, 2007. Cows were housed in a free-stall barn equipped

with fans and a sprinkler system. Overall, 15 replicates were completed with 8-30

recipients per replicate. At Farm 2, a total of 100 primiparous and multiparous lactating

Holstein cows between 75 and 376 Days in milk (mean = 161) were used as recipients

from December 7, 2007 February 1, 2008. Recipients were housed in a free stall barn

equipped with fans and sprinklers. Overall, 6 replicates were completed with 12-26

recipients per replicate. A single replicate was performed at farm 3 on December 23,

2007 using a total of 21 multiparous lactating Holstein cows between 168 and 468 Days

in milk (mean = 267). Recipients were housed in a free stall barn equipped with fans

and sprinklers.

Synchronization and timed embryo transfer

Each week, eligible cows were organized into a group (i.e. replicate) and ovulation

synchronized for embryo transfer. The timed ovulation protocol was the Ovsynch-56

procedure (Brusveen et al. 2008). Day 0 was considered the Day of expected ovulation.

Hormonal treatments consisted of 100 pg GnRH, i.m. on Day -10; 25 mg PGF2a, i.m. on

Day -3; and 100 pg of GnRH i.m. at 56 hours after PGF2a. For first-service cows only,

the timed ovulation protocol was preceded by a Presynch protocol (two injections of 25

mg PGF2a, i.m. 14 Days apart), with the last injection 14 Days before initiation of the









timed ovulation protocol. Cows were diagnosed for the presence of corpus luteum (CL)

at Day 7 after anticipated ovulation using an Aloka 500 ultrasound equipped with a 5

MHz linear array transducer. Cows diagnosed with a CL received epidural anesthesia [5

ml of 2% (w/v) lidocaine] and a single embryo transferred to the uterine horn ipsilateral

to the ovary via the transcervical route.

Effect of CSF2 and IGF1 added at Day 1 of culture on development and post-
transfer survival of bovine embryos

The experiment was conducted from June 29 through August 31, 2007 at Farm 1

in 7 replicates. At Day 1 after insemination, presumptive zygotes were randomly

distributed to 50 pl KSOM-BE2 drops with one of three treatments: 1) KSOM-BE2 alone,

2) 10 ng/ml CSF2 or 3) 100 ng/ml IGF1. Cleavage rate was assessed at Day 3 after

insemination. At Day 7 after insemination, Grade 1 morula, blastocyst and expanded

blastocyst stage embryos, considered transferable embryos, were harvested and

transferred to lactating dairy cows synchronized using the PreSynch/Ovsynch protocol.

Pregnancy was diagnosed between Days 30-35 of gestation using ultrasonography and

the incidence of calving recorded. Transfers were performed for 51-55 cows/treatment.

Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of culture on
development and post-transfer survival of bovine embryos

The experiment was conducted from September 7, 2007 through February 1, 2008

at Farms 1-3 in 15 replicates. In 8 replicates, presumptive zygotes were randomly

assigned to either 50 pl KSOM-BE2 (control) 2) 50 pl KSOM-BE2 + 100 ng/ml IGF1

added at Day 1 after insemination or 3) 50 pl KSOM-BE2 + 10 ng/ml CSF2 added at

Day 5 after insemination. Embryos assigned to the CSF2 group were placed in 45 pl

KSOM-BE2 drop at Day 1 after insemination. At Day 5 after insemination, an aliquot of

5 pl KSOM-BE2 + 100 ng/ml CSF2 was added to the drops to achieve a final









concentration of 10 ng/ml CSF2. The remaining 7 replicates used similar treatments

except the control treatment was changed so that embryos were placed in 45 pl drops

of KSOM-BE2 at Day 1 after insemination and 5 pl KSOM-BE2 were added to the drops

at Day 5 after insemination. Because there was no statistical difference between the two

types of control embryos, control data were pooled. Cleavage rate was assessed at Day

3 after insemination. At Day 7 after insemination, Grade 1 morula, blastocyst and

expanded blastocyst stage embryos were harvested and transferred to lactating dairy

cows synchronized using the PreSynch/Ovsynch-56 or Ovsynch-56 protocol. Pregnancy

was diagnosed between Days 30-35 of gestation using ultrasonography and the

incidence of calving recorded. Transfers were performed for 44-107 cows/treatment.

Statistical Analysis

Data on the percentageage of oocytes that cleaved and that became blastocysts

and transferable embryos, the percentageage of cells that were TUNEL-positive and the

ICM/TE ratio were analyzed by least-squares analysis of variance using the General

Linear Models procedure of SAS (SAS for Windows, Version 9.0, Cary, NC). Data were

transformed by arcsin transformation before analysis. The mathematical model included

main effects and all interactions. Replicate was considered as a random effect and

other main effects were considered fixed. Tests of significance were made using error

terms determined by calculation of expected mean squares. All values reported are

least-squares means + SEM. Probability values are based on analysis of arcsin-

transformed data while least-squares means are from analysis of untransformed data.

Data regarding the percentageage of cows that became pregnant after transfer

were analyzed by logistic regression using the LOGISTIC procedure of SAS. Treatment









effects were separated into individual degree of freedom comparisons using orthogonal

contrasts. Three sets of contrasts were compared. The first, testing the hypothesis that

both CSF2 and IGF1 would increase pregnancy and calving rate, were control vs IGF1

and CSF2 and IGF1 vs CSF2. The second, testing the hypothesis that only CSF2

would increase pregnancy and calving rate, were control and IGF1 vs CSF2 and control

vs IGF1. An additional contrast was also made to compare control to CSF2.

Pregnancy loss was analyzed for the first and second experiment and for all the losses

in both experiments by X2 analysis.

Results

Effects of CSF2 on Embryo Development

The experiment evaluated whether addition of CSF2 at Day 0 or 5 after

insemination would increase the percentageage of oocytes that became blastocysts.

Embryos were cultured in either high oxygen (air) or low oxygen (5%, v/v). Overall,

there was no effect of CSF2 on cleavage rate at Day 3 after insemination, with averages

varying from 73-80%. There was a tendency (P=0.09) for CSF2 to increase the

percentage of oocytes that became blastocysts on Day 7 after insemination (Figure 2-1,

Panel A). Development was higher in low oxygen (P<0.0001) but there was no

interaction between Day of treatment or oxygen concentration. On Day 8 after

insemination (Figure 2-1, Panel B), CSF2 increased (P=0.05) blastocyst development,

development was higher (P<0.0001) in low oxygen and there was a treatment x Day x

oxygen interaction (P=0.06). The observed 3 way interaction between treatment, Day

and oxygen is due to the fact that the difference in response to addition of CSF2 at Day

0 vs Day 5 depended upon oxygen concentration. For embryos cultured in low oxygen,

CSF2 increased blastocyst development to a greater extent when added at Day 5 rather









than at Day 1. For embryos in high oxygen, CSF2 increased blastocyst development to

a greater extent when added at Day 1.

Effects of CSF2 on Blastocyst Total Cell Number, Cell Differentiation and
Apoptosis

As shown in Table 2-1, blastocysts formed in the presence of CSF2 had a

tendency for an increased (P=0.066) number of ICM cells and greater ICM/TE ratio (P<

0.02) when compared with control embryos. There was no significant effect of CSF2 on

total cell number (Table 2-3) or on the percentage of blastomeres labeled as TUNEL-

positive (Table 2-4).

Effects of CSF2 and IGF1 on Pregnancy and Calving Success after Transfer to
Recipients

In the first experiment, treatments were added at Day 1 of culture and recipients

were used in the summer during heat stress (Table 2-3). There was a significant

increase in the percentageage of cleaved embryos that became transferable morulae or

blastocyst on Day 7 after insemination when embryos were treated with CSF2 (control

vs CSF2, P<0.01; control and IGF vs CSF2, P<0.02; control vs IGF and CSF2, P<0.02;

Table 2-3). Treatment did not have a significant effect on pregnancy rate at Day 30-35

of gestation or on calving rate although, numerically, pregnancy and calving rates were

higher in the IGF1 group than for the other two groups. Pregnancy loss between Day

30-35 and term was significantly lower for recipients that received a CSF2 treated

embryo compared with control embryos (CSF2 vs control, P<0.05; CSF2 and IGF1 vs

control, P<0.05; Table 2-3). Of the calves born, 85% were female.

The second experiment was conducted largely during the cool season. As for the

first experiment, IGF1 was given to embryos at Day 1 after insemination. In contrast,

CSF2 was added at Day 5 of culture. Treatment with CSF2 was modified because of









data indicating greater effects on development when added at Day 5 (see Figure 2-1)

and the failure of CSF2 treatment at Day 1 to affect pregnancy rate (Table 2-3). There

was a significant increase in the percentageage of cleaved embryos that became

transferable morulae or blastocyst on Day 7 after insemination when embryos were

treated with CSF2 at Day 5 of culture (CSF2 vs control, P<0.01; CSF2 vs IGF, P<0.01;

CSF2 and IGF vs control, P<0.02; CSF2 vs control and IGF, P<0.02; Table 2-4).

Moreover, pregnancy rate (CSF2 vs control and IGF1, P<0.05; CSF2 vs IGF, P<0.06)

and calving rate (CSF2 vs control, P<0.05; CSF2 vs control and IGF, P<0.05) were

higher for cows receiving embryos treated with CSF2 than embryos receiving control or

IGF1 treated embryos. There was no effect of treatment on pregnancy loss for any of

the treatments but again pregnancy loss was numerically lower for cows receiving

embryos treated with IGF1 or CSF2 than for cows receiving control embryos. Of the

calves born, 85% were female.

When data from the two transfer experiments were pooled, the difference in

pregnancy loss between cows receiving control embryos (9/40; 22.5%) and cows

receiving either an embryo treated with either IGF1(3/36; 8.3%) or CSF2 (8/98; 8.1%)

was significant (P<0.025).

Discussion

Experiments reported here implicate CSF2 as an important regulator of

preimplantation development. Treatment of preimplantation bovine embryos with CSF2

increased the proportion of embryos that became blastocysts, increased the cell

number in the ICM and improved the survivability of embryos after transfer to recipients.

The effect of CSF2 on post-transfer survival involved both an increase in the proportion

of embryos that established pregnancy by Day 30-35 (when treatment was from Day 5-









7 after insemination) and a reduction in the proportion of embryos at Day 30-35 which

were lost before completion of gestation (when treatment was from Day 1-7 or 5-7 after

insemination). The fact that treatment with CSF2 during such a narrow window of

development (from Day 1-7 or Day 5-7) altered embryonic function much later in

pregnancy (after pregnancy diagnosis at Day 30-35) suggests that CSF2 is exerting

epigenetic effects on the developing embryo that result in persistent changes in function

during the embryonic and fetal periods of development.

The likelihood that actions of CSF2 during the preimplantation period on survival

after Day 30-35 represent modifications of the epigenome implies that CSF2 may be

one of the molecules through which changes in maternal physiology lead to alterations

in fetal programming. An example of the importance of maternal function for concepts

development is induction of fetal overgrowth in sheep by transfer of embryos into an

advanced uterine environment (Wilmut et al. 1981) or premature elevation of

progesterone concentrations (Kleemann et al. 1994). Bovine embryos produced in

vitro, which are not exposed to most of the regulatory molecules produced by the

reproductive tract, are associated with an array of fetal abnormalities including

increased rates of abortion, fetal overgrowth, and altered metabolism (Farin and Farin

1995; Lazzari et al. 2002; Miles et al. 2005). Maternal effects on concepts

development involve epigenetic alterations in DNA methylation patterns, as has been

demonstrated for sheep exposed to nutritional stress (Sinclair et al. 2007) and for

embryos produced in vitro in cattle (Suzuki et al. 2009).

Treatment with CSF2 increased the proportion of embryos becoming blastocysts

regardless of whether it was added immediately after insemination or at Day 5 after









insemination (a time when embryos were at the morula stage of development). Similar

results were seen in an earlier experiment with bovine embryos (de Moraes and Hansen

1997a). Thus, the action of CSF2 to increase the ability of embryos to advance to the

blastocyst stage of development involves actions on the embryo during the transition

from the morula to blastocyst stage of development. Perhaps, CSF2 is mitogenic and

increases the number of embryos that have cell numbers sufficient for blastocoele

formation. Another possibility, that CSF2 increases cell number by blocking apoptosis,

is less likely. Although culture of human embryos with CSF2 decreased the number of

apoptotic blastomeres by 50% (Sjoblom et al. 2002), no effect of CSF2 on apoptosis

was observed in the present experiment.

Alternatively, CSF2 could increase one or more of the molecules involved in

blastocyst formation. The observation that blastocysts from CSF2 treated embryos had

proportionally more ICM cells relative to TE indicates the capacity of CSF2 to affect

blastocyst differentiation. In the human (Sjoblom et al. 2002) and mouse (Karagenc et

al. 2005), CSF2 treatment in culture resulted in more cells in the ICM of blastocysts.

The magnitude of the effect of CSF2 on blastocyst yield depended on the timing of

exposure and the oxygen concentration used for culture. For embryos cultured in low

oxygen, the increase in blastocyst yield caused by CSF2 was greater when the cytokine

was added at Day 5 than at Day 0. The opposite was true when embryos were cultured

in high oxygen. Differences in response between Day 0 and Day 5 may reflect

degradation of CSF2 during culture or down-regulation of CSF2 receptors. The

importance of oxygen as a determinant of the effect of timing of CSF2 precludes simple

explanations, however.









The improvement in survival of embryos after transfer to recipients also varied with

timing of CSF2 exposure. In the initial experiment, in which embryos were exposed to

CSF2 from Day 1-7 after insemination, there was no effect of CSF2 on pregnancy risk

at Day 30-35 of gestation. In the second experiment, however, when CSF2 treatment

was from Day 5-7 after insemination, pregnancy risk at Day 30-35 was greater for cows

receiving embryos treated with CSF2 than for cows receiving control embryos. Caution

must be taken when interpreting these results. The first experiment was done during

heat stress, a factor that can compromise embryonic survival (Hansen 2007) and

involved relatively small numbers of transfers so that real treatment effects may not

have been observed. What is clear is that, CSF2 reduced the loss of pregnancies

occurring after Day 30-35 of gestation regardless of whether CSF2 was administered

from Day 1-7 or 5-7 after insemination. In the mouse, as well, treatment of embryos with

CSF2 in culture enhanced fetal and postnatal growth (Sjoblom et al. 2005). To our

knowledge, CSF2 represents the only regulatory molecule shown to affect post-

placentation events when acting during the preimplantation period.

As mentioned above, the effect of CSF2 to improve survivability of the concepts

after Day 30-35 of gestation is likely the result of alterations in the concepts

epigenome. Actions of CSF2 to affect pregnancy risk at Day 30-35 could involve

changes in embryonic development related to functions important for establishment of

pregnancy. One action of CSF2 that might enhance survival after transfer is the

increase in number of ICM cells in the blastocyst. In the mouse, CSF2 increased the

number of ICM cells (Karagenc et al. 2005) and post-transfer survival of embryos

(Sjoblom et al. 2005). In the mouse, the number of cells in the ICM correlates with









viability after transfer (Lane and Gardner 1997). The ICM/TE ratio for bovine embryos

produced in vitro differs considerably from embryos generated in vivo (Iwasaki et al.

1990; Crosier et al. 2001; Knijn et al. 2003). Moreover, a high proportion of embryos

produced in vitro have no detectable embryonic disc by Day 14 of gestation (Fischer-

Brown et al. 2004; Block et al. 2007). At Day 16 of gestation, embryonic discs were

reported to be smaller for embryos produced in vitro than embryos produced in vivo

(Bertolini et al. 2002). Perhaps, CSF2 minimizes the detrimental effects of culture on

embryonic disc development or survival by increasing the ICM/TE ratio.

Another way in which CSF2 might improve post-transfer embryonic survival at Day

30-35 of gestation would be through increased secretion of the anti-luteolytic pregnancy

recognition signal IFNT by elongated embryos. At least 40% of total embryonic losses

have been estimated to occur between Days 8 and 17 of pregnancy (Thatcher et al.

1995) when the concepts ordinarily inhibits pulsatile PGF2a secretion. Treatment with

CSF2 has been reported to increase IFNT production by elongated sheep embryos

(Imakawa et al. 1993; Imakawa et al. 1997; Rooke et al. 2005) and a cell line derived

from bovine trophoblast cells (Michael et al. 2006). On the other hand, de Moraes and

Hansen (1997) found no beneficial effect of bovine CSF2 on IFNT secretion by bovine

blastocysts at Day 7-8 after insemination. CSF2 could also affect expression of other

genes during the preimplantation period. The production of embryos in vitro alters the

expression of several developmentally important genes (Bertolini et al. 2002; Rizos et

al. 2002; Sagirkaya et al. 2007; McHughes et al. 2009) and such alterations in gene

expression can persist at least through Day 25 of gestation (Moore et al. 2007).









Treatment of embryos with IGF1 represents a positive control. This treatment has

been reported to increase embryo survival when transfers to lactating cows are

performed during periods of heat stress but to be without effect on embryonic survival

when heat stress is not present (Block et al. 2003; Block and Hansen 2007). Similar

results were obtained in the present study. In the first embryo transfer study, where

cows were exposed to heat stress, pregnancy risk at Day 35 was highest for cows

receiving embryos treated with IGF1. Treatment effects were not significant but the lack

of significance may represent the small number of animals used for the study. In the

second embryo transfer study, conducted mostly outside the time of year when heat

stress is present, there was no improvement in pregnancy risk compared with the

controls.

Interestingly, like CSF2, IGF1 reduced pregnancy loss after Day 30-35 in both

studies. Thus, this growth factor may also cause changes in preimplantation

development that result in changes in post-placentation development conducive for

concepts survival.

A practical outcome of this study is that CSF2 may prove useful as an additive to

culture media for in vitro production of bovine embryos for commercial embryo transfer

programs. There is a compelling need for such treatments. Pregnancy rates following

transfer of embryos produced in vitro is lower than following transfer of in vivo derived

embryos (Farin and Farin 1995). In addition, pregnancy loss after ~Day 40 of gestation

is higher for embryos produced in vitro than for embryos produced in vivo IVP embryos

that survive the fetal period are more likeable to be lost later on (Farin and Farin 1995).









Table 2-1. Effect of CSF2 on total cell number, inner cell mass (ICM), trophectoderm
(TE) and ICM/TE ratio of blastocysts at Day 7 after insemination
Control CSF2 P-value


Total cell number 154 9.02 170 10.8
ICM 41.9 4.1 69.7 4.6
TE 112 5.6 106 6.2
Ratio ICM/TE 0.42 0.023 0.66 0.026
Values are expressed as the mean SEM
Replicates= 3; N= 18/36 embryos/group


NS
0.066
NS
0.02


Table 2-2. Effect of CSF2 on total cell number and TUNEL-positive blastomeres in
blastocysts at Day 7 after insemination


Control CSF2 P-value
Total cell number 146 5.3 154 5.5 NS
Percentage of 10.4 1 8.7 1 NS
apoptosis
Values are expressed as the mean SEM
Replicates= 3; N= 31/58 embryos/group









Table 2-3. Effect of CSF2 and IGF1 added at Day 1 of culture on embryonic
development at Day 7, pregnancy risk at Day 30-35 (based on
ultrasonography), calving rate and pregnancy loss among recipient cattle that
received embryos that were cultured in 5% 02.


Transferable
embryo yield
(percentage, %)


Control (C)


IGF1 (I)


CSF2 (Day 1; G1)


SET 1: C vs I and
G1
SET 1: I vs G1
SET 2: C and I vs
G1
SET 2: C vs I

SET 3: C vs G1
SET 3: C and G1
vs I


Pregnancy
Risk


17% 2 18/52 = 35%

18% 2 24/55 = 43%

25% 2 18/51 = 35%

Orthogonal Contrasts


Calving Rate

14/52 = 27%

22/55 = 40%

18/51 = 35%


Pregnancy
Loss

4/18 = 22%

2/24 = 8%


0/18 = 0%


<0.02

<0.06

<0.02

NS

<0.01

NS


<0.05


<0.05


aTransferable embryo yield: Grade 1 or 2 morulae and blastocysts at Day 7.









Table 2-4. Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of culture
on embryonic development at Day 7, pregnancy risk at Day 30-35 (based on
ultrasonography), calving rate and pregnancy loss among recipient cattle that
received embryos that were cultured in 5% 02.
Transferable
a Pregnancy Pregnancy
embryo yield rik Calving rate
(percentage, %)
Control (C) 10% 1 27/79 = 34% 17/74 = 23% 5/22 = 22%

IGF1 (I) 14% 2 12/44 = 27% 11/44 = 25% 1/12 = 8%
CSF2(Day5; 14% 1 47/107 = 43% 39/104 = 37% 5/44 =11%
G5)
Orthogonal Contrasts
SET 1: Cand I vs
<0.02 <0.05 <0.05 NS
G5
SET1:CvsI NS NS NS NS
SET 2: C vs I and
SET sand <0.02 NS NS NS
G5
SET 2: I vs G5 <0.05 <0.06 NS NS

SET 3: C vs G5 <0.01 NS <0.05 NS
SET 3: C and G1
NS NS NS NS
vs I
a Transferable embryo yield: Grade 1 or 2 morulae or blastocysts at Day 7.












Day 7
30 -y

C 25
-,-
S20-

15 -

S10

U5 5
4-,
0 0

SDay 8
E. 40
0

30 -
-o
0 20


S10
O

CSF2 + + +

Day 0 Day 5 Day 0 Day 5

Low 02 High 02

Figure 2-1. Percentageage of oocytes that developed to the blastocyst stage at Day 7
(Panel A) and 8 after insemination (Panel B). There was a tendency for an
increase in the percentageage of blastocysts at Day 7 (treatment, P=0.09;
treatment x Day x oxygen= NS) and a significant increase in blastocyst
development at Day 8 (treatment, P=0.05; treatment x Day x oxygen= 0.06)
when embryos were treated with CSF2. There was an effect of oxygen on the
percentageage of oocytes that became blastocysts at Days 7 and 8 on both
Days of treatment (P<0.0001).









CHAPTER 3
COLONY STIMULATING FACTOR 2 CAUSES CHANGES IN THE TRANSCRIPTOME
OF THE BOVINE PREIMPLANTATION EMBRYO INCLUDING ALTERATIONS IN
EXPRESSION OF DEVELOPMENTAL AND APOPTOSIS GENES

Introduction

Colony-stimulating factor 2 (CSF2) is an important local regulator of embryonic

function during early pregnancy in several mammalian species. It is expressed in the

oviduct (Zhao and Chegini 1994; de Moraes et al. 1999), endometrium (Chegini et al.

1999; de Moraes et al. 1999; Robertson et al. 2001; Emond et al. 2004), and decidua

(Segerer et al. 2009). Addition of CSF2 to culture medium improved the proportion of

cultured embryos developing to the blastocyst stage in the cow (de Moraes and Hansen

1997a; Chapter 2); human (Sjoblom et al. 1999), mouse (Robertson et al. 2001), and

pig (Cui et al. 2004) and also causes a preferential increase in the number of cells in the

inner cell mass(ICM) in the human (Sjoblom et al. 2002), mouse (Karagenc et al. 2005)

and cow (Chapter 2). In the mouse blastocyst, CSF2 increased expression of genes

involved in glucose transport (Robertson et al. 2001) and decreased incidence of

apoptosis (Sjoblom et al. 2002; Desai et al. 2007).

A recent report using the cow as a model indicates that changes in blastocyst

function caused by CSF2 are sufficient to increase the competence of the embryo for

sustained development through the embryonic and fetal periods of pregnancy. In

particular, addition of CSF2 to culture medium at Day 5 after insemination increased

post-transfer survival of bovine in vitro produced (IVP) embryos by increasing

pregnancy rate at Day 30-35 of gestation and decreasing loss of pregnancies after Day

30-35 (Chapter 2). The actions of CSF2 to enhance competence of the embryo for

post-blastocyst development could involve changes in the embryonic transcriptome that









alter important functions for establishment of pregnancy and for differentiation and

growth of the concepts. The objective of the present study was to determine changes

in the transcriptome of the bovine embryo caused by CSF2 and thereby identify gene

pathways and ontologies that are involved in actions of CSF2 on formation of the

blastocyst and post-transfer pregnancy success.

Materials and Methods

In vitro Production of Embryos

Embryo in vitro production (IVP) was performed as previously described (Chapter

2) with modifications described below. Cumulus oocyte complexes (COCs) from

ovaries from a mixture of beef and dairy cattle were collected in Tissue Culture Medium-

199 (TCM-199) with Hank's salts without phenol red (Hyclone, Logan UT)

supplemented with 2% (v/v) bovine steer serum (Pel-Freez, Rogers, AR) containing 2

U/ml heparin, 100 U/ml penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine.

Oocytes were allowed to mature for 20-22 h in groups of 10 in 50 pl microdrops of TCM-

199 (Invitrogen, Carlsbad, CA, USA) with Earle's salts supplemented with 10% (v/v)

bovine steer serum, 2 [tg/ml estradiol 17-P, 20 [tg/ml bovine follicle stimulating hormone

(Folltropin-V; Belleville, ON, Canada), 22 [Lg/ml sodium pyruvate, 50 [Lg/ml gentamicin

sulfate, and 1 mM glutamine.

Matured oocytes were then washed in HEPES-TALP (Parrish et al. 1986; Caisson,

Sugar City ID, USA) and transferred in groups of 50 to four-well plates containing 600

pL of IVF-TALP supplemented with 25 pL PHE (0.5 mM penicillamine, 0.25 mM

hypotaurine, and 25 pM epinephrine in 0.9% [w/v] NaCI), and fertilized with 30 pL

Percoll-purified spermatozoa (~ 1x106 sperm cells). Sperm were prepared from a pool









of frozen-thawed semen from three different bulls; a different set of bulls was generally

used for each replicate). Fertilization proceeded for 18-20 h at 38.50C in a humidified

atmosphere of 5% (v/v) CO2 in humidified air. Putative zygotes were removed from

fertilization plates, denuded of cumulus cells by vortexing in HEPES-TALP, and placed

in groups of 30 in 45-[Ll microdrops of KSOM-BE2 (Soto et al. 2003).

Embryos were cultured at 38.50C in a humidified atmosphere of 5% CO2 or 5%

CO2, 5% 02, and 90% N2 (v/v). Cleavage rate was assessed at Day 3 after

insemination. At Day 5 after insemination, 5 pl of KSOM-BE2 or 5 pl of KSOM-BE2

containing 100 ng/ml CSF2 (a gift from Novartis, Basle Switzerland) were added to each

drop to achieve a final CSF2 concentration of 0 or 10 ng/ml. The concentration of CSF2

was one that increased blastocyst yield and post-transfer pregnancy rates when added

at Day 5 after insemination (de Moraes and Hansen 1997a; Chapter 2).

On Day 6, 24 hours after treatment, morulae and early blastocysts were selected

and washed three times in 50 pl microdrops of 10 mM PO4 buffer, pH 7.4 containing

0.9% (w/v) NaCI and 1 mg/ml polyvinylpyrrolidone (PBS-PVP) by transferring the

embryos from microdrop to microdrop. Embryos were frozen at -800C in PBS-PVP in

groups of 50. A total of 4 groups of embryos from each treatment were prepared in a

total of 6 replicates of in vitro production.

RNA Purification and Processing

Total cellular RNA was extracted from embryos with the RNeasy Plus Micro kit

(Qiagen-Inc, CA, USA) following manufacturer's instructions. Concentration of the input

RNA was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific,

Waltham, MA, USA) and RNA integrity was determined by Agilent 2100 Bioanalyzer









with RNA 6000 Nano LabChip kit (Agilent Technologies, Santa Clara CA, USA). Only

samples that showed RNA integrity > 7 were used for the microarray hybridization and

quantitative PCR analysis. Extracted RNA was stored at -800C until microarray analysis.

Microarray Hybridization

The effect of CSF2 on gene expression was assessed using the Bos taurus Two

Color Microarray Chip from Agilent v1 (Agilent Technologies). This array contains

21,475 unique 60-mer probes, representing approximately 19,500 distinct bovine genes

arranged on a slide as 4 arrays in a 4 x 44K format. The probes were developed by

clustering more than 450,000 mRNA and EST sequences of the bovine genome (btau

2.1). A total of four separate samples of RNA from CSF2 treated embryos and four

separate samples of RNA from control embryos were subjected to microarray analysis.

All microarray protocols were carried out by Mogene LLC (St. Louis MO, USA), an

Agilent Certified Service Provider.

Prior to labeling, samples of RNA (1.5-3.0 pg/pl) were concentrated to 5 pl using a

Savant SpeedVac (Therno Scientific) at low heat and amplified into ss-cDNA using the

NuGEN WT-Ovation Pico RNA amplification System (NuGen Technologies, Inc, San

Carlos, CA, USA) following manufacturer's instructions. Amplified ss-cDNA was purified

using Zymo Spin IIC columns (Zymo Research, Orange, California, USA) and stored at

-20 OC overnight. Product yield and purity were determined by the Nanodrop 1000

spectrophotometer assuming that 1 absorbance unit at 260 nm of ss-cDNA is equal to

33 pg/ml. All 260/230 values were greater than 2.

Aliquots containing 2 pg of ss-cDNA were labeled with the Agilent Genomic DNA

Enzymatic Labeling kit to incorporate cyanine 3- or 5-labeled CTP. For half the









replicates, ss-cDNA from control embryos was labeled with Cy3 and ss-cDNA from

CSF2-treated embryos was labeled with Cy5. For the other replicates, control embryos

were labeled with Cy 5 and CSF2-treated embryos with Cy3.

Hybridizations were set up using 2 pg of each sample (Cy3 and Cy5 components)

for a total of 4 pg per array. Volumes were brought to 44 pl with Diethylpyrocarbonate

(DEPC)-water and then 11 pl of Agilent 10x GE Blocking Agent was added. The

mixtures were incubated at 980C for 3 minutes and then cooled to room temperature for

5 minutes before adding 55 pl of Agilent 2x Hi-RPM Hybridization buffer. Tubes were

flash-spinned on a microfuge and lightly vortexed before loading 100 pl of the

hybridization mixture onto each array. Hybridization was carried out for 17 h at 65C

and 10 rpm in a SureHyb gasket slide (Agilent). Washing and scanning procedures

were carried out using standard Agilent guidelines for gene expression microarray

processing.

At the end of hybridization, microarray slides were sequentially washed using

standard Agilent guidelines for gene expression microarray processing. Microarray

slides were scanned immediately using an Agilent G2505B scanner.

Analysis of Microarray Data

Images were extracted and pre-processed using the Agilent Feature Extraction

Software v 9.5 with default analysis parameters for the initial extraction, signal

quantifications, and scaling of the generated data. The software produces background

adjustment and normalizations for the dye of individual genes.

The intensity of each spot was summarized as the median pixel intensity. All the

generated values were then transformed to log2. The Lowess method was used for

intensity normalization within each array. JMP Genomics 3.1 for SAS 9.1.3 software









(SAS Inst., Inc., Cary, NC) was used for data global normalization and identification of

differentially expressed genes. The PROC ANOVA procedure was used for

simultaneous comparisons and the quantile method for intensity normalization. The

model included replicate and treatment. Replicate (array) was considered random and

treatment was considered fixed. Correction for false discovery rate was performed by

the Benjamini and Hochberg method (Benjamini and Hochegerg 1995). Only genes with

median pixel intensity of at least 2.8 were considered to be expressed. To increase the

reliability of the results, only genes with a 1.5-fold difference and false discovery rate 5

0.01 were considered differentially expressed.

David Bioinformatics Database (http://david.abcc.ncifcrf.gov/; Huang et al. 2009)

was used to categorize the genes into different ontologies. The Ingenuity pathway

analysis software (http://www.ingenuity.com) was used to determine differentially

regulated pathways and to modify existing canonical pathways to better fit the findings

from the literature. The significance of the association between the list of genes and the

canonical pathway was measured by the ratio of the number of molecules from the data

set that mapped to the pathway divided by the total number of molecules in the pathway

and a p-value calculated by Fisher's exact test that determined the probability that the

association between the genes and the pathway is explained by chance alone. To more

completely fit differentially-expressed genes into pathways, analyses of each gene

based on available literature was used to modify certain canonical pathways to include

additional genes.

Quantitative Real Time PCR

Quantitative Real Time PCR analysis (qPCR) of 16 differentially-expressed genes

was performed to confirm microarray results. Also, qPCR was performed on one









housekeeping gene (GAPDH) for use as an internal control. Specific primers (Table 3-1)

were designed using Integrated DNA Technologies software (http://idtdna.com).

The qPCR analysis was carried out by Mogene LLC with the Applied Biosystems

Taqman Gene Expression Mastermix (Foster City, CA, USA). Each reaction consisted

of a total volume of 25 pl, 25 ng of template cDNA and with the final concentration of

each primer and probe being 200 nM. All probes had a 5' 6-Fam label and a 3' IA-Black

Quencher. Following incubation at 95C for 10 min, 40 cycles of denaturation (95C for

15 s) and annealing/synthesis (600C for 1 min) were completed. Each RNA sample was

analyzed in triplicate. Responses were quantified based on the threshold cycle (CT).

All CT responses from genes of interest were normalized to the housekeeping

GAPDH gene using the ACT method. The AACT for each sample was calculated by

subtracting the ACT of CSF2-treated group from the control. Fold change of genes inthe

CSF2-treated group was determined by solving for 2-AACT relative to the controls.

Treatment effects were analyzed by the median scores procedure of SAS (SAS for

Windows, Version 9.0, Cary, NC, USA).

Regulation of Apoptosis by CSF2

This experiment was designed to verify whether CSF2 could regulate apoptosis by

testing whether treatment of embryos with CSF2 reduced the induction of apoptosis

caused by heat shock. The experimental design involved a 2 x 2 factorial arrangement

of treatments (control vs heat shock and 0 or 10 ng/ml CSF2). Putative zygotes were

randomly distributed in 45 pl microdrops of KSOM-BE2 at 38.50C in a humidified

atmosphere of 5% CO2, 5% 02, and 90% (v/v) N2. At Day 5 after insemination, 5 pl of

vehicle (KSOM-BE2) or CSF2 (to create a final concentration of 10 ng/ml) were added

to the drops. On Day 6 (24 h after treatment), morulae and blastocysts were selected,









placed in previously-conditioned microdrops of the same treatment and then cultured at

either 38.5C or 42C for 15 h in an atmosphere of 5% C02. Embryos were then

returned to an environment of 38.5C in a humidified atmosphere of 5% C02, 5% 02,

and 90% (v/v) N2 for 9 h back. Embryos were then removed from culture and washed

three times in 50-tl microdrops of PBS-PVP. Embryos were fixed in a 50-tl microdrop

of 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature, washed twice in

PBS-PVP, and stored in 600 [il of PBS-PVP at 4C until analysis for terminal

deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using the in situ Cell

Death Detection Kit TMR red (Roche Diagnostics, Indianapolis, USA) to determined

apoptotic nuclei as described previously (Loureiro et al. 2007). The experiment was

replicated 2 times using 65-81 embryos/group.

Results

Transcriptomal Profile

As shown in a Venn diagram (Figure 3-1A), there were a total of 17884 genes that

were expressed by embryos from both treatments (control and CSF2), 276 genes

expressed exclusively expressed in CSF2-treated embryos and 282 genes present only

in control embryos (intensity cutoff for expression= 2.8). Hierarchical analysis

(Figure3.1 B) of microarray results indicated that the four CSF2 samples formed a

distinct cluster from the four control samples.

Differentially expressed genes were determined as those genes where there were

at least a 1.5-fold difference in expression and a significance of P < 0.05 (adjusted to a

false discovery rate of 0.01). A total of 214 genes met these criteria and 160 of these

could be annotated (67 genes upregulated by CSF2 and 93 genes downregulated).









The five most upregulated genes in terms of fold-change were calcium channel,

voltage-dependent, alpha 1G subunit (CACNA1G; 9.2 fold increase), stearoyl-

coenzyme A desaturase (SCD; 6.5 fold increase), Kruppel-like factor 8 (KLF8; 5.8 fold

increase), secreted frizzled-related protein 4 (SFRP4; 4.0 fold increase), and alcohol

sulfotransferase (SULT2A1; 3.2 fold increase). The five most downregulated genes, in

terms of fold change were olfactomedin 4 (OLFM4; 4.2 fold decrease), wingless-type 16

(WNT16; 3.8 fold decrease), neural precursor cell expressed, developmentally down-

regulated 4 (NEDD4; 3.8 fold decrease), MAP-kinase activating death domain (MADD;

3.7 fold decrease), and coiled-coil domain containing 103 (CCDC103; 2.9 fold

decrease).

Biological Process Ontologies Affected by CSF2

Analysis using the David software categorized the differentially expressed genes

into 13 biological process ontologies (Table 3-2). These ontologies could be grouped

into four functional groups. One was for genes involved in development and

differentiation. There were 42 differentially expressed genes in the developmental

process ontology (26% of the differentially expressed annotated genes), 32 genes in the

multicellular organ development ontology, 26 genes in the system development

ontology, 26 genes in the anatomical structure development ontology, 23 genes in the

cellular developmental process-cell differentiation ontology and 5 genes in the pattern

specification process. The second was for differentially expressed genes involved in

signal transduction and cell communication. There were 45 differentially expressed

genes in the cell communication ontology (28% of the differentially expressed genes),

44 differentially expressed genes in the signal transduction ontology, and 25 genes in

the cell surface receptor linked signal transduction ontology. The third was for genes









involved in apoptosis with 9 differentially expressed genes in the regulation of apoptosis

ontology and 6 genes in the induction of programmed cell death ontology. The fourth

group was for genes involved in cell adhesion with 13 differentially expressed genes in

the biological adhesion-cell adhesion ontology. There was no pattern for CSF2 to

regulate cell adhesion and migration genes in a way that would consistently promote or

inhibit cell adhesion or migration.

Genes Involved in Cellular Development and Differentiation

Many of the differentially-regulated genes identified by DAVID as being involved in

developmental processes were involved in neurogenesis, mesoderm or muscle

formation, and pluripotency. A total of 13 genes identified as being involved in

neurogenesis were differentially regulated by CSF2, with 4 upregulated genes

(MAB21L2, 3.0-fold increase; HUWE, 2.5-fold increase; NOTCH2, 2.2-fold increase

RTN4, 1.6-fold increase) and 8 downregulated genes (DTX3, 2.2 fold decrease; HUNK,

2.1-fold decrease; CELSR, 1.6-fold decrease; SEMA4, 1.6 fold decrease; ARSA, 1.6-

fold decrease; GREM1, 1.6-fold decrease; GLIS2, 1-6 fold decrease; CHURC1, 1.5-fold

decrease; and RGS12, 1.5 fold decrease). A total of 8 differentially regulated genes

were identified as being associated with mesenchyme, mesoderm or muscle cells,

including regulation of the epithelial-mesenchymal transition. Upregulated genes were

KLF8 (5.8-fold increase), MYF6 (3.1 fold increase), HOXA5 (2.2 fold increase),

NOTCH2 (2.2 fold increase), CD73/NT5E (2.2 fold increase) and FHL1 (2.1 fold

increase) while DTX3 (2.2 fold decrease) and GREM1 (1.7-fold decrease) were

downregulated by CSF2. Two of these genes are involved in hematopoiesis (KLF8 and

NOTCH2). In addition, CSF2 upregulated expression of two other genes involved in

hematopoiesis, CCL23 (2.5 fold increase) and HOXA5 (2.2 fold increase), and









decreased expression of another gene involved in hematopoiesis (CXCL12, 1.9 fold

decrease).

Genes Involved in Signal Transduction and Cell Communication

Analysis of differentially-regulated genes in signal transduction and cell

communication ontologies (Table 3-2) indicates a broad range of effects of CSF2 on

expression of genes encoding ligands (TNFSF8, CXCL2, CXCL12), receptors

(OR51E1, CCRL1, GIPR,, NCOA7, GRID1, OR2T11, ITPR2, PTGER4, GPR143),

receptor tyrosine kinases (ROR2, PTPRK, RIPK3), other protein kinases (PTPN22,

MARK2, PRKA2B, PTPRK, DAPK1, CSNK2B, MADD), transcription factors (KLF8,

MYF6) and cAMP regulators (NOTCH2, PDE7B, CREM, PTGER4).

Genes Involved in WNT Signaling

One signaling system that was represented often in the list of differentially genes

regulated in the most consistent manner was the WNT system. As shown in Table 3-3,

expression of a total of 10 genes involved in WNT signaling were affected by CSF2. Of

the 6 upregulated genes, four (SFRP4/FrpHE, NOTCH2, PPP2R3A, and PCDH24) are

inhibitory to P-catenin-dependent signaling. Of the 4 downregulated genes, two activate

the p-catenin-dependent signaling pathway (WNT16 and CSNK2B) and all 4 activate at

least one p-catenin-independent signaling pathway (WNT16, CSNK2B, ROR2, and

CELSR2).

The data set of differentially expressed genes was also examined for 70 genes

reported to be upregulated by WNT signaling (Ambrosetti et al. 2008; Segditsas et al.

2008; Chien et al. 2009). Only three genes in this list were found to be differentially









regulated by CSF2. GREM1 (1.7 fold decrease), SEMA4 (1.6 Fold decrease) and

MAB21L2, (3.0 fold increase).

Genes Involved in Apoptosis Signaling Pathway

Effects of CSF2 on genes involved in apoptosis signaling are shown in Table 3-4.

A total of 16 genes were differentially regulated. Of the 7 upregulated genes, 3 are anti-

apoptotic (TNFSF8, PRKAR2B, CD73/NT5E, and PGR), one (NOTCH2) is usually, but

not always, anti-apoptotic, and 2 (CASP7 and RTN4) are pro-apoptotic. Of the 9 genes

downregulated by CSF2, 6 are pro-apoptotic (NOD2, PIK31P1, RIPK3, MADD, DAPK1,

and CREM) and 3 are anti-apoptotic (RNF7, CXCL12, and PLD2).

Quantitative Real Time PCR

Quantitative real time PCR (qPCR) was used to confirm the findings of the

microarray analysis (Figure 3-2). For 11 of the 16 genes examined, the fold change

caused by CSF2 as determined by qPCR microarray was in the same direction as the

fold change as determined by microarray hybridization. The effect of CSF2 as

determined by qPCR was significant (P<0.001) for one of these genes (SLC16A10),

approached significance (P=0.09) for 7 genes (PPP2R3A, PMM2, ANKRD37, NOTCH2,

MAB21L2, MRSPS12,and MADD) and was not significant for three genes (VGLL2,

ECE1, and TNFSF8). One gene, WNT16. whose expression was decreased by CSF2

as determined by microarray hybridization was increased by CSF2 (P<0.001) as

determined by qPCR. While not significant, the same trend was apparent for RIPK3

and HK1. Two other genes affected by CSF2 as determined by microarray, had a fold

change near 1.0 as determined by qPCR (MASP2 and CSNK2B).









Actions of CSF2 to Block Heat-Shock Induced Apoptosis

As another validation of the microarray results, an experiment was performed to

test whether embryonic function was changed by CSF2 in a way predicted from the

microarray results. In particular, since CSF2 altered gene expression in a way that

would inhibit apoptosis, it was tested whether culture of embryos with CSF2 beginning

at Day 5 after insemination would block induction of apoptosis caused by heat shock

initiated at Day 6. As shown in Figure 3-3E, culture at 42C for 15 h increased the

percentage of blastomeres that were TUNEL-positive and reduced total cell number

(P<0.0001). While CSF2 did not affect the percentage of cells that were TUNEL positive

at 38.50C, it blocked the increase in TUNEL labeling caused by heat shock (CSF2,

P<0.005).

Discussion

Colony stimulating factor 2 is an important regulator of preimplantation embryonic

development. In the cow, it can increase the proportion of embryos that develop to the

blastocyst stage, increase the number of cells in the ICM, improve the competence of

the embryo to establish pregnancy after transfer to a recipient female, and reduce the

probability of fetal loss after Day 30-35 of pregnancy (de Moraes and Hansen 1997a;

Chapter 2). As indicated by changes in the embryonic transcriptome described in this

paper, these actions of CSF2 involve changes in expression of genes controlling

developmental and apoptotic processes. Changes in expression of genes controlling

development are likely to lead to a blastocyst whose developmental trajectory favors

embryonic survival in the embryonic and fetal period. Actions of CSF2 to increase

resistance to induction of apoptosis could conceivably contribute to growth of the

embryo and survival after transfer into a recipient with suboptimal uterine environment.









A large number (42) of the genes whose expression was altered by CSF2 are

genes implicated in developmental processes. A total of 26% of annotated genes that

were differentially expressed were in the developmental process ontology. Thus, it is

likely that CSF2 acts on the embryo to control cell fate and differentiation in the

blastocyst. Two major actions of CSF2 on development could be inferred by

examination of the developmental genes regulated by CSF2. The first was regulation of

genes involved in neurogenesis in a manner that would act to inhibit neural cell

formation and differentiation. The second was stimulation of genes involved in formation

or differentiation of mesoderm or mesoderm derived cells.

That CSF2 acts to inhibit processes involved in neurogenesis is evident from an

examination of the 4 genes involved in neurogenesis increased by CSF2 (MAB21L2,

HUWE, NOTCH2 and RTN4) and the 9 genes that were downregulated by CSF2

(D7X3, HUNK, CELSR, SEMA4, ARSA, GREM1, GLIS2, CHURCH, and RGS12). Two

of the upregulated genes are required for neurogenesis. Use of antisense

oligonucleotides to deplete MAB21L2 impaired notochord and neural tube differentiation

(Wong and Chow 2002). HUWE1 causes ubiquitination of N-myc and induces ES cells

to differentiate into neural tissue (Zhao et al. 2009). Conversely, RTN4 is a neural stem

cell marker that prevents neurite outgrowth (Zheng et al. 2010) and NOTCH signaling

can inhibit mitosis of neuronal precursors (le Roux et al. 2003). However, it is not clear

whether NOTCH signaling is enhanced in CSF2 treated embryos, since one of the

genes downregulated by CSF2, DTX3, is a component of the NOTCH signaling

pathway (Pampeno et al. 2001). Moreover, NOTCH signaling is involved in a wide array

of developmental events and the phenotype caused by activation of NOTCH pathways









depends upon inputs from multiple inputs (Lewis et al. 2009). The other 8 genes

involved in neurogenesis that were downregulated by CSF2 are all involved in neural

cell function. Two of these genes, the bone morphogenic protein (BMP) inhibitor

GREM1 (Wordinger et al. 2008) and the transcription factor CHURC1, participate in

neurulation. Early in development, ectoderm is inhibited from differentiating into neural

cells by the actions of BMP4. Secretion of BMP antagonists relieves this inhibition and

contributes to neural induction (Hemmati-Brivanlou and Melton 1997). Neural formation

also requires actions of fibroblast growth factors, which act through the transcription

factor CHURC1 to enhance actions of BMP antagonists and activate expression of

SOX2 (Sheng et al. 2003). Inhibition of GREM1 and CHURC1 expression by CSF2

could, therefore, result in maintenance of the inhibition of neural cell formation. The

other genes involved in neurogenesis inhibited by CSF2 are the transcription factor

GLIS2 (Zhang et al. 2002), RGS12, a component of the signaling system for nerve

growth factor (Willard et al. 2007), CELSR, a cadherin involved in planar polarity and

neural tube formation (Curtin et al. 2003; Zhou et al. 2007a), SEMA4, a member of the

semaphorin family of axon guidance proteins (Pasterkamp and Giger 2009), HUNK, a

protein kinase expressed in fetal brain (Gardner et al. 2000), and ARSA, a lysosomal

enzyme involved in myelin formation (Gieselmann et al. 1991).

In contrast to the apparent inhibition of neural formation caused by CSF2, there

was evidence that CSF2 regulated gene expression in a way that would increase

mesoderm formation and differentiation. One gene increased by CSF2 was KLF8, a

Kruppel-like transcription factor that can induce epithelial-mesenchymal transition

(Wang et al. 2007). The process of epithelial-mesenchymal transition occurs at several









developmental events including gastrulation and leads to epithelial cells becoming

migratory and differentiating into mesenchymal cells (Baum et al. 2008). CSF2 also

caused upregulation of HOXA5, a mesenchymally-restricted morphogen involved in

pattern formation (Boucherat et al. 2009), MYF6, a member of the basic helix-loop-helix

family of transcription factors that activates genes involved in muscle cell formation

(Rescan 2001), CD73/NT5E, an extracellular nucleotidase and mesenchymal stem cell

marker which can induce proliferation, migration, invasion, and adhesion of breast

cancer cells (Barry et al. 2001; Babiychuk and Draeger 2006; Zhou et al. 2007b; Wang

et al. 2008b), and FHL1, a four-and-a-half-LIM only protein involved in muscle

development (McGrath et al. 2003) (2.1 fold increase). One effect of CSF2 that was

inconsistent with promotion of differentiation of mesoderm derivatives was the inhibition

of GREM1 expression because this BMP antagonist promotes the epithelial-

mesenchymal transition in kidney and limb bud (Michos et al. 2004).

The first differentiation event in the embryo after formation of the blastocyst is

gastrulation whereby the three germ layers (ectoderm, mesoderm, and endoderm) are

formed. In the cow, this event occurs as early at Day 9 of development when mesoderm

can be identified (Maddox-Hyttel et al. 2003). Neurulation occurs later in development

and the neural groove forms between Day 14 and 21 (Maddox-Hyttel et al. 2003).

Taken together, the net result of actions of CSF2 on expression of genes involved in

developmental processes could be a blastocyst in which formation of mesoderm and

mesoderm-derived cell types is enhanced while differentiation of the ectoderm into the

neural plate is delayed. Perhaps, CSF2 favors embryonic survival by ensuring allocation









of a sufficient number of ICM cells towards mesoderm early in development and by

inhibiting neurulation until the appropriate stage of development.

A total of 10 genes involved in WNT signaling were differentially expressed by

CSF2. Given the involvement of WNT in epidermal fate determination, BMP4 regulation

and endoderm formation (Wilson et al. 2001; Hansson et al. 2009), such changes could

conceivably contribute to changes in the process of gastrulation. Analysis of the WNT-

related genes regulated by CSF2 would indicate inhibition of p-catenin dependent WNT

signaling. Four genes inhibitory to this pathway (SFRP4/FrpHE, NOTCH2, PPP2R3A,

and PCDH24) were upregulated by CSF2 and two of the genes downregulated by CSF2

(WNT16 and CSNK2B) promote p-catenin dependent signaling. Actions of proteins

involved in WNT signaling are complex, however, and depend upon the array of other

WNT signaling proteins present in the cell (Chien et al. 2009). Moreover, all four of the

WNT-signaling genes that were downregulated by CSF2 are involved in one or more 3-

catenin-independent signaling pathways (WNT16, CSNK2B, ROR2, and CELSR2) and

activation of these pathways leads to an inhibition of p-catenin dependent gene

expression (Chien et al. 2009). Expression of only 3 of 70 WNT-induced genes were

significantly affected by CSF2 and only two of those (GREM1 and SEMA4) experienced

the decrease in expression expected if CSF2 decreased p-catenin dependent gene

expression. Further research is required to delineate regulation of WNT signaling by

CSF2 in the preimplantation embryo.

It has been demonstrated previously that CSF2 can reduce apoptosis in

preimplantation mouse embryos (Sjoblom 2002). Similar results were obtained in the

present experiment with bovine embryos. Expression of three genes (CD73/NT5E,









PGR, and PRKAR2B) that inhibit apoptosis were increased by CSF2. The ecto-

nucleotidase CD73/NT5E blocks TRAIL-induced apoptosis, possibly through interaction

with death receptor 5 (Mikhailov et al. 2010). Ligand dependent action of PGR leads to

inhibition of apoptosis through an unknown mechanism (Friberg et al. 2009). The cAMP-

dependent kinase PRKAR2B can inhibits apoptosis through a p53-dependent

mechanism (Srivastava et al. 1999). CSF2 also increased expression of two genes that

can be pro- or anti-apoptotic, NOTCH2 (Quillard et al. 2009; Yoon et al. 2009) and the

TNF-receptor superfamily member TNFSF8 (CD30) (AI-Shamkhani 2004). Treatment

with CSF2 also inhibited expression of 6 pro-apoptotic genes (MADD, RIPK3, PIK31P,

NOD2, DAPK1 and CREM1). MADD is a death domain-containing adaptor protein,

DAPK1 is a death associated protein kinase 1 (Martoriati et al. 2005; Okamoto et al.

2009) and NOD2 is a caspase recruitment domain family (Geddes et al. 2009). PIK31P1

(He et al. 2008) and RIPK3 are kinase proteins that can trigger apoptotic signaling

(Declercq et al. 2009) and CREM decreases amounts of the anti-apoptotic protein BCL2

(Jaworski et al. 2003; Mioduszewska et al. 2003)).

Consistent with the idea that CSF2 inhibits apoptosis was the finding that induction

of apoptosis in Day 6 embryos by heat shock was reduced by treatment with CSF2.

Heat shock induces apoptosis in preimplantation embryos through mitochondrial

depolarization and activation of group III caspases such as caspase 9 (Loureiro et al.

2007).

It is possible that the increased number of ICM cells and increased ICM/TE ratio

seen in CSF2 treated embryos (Chapter 2) is a result, at least in part, in inhibition of

apoptosis responses because the incidence of apoptosis is greater in ICM than TE









(Knijn et al. 2003; Fouladi-Nashta et al. 2005; Pomar et al. 2005). Increased survival

after transfer to recipients (Chapter 2) could also be due, in part, to increased resistance

of embryos to adverse maternal environments that could induce blastomere apoptosis

after transfer.

Quantitative PCR was used to confirm the microarray results. While 11 of 16

genes showed fold changes in the same direction as for microarray analysis,

differences were significant in one case only and approached significance (P=0.09) in 7

other cases. Lack of significance was likely due to part to the small sample size and

between-sample variation. It could also be, for some genes, the microarray and the

qPCR probes recognize different splice variants. The most notable discrepancy

between PCR and microarray results was for WNT16, where expression was reduced

by CSF2 as determined by microarray hybridization and increased by CSF2 as

determined by qPCR. This gene was not very abundant and the large fold-change

differences may reflect sampling error.

While the lack of uniform agreement between microarray and qPCR results

means that definitive conclusions regarding effects of CSF2 on individual genes is not

possible, it is likely that systems and pathways found by microarray hybridization to be

regulated by CSF2 reflect biological actions of CSF2. Indeed, the experiment on

induction of apoptosis by heat shock represents a biological confirmation of the results

from microarray analysis with respect to regulation of genes involved in apoptosis. Even

though a small percentageage of genes were confirmed in the qPCR we were able to

confirm the regulation of apoptosis by CSF2 evaluating the percentageage of apoptotic

blastomers by a well characterized assay.









It is concluded from the present study that CSF2 can regulated embryonic

development by altering the expression of genes controlling developmental process and

apoptosis. Results indicate that CSF2 act to increase expression of genes regulating

epithelial to mesenchymal transition and decrease neural cell differentiation. Other

CSF2 effects include a decrease in genes involved in apoptosis and an increase in

genes that regulate cell survival. Perhaps this change in cellular fate and the decrease

in cellular death are responsible for the higher pregnancy rates at Day 35 and the lower

embryonic losses seem on recipients that receive a CSF2 treated embryo.









Table 3-1. Primers and Probes used on qPCR.

Gene Name Accession Primer/Probe Sequence (5'-3')


Ankyrin repeat domain 37
(ANKRD37)


XM 586036


Casein kinase 2, beta polypeptide XM_585826
(CSNK2B)


Endothelin converting enzyme 1
(ECEI)



Hexokinase 1 (HKI)



Mab-21-like 2 (MAB21L2)



MAP-kinase activating death
domain (MADD)


NM_181009




NM_001012668



XM_585738



XM_867337


Forward

Probeb
Reverse
Forward

Probeb
Reverse
Forward

Probeb
Reverse
Forward
Probeb
Reversec
Forward
Probeb
Reverse
Forward

Probeb
Reverse


GTG GGA AAA GGA AGT GTT GAT G

TGG TCA TGA AGA GGT GAG GAG AAG GT
CTT GTA GCT GAA CGG TAG ACC
GTT TCC CTC ACA TGC TCT TCA

TGG ATC TTG AAA CCG TAA AGC CTG GG
TCA CTG GGC TCT TGA AGT TG
CAT TCT ACA CCC GCT CTT CAC

CCG ATG CCG CCG AAG TTT AAG G
GTT CCC ATC CTT GTC GTA CTC
CCA AAG TGT AAT GTG TCC TTC C
TGC CGC TGC CGT CTT CAG ATA A
GAA GAG AGA AGT GCT GGA AGG
TCT CAC CAA TCC CAA AAG CC
AGC AGT CCC CAG CAC CCT ATA GT
TCT GGA GTT CTC GCA GTT TG
AGT GCA ATA CAG TCC GAG G

AGT ACA AGA CAC CGA TGG CCC AC
CAA CAC GGA GTA GCA GAT C









Table 3-1. Continued

Gene Name Accession Primer/Probe Sequence (5'-3')


Mannan-binding lectin serine
peptidase 2 (MASP2)



Phosphomannomutase 2 (PMM2)



Notch homolog 2 (NOTCH2)


XM_582170




NM_001035095



XM 867242


Protein phosphatase 2, regulatory XM_599849
subunit B, alpha 1 (PPP2R3A)


Receptor-interacting serine-
threonine kinase 3 (RIPK3)



Solute carrier family 16, member
10 (SLC16A10)


XM_584025




XM_615239


Forward

Probeb
Reverse
Forward
Probeb
Reverse
Forward
Probeb

Reversec
Forward

Probeb

Reversec
Forward

Probeb
Reversec
Forward

Probeb
Reverse


TGG TTT GTG GGA GGA ATA GTG

AGT ACA AGA CAC CGA TGG CCC AC
CAA CAC GGA GTA GCA GAT C
TCT AAC CCA GTC TCC CCT C
CCT CCT GCA AGT TCC TGT GGC T
GGG ACC AAA GCT GAA CAA TG
ACA CAT GTC TGA GCC ACC
FAM/TCT ATG CAT GAA AGA GTC TGC CTC
CA
TTT CCC GGA TGA CCT TCA
CAA GAT GAC CAG CAC AGT

TGA TCC CAG AAC TCT AAA AGA TGT CCA
GC
GTT AGT GGC TGC GAT TGA
CTG ACA GAT TTG ATG CAG AAG TG

AAG GGC TTT CTT GGT GTT TAT TCG GC


GCT CGG ATT CAT GTC TAT ACC C

TGA TGT GGC CTT CTA CCT CGC TG
AAC AGC ACC TCC AAT AAG GG









Table 3-1. Continued

Gene Name Accession Primer/Probe Sequence (5'-3')

Tumor necrosis factor (ligand) NM_001025207 Forwarda GCA AAA CTG ACC ATC CTG AAT C
superfamily, member 8 (TNFSF8)
Probeb CTG CCG GAA CTG AGA CTG ACA ATA AGA
C
Reversec CTG ACG GGA AAC AAA AGC TG
Vestigial like 1 (Drosophila) XM_872858 Forwarda AGC TCT GGG CAA TGT CAA G
(VGLL1)
Probeb AGT GGC GTT TCT CTG CTC CGT
Reverse GCA AAA GAT ACT TCC GGC TG
Wingless-type MMTV integration NM_016087 Forwarda GAG GTG TGA AAG CAT GAC TG
site family, 16 isoform 1 (WNT16)
Probeb TGC CGC TGC CGT CTT CAG ATA A
Reverse GAA GAG AGA AGT GCT GGA AGG
Mitochondrial ribosomal protein NM_01077101 Forwarda TCT AGA AGT AGC TGG TCT GGG
S12 (MRPS12)
Probeb CTA TGG GTT AAG GGT CCA CTG GGC
Reversec GAT GGC AGT ACA GAG TCT TGT C


aForward=sense (5') primer
bEach probe was synthesized with a 5' 6-FAM
CReverse=antisense (3') primer


reporter dye and 3' IA-Black quencher









Table 3-2. Gene ontologies in the biological process category that were regulated by CSF2a.

Ontology Effect of Genes
CSF2


GO:0032502:
Developmental process



GO:0007275: Multicellular
organism development


GO:0048731: System
development


GO:0048856: Anatomical
structure development


GO:0007389: Pattern
specification process


Up

Down


Up

Down

Up

Down

Up

Down

Up

Down


MAB21, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTCH2, PCDH1, PTPN2,
CD2, RTN4, MYF6, FHL1, SFRP4, SLC26, FGD3, TNFSF8
CYLC1, CELSR, ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2,
PHGDH, GLIS2, GYPC, PTGER, HUNK, CREM, WNT16, BOLL, CHURCH,
RIPK3, CENPI, MADD, NOD2, RNF7, GPR14, DAPK1
MAB21, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTCH2, PCDH1, PTPN2,
CD2, RTN4, MYF6, FHL1, SFRP4
CYLC1, CELSR2, ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2,
PHGDH, GLIS2, GYPC, PTGER, HUNK, CREM, WNT16, BOLL, CHURCH
MAB21L, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTCH2, PCDH1, PTPN2,
CD2, RTN4, MYF6, FHL1, SLC26, FGD3
ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2, PHGDH, GLIS2,
GYPC, PTGER4,
MAB21L, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTCH2, PCDH1, PTPN2,
CD2, RTN4, MYF6, FHL1, SLC26, FGD3
ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2, PHGDH, GLIS2,
GYPC, PTGER4
HOXA5, NOTCH2, MYF6

GREM1, ROR2









Table 3-2. Continued

Ontology Effect of Genes
CSF2


GO:0048518: Positive
regulation of biological
process


GO:0007165: Signal
transduction


GO:0007154: Cell
communication


GO:0007166: Cell surface
receptor linked signal
transduction


Up


Down


Down


Down


Down


CD2, MASP2, MYF6, NOTCH2, PTPN2, RIPK3, RNF7, TNFSF8


BOLL, CHURCH, DAPK1, GLIS2, NOD2, STUB1, VAV1


CASP7, CD2, CCRL, FGD3, GIPR, MARK, NOTCH2, NXPH4, PDE3B,
PRKAR, PTPN2, PDE7B, PGR, PYGO1, OR51E,TNFSF8
CREM, CELSR2, CXCL2, CXCL1, CSNK2, DTX3, DAPK1, ECE2, FYB,
GPR14, HUNK, ITPR2, LRRN2, MADD, MPP2, RIPK3, NOD2, OR2T1, PLD2,
STUB1, TBCID2, VAV1, WNT16, SFRP4, PTGER, RALGP, ROR2, RGS12
CASP7, CD2, CCRL1, ECE1, FGD3, GIPR, MARK, NOTCH2, NXPH4,
PDE3B, PRKAR, PTPN2, PDE7B, PGR, PYGO1 SFRP4, TNFSF8, OR51E
CREM, CELSR2, CXCL2, CXCL1, CSNK2, DTX3, DAPK1, ECE2, FYB,
GREM1, GPR14, HUNK, ITPR2, LRRN2, MADD, MPP2, NOD2, OR2T1,
PLD2, STUB1, VAV1, WNT16, PTGER, RIPK3, RALGP, ROR2, RGS12
CD2, CCRL1, GIPR, NOTCH2, NXPH4, PTPN2, PYGOI, SFRP4, OR51E


CELSR2, CXCL2, CXCL1, CSNK2, DTX3, ECE2, GPR14, MADD, OR2T1,
PLD2, STUB1, VAV1, WNT16, PTGER, ROR2, RGS12









Table 3-2. Continued

Ontology Effect of Genes
CSF2

GO:0012502: Induction of Up CD2, TNFSF8, NOTCH2
programmed cell death
Down RNF7, DAPK1, RIPK3
GO:0043065: Regulation Up CD2, TNFSF8, NOTCH2, RTN4
of apoptosis
Down RNF7, DAPK1, RIPK3, MADD, NOD2
GO:0022610: Biological Up CLDN2, IBSP, PCDH15, CD2, THBS3
adhesion-cell adhesion
Down CELSR2, LRRN2, VAV1, F5, OLFM4, ROR2, CXCL12, PPFIBP1
GO:0048869: Cellular Up TNFSF8, SFRP4, CASP7, MYF6, NOTCH2, PCDH1, FHL1, CD2, RTN4,
developmental process- PTPN2
cell differentiation
Down CYLC1, CREM, ECE2, NOD2, DAPK1, RIPK3, BOLL, SEMA4, FEZF1,
MADD, ROR2, GLIS2, RNF7


a Gene ontologies are from the


David Bioinformatics Database GO and were those where the p value 5 0.05









Table 3-3. Differentially-regulated genes involved in WNT signaling.
Least-squares
mean
Fold P-
Symbol CSF2 Control Change value Role in WNT signaling Reference
Upregulated


SFRP4/FrpHE

NOTCH2

PDE7B

PYGOI

PPP2R3A
(PP2A)
PCDH24

Downregulated

WNT16

CSNK2B (CK2)




ROR2

CELSR2
(Flamingo)


13.07 3.26

84.22 38.10

4.88 2.89

135.6 82.08

28.53 18.09

320.2 211.28


3.76 14.28

119.2 256.51




34.89 63.48

206.1 316.26


4.00 0.04 Binds to and inhibits WNT

2.21 0.03 Blocks induction of WNT-induced
genes
1.69 0.02 Promotes p-catenin independent
and dependent signaling
1.65 0.01 Nuclear cofactor for p-catenin

1.58 0.04 Phosphatase that inhibits p-catenin
dependent signaling
1.52 0.01 Protocadherin that inhibits activation
of p-catenin


-3.80 0.01 WNT ligand for p-catenin dependent
and independent pathways
-2.20 0.02 Enhances activation of p-catenin
dependent transcription; enhances
activation of planar cell polarity;
inhibits RAC-1 mediated effects of
WNT
-1.80 0.01 WNT receptor or co-receptor for
planar cell polarity pathway
-1.60 0.01 Cadherins that interact with WNT
and activate planar cell polarity
signaling


Chien et al., 2009

Walsh and Andrews, 2003

Ahumada et al., 2002; Li et
al., 2002
Jessen et al., 2008

Creyghton et al., 2005

Ose et al., 2009


Mazieres et al., 2005; Binet
et al., 2009
Wang and Jones, 2006;
Bryja et al., 2008



Chien et al., 2009

Saburi et al., 2005









Table 3-4. Differentially-regulated genes involved in apoptosis.


Symbol


Least-squares mean

Fold
CSF2 Control Change P-value Role in apoptosis


Symbol
Upregulated
NOTCH2
CD73/NT5E
PGR
TNFSF8
(CD30)
CASP7
PRKAR2B
RTN4

Downregulated
MADD
RIPK3
PIK31P1

CXCL12

NOD2
DAPK1
CREM
RNF7
PLD2


84.22
27.11
63.08
64.96

82.11
182.85
492.97


8.18
43.02
34.58
75.16
5.49
20.42
53.9
95.80
308


38.10
12.20
34.46
37.64

49.07
119.97
311.20


29.55
85.65
62.71
138.34
9.77
31.88
83.1
155.50
493.9


2.21
2.22
1.83
1.73

1.67
1.52
1.58


-3.60
-2.00
-1.80
-1.80
-1.80
-1.60
-1.60
-1.60
-1.60


0.03
0.03
0.05
0.03

0.03
0.04
0.02


0.03
0.03
0.01
0.02
0.05
0.02
0.02
0.03
0.02


anti/pro-apoptotic
anti-apoptotic
anti-apoptotic
anti/pro-apoptotic

pro-apoptotic
anti-apoptotic
pro-apoptotic


pro-apoptotic
pro-apoptotic
pro-apoptotic
anti-apoptotic
pro-apoptotic
pro-apoptotic
pro-apoptotic
anti-apoptotic
anti-apoptotic


















A W2 .. B CM CM












NS S S S -
0 ) 0 ) 0 ) 09
o o o o 0 0 0 0
0 0 0 0




Figure 3-1. Genes expressed in control and CSF2-treated embryos at Day 6 of
development. Shown in panel A is a Venn diagram of genes expressed in
embryos of both treatments (brown), only in control embryos (red) or only in
CSF2-treated embryos (green). Intensity cutoff for expression = 2.8. Shown
in panel B is the hierarchical cluster of differentially-expressed genes.




















100











0
WNT16

5

04
Q-. 4


S3
8 PPP2R3A
0
5 2
cc TNFSF8 0
U 0 RIPK3 SLC16A10
1 ANKRD37 NOTCH2
'- PMM2 *
cc oHK1 0 *
-- ECE1 MAB21L2
O
^-o 0 CSNK2B
0 MASP2
OVGLL1
MRPS12
-1 -
MADD

-2
-4 -3 -2 -1 0 1 2 3 4
Fold change relative to control microarray




Figure 3-2. Validation of microarray results using quantitative PCR. Shown are the
fold-change increases (positive) or decreases (negative) in expression
caused by CSF2 as determined by microrarray hybridization (x-axis) and
qPCR 9y-axis). Probability values for CSF2 effects in the PCR analysis are
designated by the color of the circle (red, P<0.001; black, P=0.09; open,
P>0.10).


101












20 0
175 E
150
0_ 125
4 C100
O0) 75
0 50
25
00
120
I 100
E 80
60
0 40
20
0
38.5 42.0 38.5 42.0
Control CSF2


Figure 3-3. Regulation of heat-shock induced apoptosis in Day 6 bovine embryos by
CSF. Panels A-D are representative photomicrographs illustrating the
frequency of apoptotic nuclei in embryos at 38.5C (A, D) and 42C (B, E). as
affected by heat shock at 42C and treatment with colony stimulating factor 2
(CSF2). Detection of apoptosis was by TUNEL analysis using TMR red-
conjugated dUTP to identify apoptotic nuclei (red) and Hoescht 33342 to
identify all nuclei (blue). Note that exposure of embryos to 42C for 15 h (B)
increased the frequency of TUNEL-positive cells compared with embryos
cultured at 38.5C (A). In the presence of CSF2, however, there was no
difference in TUNEL labeling between embryos cultured at 38.5C (C) or
42C (D). Panel E shows least-squares means for the percentage of cells that
were apoptotic (top panel) and total cell number (bottom panel). Data
represent results from 65-81 embryos per treatment. Heat shock at 42C for
15 h increased the proportion of cells that were TUNEL-positive (P<0.0001)
and decreased the total cell number (P<0.0001). Treatment with CSF2
decreased the percentage of cells that were TUNEL-positive (P<0.005) but
did not affect total cell number.


102


3-i r.


A20o









CHAPTER 4
CONSEQUENCES OF EMBRYONIC EXPOSURE TO CSF2 FROM DAY 5 TO 7
AFTER INSEMINATION ON TROPHOBLAST ELONGATION, INTERFERON-TAU
SECRETION AND GENE EXPRESSION IN THE EMBRYONIC DISC AND
TROPHECTODERM

Introduction

Colony-stimulating factor 2 (CSF2) is an important regulator of embryonic

development in the cow. It improves the proportion of cultured embryos that develop to

the blastocyst stage (de Moraes and Hansen 1997a; Chapter 2), increases the number

of cells in the inner cell mass (Chapter 2), and decreases the percentage of

blastomeres undergoing apoptosis in response to heat shock (Chapter 3). Moreover,

addition of CSF2 to culture medium Days 5 to 7 after insemination improved the

competence of in vitro produced embryos to establish pregnancy after transfer to a

recipient female while also reducing the probability of fetal loss after Day 30-35 of

pregnancy (Chapter 2).

The primary objective of this study was to evaluate possible mechanisms by which

CSF2 acts during Day 5 to 7 of development to improve embryonic and fetal survival.

One hypothesis was that CSF2 causes increased secretion of interferon-tau (IFNT) by

the trophoblast of elongated conceptuses. CSF2 has been shown to stimulate

production of IFNT by sheep trophoblast (Imakawa et al. 1993; Imakawa et al. 1997;

Rooke et al. 2005) and by a bovine trophoblast cell line (CT-1 cells) (Michael et al.

2006). CSF2 might also affect elongation of the trophoblast because of evidence that

treatment of embryos from Day 5-6 with CSF2 caused differential regulation of a large

number of genes involved in developmental processes (Chapter 3). Analysis of the

genes involved in developmental processes regulated by CSF2 indicates a propensity

for CSF2 to upregulate genes involved in mesoderm formation or differentiation while


103









decreasing expression of genes involved in neurogenesis (Chapter 3). This finding

raises the possibility that CSF2 causes changes in gastrulation that could be reflected in

changes in gene expression in the embryonic disk or trophoblast. A large proportion

(~25%) of in vitro produced embryos that survive to Day 14-15 of gestation lose the

embryonic disk (Fischer-Brown et al. 2004; Fischer-Brown et al. 2004; Block et al. 2007)

and CSF2 might act to improve survival of the embryonic disc, either because of its

antiapoptotic actions (apoptosis being more frequent in inner cell mass) (Knijn et al.

2003; Fouladi-Nashta et al. 2005; Pomar et al. 2005) or because of regulation of genes

involved in developmental processes.

As part of this study, methods were developed to divide the elongated concepts

into 1) the embryonic disk and a small amount of adjacent trophoblast and 2) tissue

containing trophoblast only. As a result, over 500 genes were identified that were

preferentially expressed in the embryonic disk. These genes, or the proteins they

encode, represent candidates markers for embryonic disk that should prove useful for

studying the differentiation of the bovine concepts through the periattachment period.

Materials and Methods

In vitro Production of Embryos

Embryo production was performed as described in Chapter 3 with modifications

described below. Cumulus oocyte complexes (COCs) from ovaries from a mixture of

beef and dairy cattle were collected in Tissue Culture Medium-199 (TCM-199) with

Hank's salts without phenol red (Hyclone, Logan UT) supplemented with 2% (v/v)

bovine steer serum containing 2 U/ml heparin (Pel-Freez, Rogers, AR), 100 U/ml

penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocytes were allowed to

mature for 20-22 h in groups of 10 in 50 [l microdrops of TCM-199 (Invitrogen,


104









Carlsbad, CA, USA) with Earle's salts supplemented with 10% (v/v) bovine steer serum,

2 [Lg/ml estradiol 17-P, 20 [Lg/ml bovine follicle stimulating hormone (Folltropin-V;

Belleville, ON, Canada), 22 [Lg/ml sodium pyruvate, 50 [Lg/ml gentamicin sulfate, and 1

mM glutamine. Matured oocytes were then washed in HEPES-TALP (Parrish et al.

1986) (Caisson, Sugar City ID, USA) and transferred in groups of 50 to four-well plates

containing 600 pL of IVF-TALP supplemented with 25 pL PHE (0.5 mM penicillamine,

0.25 mM hypotaurine, and 25 pM epinephrine in 0.9% [w/v] NaCI), and fertilized with 30

pL Percoll-purified spermatozoa (~ 1x106 sperm cells). Sperm were prepared from a

pool of frozen-thawed semen from three different bulls; a different set of bulls was

generally used for each replicate). Fertilization proceeded for 18-20 h at 38.50C in a

humidified atmosphere of 5% (v/v) CO2 in humidified air. Putative zygotes were

removed from fertilization plates, denuded of cumulus cells by vortexing in HEPES-

TALP, and placed in groups of 30 in 45-tl microdrops of KSOM-BE2 (Soto et al. 2003).

Embryos were cultured at 38.50C in a humidified atmosphere of 5% CO2 or 5%

CO2, 5% 02, and 90% N2 (v/v). Cleavage rate was assessed at Day 3 after

insemination. At Day 5 after insemination, 5 pl of KSOM-BE2 or 5 pl of KSOM-BE2

containing 100 ng/ml recombinant bovine CSF2 (a gift from Novartis, Basle Switzerland)

were added to each drop to achieve a final CSF2 concentration of 0 or 10 ng/ml.

Transfer Into Recipients

Morula, blastocyst and expanded blastocyst stage embryos (6, 9 and 5 in the

control group and 5, 5 and 5 in the CSF2 group, respectively) classified as Grade 1

(Robertson and Nelson, 1998) were harvested on Day 7 after insemination and loaded

into 0.25 ml straws in 250 pl HEPES-TALP supplemented with 10% (v/v) fetal calf


105









serum and 50 pM dithiothreitol (Sigma-Aldrich, St. Louis MO, USA). Straws containing

selected embryos were then placed horizontally into a portable incubator (Cryologic,

Mulgrave, Vic, Australia) at 38.50C and transported to the farm.

Thirty five multiparous lactating Holstein cows were used as recipients. Cows were

housed in a free-stall barn equipped with fans and a sprinkler system at a commercial

dairy in Bell, Florida (29.75578N, 82.86188W). Overall, 4 replicates were completed

with 5-11 recipients per replicate from June to October of 2009. For each replicate,

eligible cows were synchronized for embryo transfer using the Ovsynch-56 procedure

(Chapter 2). Day 0 was considered the Day of expected ovulation. Hormone treatments

consisted of 100 pg gonadotrophin releasing hormone (GnRH; Merial; Duluth, GA, USA)

i.m. on Day -10; 25 mg PGF2a (Pfizer; New York, NY, USA), i.m. on Day -3; and 100 pg

of GnRH i.m. 56 hours after PGF2a. Cows were diagnosed for the presence of a corpus

luteum (CL) at Day 7 after anticipated ovulation (Day of ovulation was considered Day

0) using an Aloka 500 ultrasound equipped with a 5 MHz linear array transducer. Cows

diagnosed with a corpus luteum were given epidural anesthesia [5 ml of 2% (w/v)

lidocaine] and a single embryo transferred to the uterine horn ipsilateral to the ovary via

the transcervical route.

Embryo Recovery and Evaluation

At Day 15 after expected ovulation, the ovary ipsilateral to the uterine horn that

received the embryo was examined using ultrasonography to confirm the presence of

the corpus luteum. Cows that did not have a visible CL were not flushed. Embryos were

recovered transcervically with a 20 French Foley catheter inserted with a stainless steel

stylet and held in position by inflating the cuff at the end of the catheter. The uterine

horn ipsilateral to the CL was flushed with DPBS (Sigma-Aldrich St. Louis MO, USA)


106









with 1% (v/v) polyvinyl alcohol (PVA). Flushing involved multiple injections and recovery

of 60 ml DPBS using a 60 cc syringe attached to the Foley catheter. The procedure was

continued until either an embryo was identified in the flush or 180 to 240 ml of DPBS-

PVA had been flushed. The flushings recovered from the first 60 ml flush were

centrifuged at 500 rpm for 10 min and stored at -200C for analysis of antiviral activity.

Following recovery, each embryo was assessed for length, stage, and the

presence or absence of an embryonic disc (ED) by light microscopy using a

stereomicroscope. Stage of development was classified based on shape as ovoid,

tubular, or filamentous. After all measurements were recorded, embryos were washed

once in DPBS-PVA and the embryo was dissected to produce a piece of tissue

containing the ED and some nearby trophoblast (termed ED) and two pieces of

trophoblast (Tr) that were on either end of the ED (Figure 4-1). Tissues were

immediately snap frozen separately in liquid nitrogen.

Antiviral Assay

The quantity of biologically active IFNT in uterine flushings was determined

indirectly using an antiviral assay based on the inhibition of vesicular stomatitis virus

(VSV) induced lysis of Madin-Darby bovine kidney (MDBK) cells. The assay was

performed as described by Micheal et al. (2006). Briefly, 1:3 serial dilutions of the

uterine flushings was performed using DMEM containing 10% (v/v) FBS in 100 pl

volume in 96 well plates. MDBK cells were resuspended in DMEM-FBS and 50 pl of cell

preparation (~500.000/ml) was added to each well. The plate was incubated at 37C

and 5% C02 in air for 24 h. Medium was then aspirated from wells and VSV diluted in

DMEM (without FBS) was added to each well. The plate was incubated for 1 h at 370C.


107









The VSV solution was aspirated, replaced by 100 [l DMEM-FBS and the plate was

incubated for 24 h at 37C. After aspiration of medium, cells were fixed with 70% (v/v)

ethanol and stained with 0.5% (w/v) gentian violet stain in 70% (w/w) ethanol (Fisher

Scientific, Pittsburgh, PA, USA). Plates were washed two times in water and allowed to

dry. Antiviral activity of each sample was assessed visually based on inhibition of lysis

of MDBK cells. The antiviral activity (units/ml) was defined as the dilution of flushing that

reduced by 50% the destruction of the monolayer by virus. Antiviral units were

converted to IFN (IU/ml) using a standard curve with known amounts of human IFN-a

standard included in the assay (EMD Biosciences, San Diego, CA). All samples (from

cows with and without an embryo) were analyzed in duplicate.

Analysis of the Transcriptome of Trophectoderm and Embryonic Disc

Microarray analysis was performed using a subset of 8 ED and 8 Tr samples that

were randomly selected from among the filamentous embryos. Tissues were disrupted

by vortexing for 30 seconds and passed through a 20 gauge needle. Samples were

homogenized using a QIAshredder (Qiagen-lnc, Valencia,CA, USA). Total cellular RNA

was extracted with the RNeasy Plus Micro kit (Qiagen-lnc) following manufacturer's

instructions. Concentration of the input RNA was determined by Nanodrop 2000

spectrophotometer (Thermo Scientific, Waltham, MA) and RNA integrity was

determined by use of the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa clara,

CA, USA). Only samples that showed high RNA integrity (RIN > 7) were used for the

microarray hybridization and quantitative PCR analysis. Extracted RNA was stored at -

800C until microarray analysis.


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Microarray Hybridization

Procedures were performed by the University of Florida Gene Expression Division

of the Interdisciplinary Center for Biotechnology Research. Labeled aRNA was

generated using Amino Allyl MessageAmp II Amplification kit (Ambion Inc, Austin, TX,

USA). First strand cDNA was synthesized from 425 ng of total RNA. Half of the first

strand cDNA was used to generate labeled aRNA according to the manufacturer's

protocol and the remainder was reserved for quantitative PCR (qPCR) verification. The

microarray analysis was performed using the Bos taurus Two Color Microarray Chip

from Agilent v 2 (Agilent Technologies, Santa Clara CA, USA). The 16 samples were

distributed between the 2 microarray chips and the two dyes to avoid location biases. A

total of 825 ng of labeled aRNA per sample was used for the hybridization. Hybridization

and washing were performed according to the manufacturer's protocol using the Gene

Expression Hybridization Kit and Gene Expression Wash Buffers (Agilent). Arrays were

scanned using a dual-laser DNA microarray scanner (Model G2505C, Agilent).

Analysis of Microarray Data

The microarray images were first analyzed with Agilent Feature Extraction

Software v10.1 (Agilent Technologies, Inc). Spot signal intensities were adjusted by

subtracting local background and normalizing using within-array lowess approach for

dye-bias correction. The quantile approach was then used for between array

normalization. Statistical tests were performed using BioConductor statistical software

(http://www.bioconductor.org/), which is an open source and open development

software project for analysis of microarray and other high-throughput data based

primarily on the R programming language (Gentleman et al. 2004).


109









Differentially expressed genes were identified using Limma, a software package

that implements linear models for microarray data (Smyth et al. 2005).In Limma, p-

values are obtained from moderated t statistics or F statistics using empirical Bayesian

methods. The primary analysis comparing differential expression was a 2 x 2 factorial

design with tissue (ED, Tr) and treatment (control, CSF2) as main effects and the tissue

x treatment interaction as another effect. Pairwise comparisons of interest were also

performed to test for significant differential expression. The Benjamini-Hochberg

procedure (Benjamini and Hochberg 1995) was used to control false discovery rate

(FDR) at the 0.01 level. Genes meeting this statistical threshold and showing a fold

change equal or greater than 2 were considered as differentially expressed.

Ingenuity pathway analysis software (http://www.ingenuity.com) was used to

identify canonical pathways associated with differentially expressed genes. Fisher's

exact test was used to determine the probability that the association between the genes

and the pathway was explained by chance alone.

Quantitative Real Time PCR

Quantitative Real Time PCR analysis (qPCR) of 11 differentially-expressed genes

and one housekeeping gene (GAPDH) was performed to confirm microarray results.

Specific primers (Table 4-1) were designed using Integrated DNA technologies software

(http://idtdna.com). The SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules,

CA, USA) reaction chemistry and a CFX 96 Real-Time PCR Detection System (Bio-

Rad) were used to quantify mRNA concentrations. After an initial activation step (950C

for 30 sec), 45 cycles of a two-step amplification protocol (950C for 5 sec; 600C for 5

sec) were completed.


110









The amplification of a single product was verified by performing dissociation curve

analysis (65-950C) in the thermocycler. In additional, agarose gel electrophoresis of the

PCR product was performed. Identity of the amplicon was confirmed by sequencing the

PCR product of the band product. The DNA was extracted from the gel fragment using

the PureLink Quick gel Extraction and PCR Purification Combo Kit (Invitrogen Carlsbad,

CA, USA). First, the gel with the DNA fragment was dissolved using Solubilization

Buffer for 15 min at 500C. The gel mixture was centrifuged for 1 min at 10.000 x g in a

PureLink clean up spin column. After a wash, the DNA was extracted from the tube

membrane by centrifuging the column with Elution Buffer for 1 min at 10.000 x g. The

purified DNA was sequenced by the University of Florida Gennomics Division of the

Interdisciplinary Center for Biotechnology Research.

Each sample was analyzed in duplicate reactions. All CT values for genes of

interest were normalized to the housekeeping gene GAPDH using the ACT method.

The AACT for each sample was calculated by subtracting the average ACT of the gene

of interest for control embryos from the value for each individual embryo. Fold change

was determined by solving for 2-AACT

Statistical Analysis

Categorical data were analyzed by logistic regression using the LOGISTIC procedure of

the Statistical Analysis System (SAS 9.1.3, SAS Inst., Cary, NC, USA) with backward

selection (P=0.2). The statistical model included replicate, stage of the embryo at the

time of transfer, and treatment. Effects on continuous variables were analyzed by least-

squares analysis of variance using the General Linear Models procedure of SAS.

Various mathematical models were utilized. All included treatment and, in addition,

included replicate, stage of the embryo at the time of transfer, stage of the embryo at


111









Day 15 and, for embryo length, whether the embryo was intact. Data for embryo length

and antiviral activity were subjected to log transformation before analysis to account for

heterogeneity of variance. In addition, these data were analyzed using the Wilcoxon's

and median nonparametric tests with the NPARWAY1 procedure of SAS.

Results

Embryo Survival After Transfer

Results are presented in Table 4-2. A total of 80% of cows receiving a control

embryo and 93% of cows receiving a CS2F-treated embryo had a CL detected by

ultrasound. For the other cows, the CL was either absent or small and regressing. Cows

without a CL represent either cows that were not successfully synchronized, so that the

presumed Day 15 was actually later in the estrous cycle, or cows in which the CL had

undergone luteolysis by Day 15. When all cows were considered, including those

without a large corpus luteum, there was a tendency (P=0.07) for the proportion of cows

with a recovered embryo to be higher for those receiving a CSF2 treated embryo (35%

for control vs. 66% for CSF2). The same trend was apparent when only cows with a CL

were considered (44% for control vs 71% for CSF2) but the difference did not approach

significance.

One cow in the control group did not have a detectable embryo but there was

abundant antiviral activity in uterine flushings (59.049 IU IFN/ml). This cow either lost its

pregnancy after the embryo initiated large scale IFNT secretion or the embryo was not

recovered in the flushing. When this cow was considered pregnant, differences in

survival between control and CSF2 groups remained but did not approach significance

(Table 4-2).


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Embryonic Growth and Development

Five of 7 control embryos (71%) and 8 of 10 (80%) CSF2-treated embryos were

filamentous. The embryonic disc was visible in 6 of 7 (83%) of control embryos and 6 of

10 (60%) of CSF2-treated embryos. Differences between treatments in these two

variables were not significant.

As illustrated in Figure 4-2, there was great variability in embryo length, even if the

dataset was restricted to embryos that were filamentous. While there was no significant

effect of treatment, CSF2-treated embryos tended to be longer than control embryos

regardless of whether all embryos were considered, only those recovered intact were

considered (4 embryos were recovered in pieces and were likely to be larger than

measured), or only filamentous embryos were recovered. Overall, the average length

was 39 mm for control embryos and 62 mm for CSF2-treated embryos.

Antiviral Activity in Uterine Flushings

Antiviral activity was also highly variable, ranging from non-detectable amounts

(one cow in each group) to almost 9,000,000 IU IFN/ml (Figure 4-2). For all cows, there

was a nonsignificant trend for antiviral activity to be higher for cows receiving CSF2-

treated embryos than for cows receiving control embryos. This difference approached

significance (P=0.07) if only cows with detectable antiviral activity were considered.

Changes in the Transcriptome of Embryonic Disc and Trophectoderm

To test the hypothesis that CSF2 alters the transcriptome of the filamentous

embryo, gene expression was evaluated in two tissues embryonic disc (which also

contains adjacent trophoblast) and trophoblast using microarray technology. All 43.803

probes on the array produced signal above background. These probes represent

19,500 distinct genes or 88% of the bovine genome (btau 4.0). Hierarchical analysis


113









(Figure 4-3) indicated that the 8 ED samples formed a distinct cluster separated from

the Tr. There was, however, no such distinction between samples from control and

CSF2-treated embryos. Using a FDR 50.01 and a 2-fold difference as criteria, there

were no genes affected by treatment or the treatment x tissue type interaction. There

was, however, a total of 627 genes that were differentially expressed between ED and

Tr and 576 of these could be annotated. Of the annotated genes, 538 genes were

upregulated in ED and 38 genes were upregulated in Tr. One would expect a

preponderance of differentially regulated genes that were upregulated in ED, and not in

Tr, because the former tissue contains some Tr while the Tr sample was free of ED

tissue.

Characteristics of Genes Differentially Expressed Between Embryonic Disc and
Trophoblast

Ingenuity software was used to identify pathways that were significantly

overrepresented in the set of genes that were differentially expressed between ED and

Tr. Determination of significant pathways is based on the ratio of the number of genes in

the differentially expressed dataset divided by the number of genes in the pathway and

the P-value. A total of 31 canonical pathways were identified as above the threshold

(P<0.05; Table 4-3). The pathway with the highest score (smallest P-value) was

Embryonic Stem Cell Pluripotency. Of the 148 genes in this pathway, 16 (RAC2,

NODAL, FGF2, FGFR1, SMAD6, FZD1, LEFTY2, TCF7, SOX2, NANOG, SMO,

PDGFRA, BMP6, WNT11, POU5F1, FZD7) were represented in the list of differentially

expressed genes. Other pathways related to pluripotency were also significant,

including Role of Oct4 in Mammalian Embryonic Stem Cell Pluripotency, with 7 of 45


114









genes in the pathway being differentially expressed, and Role of NANOG in Mammalian

Embryonic Stem Cell Pluripotency, with 9 of 114 genes being differentially expressed.

Several pathways that promote cellular differentiation were identified including

pathways for Factors Promoting Cardiogenesis in Vertebrates, responsible for

differentiation of the mesoderm into myocardium and endocardium, with 11 of 89 genes

in the pathway being differentially expressed, Wnt/p-catenin Signaling (responsible for

many differentiation events including cell polarity, neural tube patterning and

cardiogenesis), with 15 of 168 genes being differentially expressed and Axonal

Guidance Signaling, responsible for formation of neuronal connections, with 22 of 403

genes being differentially expressed.

Another type of pathway that was well represented was for cell signaling. Among

these were pathways for Basal Cell Carcinoma Signaling, with 8 of 68 genes being

differentially expressed, Ovarian Cancer Signaling, with 10 of 135 genes, Leukocyte

Extravasation Signaling, with 12 of 194 genes, Prolactin Signaling, with 6 of75 genes,

CTLA4 Signaling in Cytotoxic T Lymphocytes, with 7 of 94 genes, G-Protein Coupled

Receptor Signaling, with 12 of 220 genes, HER-2 Signaling in Breast Cancer, with 6 of

79 genes, TGFB Signaling, with 6 of 83 genes, Acute Phase Response Signaling, with

10 of 178 genes, and Colorectal Cancer Metastasis Signaling, with 12 of 249 genes

represented.

Identification of Likely Candidate Genes for Use as Embryonic Disc Markers

It is likely that many of the genes that are differentially expressed between ED and

Tr will prove useful in future studies for identifying ED and Tr. Two different criteria were

used to identify some of the 576 differentially expressed genes that might be particularly


115









useful as markers. The first criterion was to identify genes with the highest fold change

between ED and Tr. The 15 genes with the highest fold difference for genes

overexpressed in ED and the 15 genes with the highest fold difference for genes

overexpressed in Tr are shown in Table 4-4. The fold difference for the 15 highest

genes overexpressed in ED ranged from 120-fold to 45-fold, with the most

overexpressed gene being fatty acid binding protein 1 (FABP1). Other genes with large

fold increase for ED compared with Tr were transforming growth factor-p induced

protein IG-H3 (TGFBII) (95.2 fold increase), sepallata3 (SEP3) (89.9 fold increase) and

nanog homeobox (NANOG) (75.7 fold increase). In contrast, the fold-difference for the

15 highest genes overexpressed in Tr ranged from -8.3 to -2.5, with the greatest fold

change for ribosomal protein L36a (RPL36A) (-8.3 fold), ribosomal protein L10a (-5.2

fold) RAR-related orphan receptor B (RORB) (-4.7 fold).

The second approach to identify candidate gene markers was to identify the

differentially expressed genes that were the most abundant. The rationale was that

abundant genes, or their protein products, would be more easy to identify using a

variety of molecular and immunochemical procedures. A list of the 15 most abundant

genes for ED and Tr, based on signal intensity on the microarray, is presented in Table

4-5.

Validation of Microarray Results Using qPCR

A total of 11 genes that were differentially expressed between ED and Tr based

on microarray analysis, were subjected to analysis by qPCR. For all 11 of the genes,

the fold change for ED relative to Tr was in the same direction for qPCR as the fold

change as determined by microarray hybridization. Differences between ED and Tr


116









were significant for 10 of the 11 genes, the exception being GJC1. For 7 genes

(BMPER, CLDN11, FGF2, GATA5, GDF3, IGFL1 and NODAL), the fold-change was of

greater magnitude as determined by qPCR than as determined by microarray

hybridization. There was no effect of treatment (control vs CSF2) or treatment x tissue

interaction on mRNA abundance as determined by qPCR.

Discussion

Exposure of bovine embryos to CSF2 from Day 5 to 7 of development can have a

profound effect on the subsequent developmental fate of the embryo. In particular, a

greater percentage of embryos result in pregnancies at Day 30-35 of pregnancy and

fewer of the pregnancies established at that point are lost thereafter (Chapter 2). The

purpose of this experiment was to understand the mechanism by which CSF2 increases

embryonic survival as measured at Day 30-35 and reduces fetal mortality thereafter.

Results suggest that higher pregnancy rates at Day 30-35 represent increased

embryonic survival before Day 15 and a greater capacity of the embryo to elongate and

secrete IFNT at Day 15. Analysis of gene expression in filamentous embryos indicates

little difference in transcription among this subset of embryos that survived to Day 15

and elongated successfully. Therefore, the reduction in embryonic and fetal loss after

Day 30-35 caused by CSF2 is probably not a direct reflection of altered gene

expression at Day 15.

Conclusions regarding embryonic survival, growth and IFNT secretion at Day 15

must be tentative because differences between control and CSF2 were largely non-

significant. Nonetheless, it seems more likely that these traits were affected by CSF2.

The proportion of transferred embryos that were recovered at Day 15 was twice as high

for cows receiving CSF2 embryos (35% in control vs 66% in CSF2; P<0.07). Embryonic


117









length and antiviral activity in the uterus (a measure of IFNT bioactivity) (Maneglier et al.

2008) were highly variable, as is typical at this stage of pregnancy (Bilby et al. 2004;

Bilby et al. 2006), but the largest embryos were in the CSF2 group and antiviral activity

among cows that had detectable activity (excluding cows with tubular embryos where

IFNT secretion would be low (Short et al. 1991) tended (P<0.07) to be greater for cows

receiving CSF2 embryos.

Based on these findings, it can be postulated that CSF2 improves embryonic

survival at two periods. The first is in the 8 Day period between transfer at Day 7 and

flushing at Day 15. Embryos treated with CSF2 have large inner cell mass at Day 7

(Chapter 2) and are less prone to undergo apoptosis in response to heat shock

(Chapter 3). The larger size of the inner cell mass and resistance to stress could

promote early survival of the embryo. The second period is the period of maternal

recognition of pregnancy, around Day 15-18, when the embryo acts to prevent luteolysis

through secretion of IFNT (Thatcher et al. 2001) The larger size and greater IFNT

secretary activity of the CSF2-treated embryos (as determined by uterine antiviral

activity) would make the CSF2 embryo better able to prevent luteolysis and allow

continued development of the embryo. Size of the embryo at this period does affect

IFNT secretion (Bilby et al. 2004; Bilby et al. 2006). Indeed, the trend for a lower

incidence of cows without functional CL (based on ultrasound) in cows receiving CSF2-

treated embryos may reflect the greater antiluteolytic capacity of the CSF2-treated

embryo.

The mechanism by which CSF2 treatment from Day 5-7 results in greater

embryonic growth and IFNT secretion at Day 15 is not known. However, CSF2 can


118









increase expression of IFNT in sheep trophoblast (Imakawa et al. 1993; Imakawa et al.

1997; Rooke et al. 2005) and bovine trophoblast cells (CT-1 cells) in vitro (Michael et al.

2006). In addition, embryos treated with CSF2 at Day 5 had altered expression of genes

involved in developmental processes at Day 6 (Chapter 3) characterized by an increase

in expression of genes involved in mesoderm, mesenchyme and muscle formation and

a decrease in genes involved in neurogenesis. Perhaps, effects of CSF2 on gastrulation

lead to increased growth of trophoblast between Day 7 and 15 of development.

A relatively large number of embryos at Day 14 -15 do not have a detectable

embryonic disc (Bertolini et al. 2002; Fischer-Brown et al. 2002; Fischer-Brown et al.

2004) and these embryos all eventually die (Fischer-Brown et al. 2004). Despite its

effects on inner cell mass number, however, CSF2 did not affect the incidence of

embryos without a disk and, numerically, the percentage of embryos without a disk was

lower in the CSF2 group. It can be concluded from these observations that CSF2 is not

improving embryonic survival by increasing the likelihood that the embryonic disc

survives to Day 15.

There was no effect of CSF2 on expression of genes in either the ED or Tr at Day

15. The embryos used for this analysis were all filamentous embryos. Thus, CSF2 has

no discernable effect on embryonic gene expression at Day 15 for the subset of

embryos that has developed normally up to that point. It is unlikely, therefore, that the

reduction in embryonic or fetal loss caused by CSF2 is the result of global changes in

transcription. Instead, perhaps, altered trajectory of development caused by CSF2, as

characterized by changes in expression of developmentally important genes at Day 6

(Chapter 3) could lead to formation of a fetus that is less likely to experience a


119









developmental defect leading to pregnancy loss after Day 35. It is also possible that

CSF2 causes epigenetic changes in the developing embryo that result in altered gene

expression at time points later than studied here. These changes would affect late

embryonic and fetal loss.

One benefit of the transcriptomal analysis was that a large number of genes that

are overexpressed in ED were identified. These genes represent good candidates for

markers of the embryonic disc. The developmental processes involved in

developmental events after embryonic hatching, including gastrulation and organ

development, are not well understood in cattle and is the subject of investigation by

several groups (Maddox-Hyttel et al. 2003; Alexopoulos et al. 2005; Tveden-Nyborg et

al. 2005; Vejlsted et al. 2006). Identification of markers of the embryonic disc could

simplify the study of post-hatching development. The genes that were differentially

regulated between ED and Tr included large numbers of pluripotency genes, genes

controlling differentiation and development and cell signaling genes. This is to be

expected given the nature of the developmental processes underway in the ED at Day

15. Three genes involved in pluripotency serve as examples of the functional

importance of the genes overexpressed in ED. NANOG was overexpressed 75 fold in

the ED. In the mouse and human blastocyst, NANOG expression is limited to inner cell

mass and epiblast and is necessary for pluripotency maintenance (Marikawa and

Alarc6n 2009; Guo et al. 2010). NANOG is downregulated in trophoblast by CDX2

(Strumpf et al. 2005). In the bovine, NANOG mRNA and protein are found in both inner

cell mass and trophectoderm of the blastocyst, although NANOG expression is greater

in the ICM and epiblast (Degrelle et al. 2005; Muioz et al. 2008). Another gene that


120









serves as a marker of ICM and epiblast in mouse, human, and bovine,SOX2 (Degrelle

et al. 2005; Guo et al. 2010), was upregulated 57 fold in the ED compared with Tr in the

present study. SOX2 is the first transcription factor to appear in the late morula and is a

key factor for maintenance of pluripotency and for reprogramming of differentiated cells

into induced stem cells (Takahashi and Yamanaka 2006). Finally, POU5F1, also known

as Oct4, was increased 29 fold in ED compared with Tr. Expression of POU5F1 is

limited to the ICM in human and mouse blastocysts (Zernicka-Goetz et al. 2009) but is

in both ICM and Tr of in vitro and in vivo produced bovine blastocysts through Day 10 of

development (Eijk et al. 1999; Kirchhof et al. 2000; Degrelle et al. 2005). POU5F1 is a

transcription factor necessary for the maintenance of the pluripotency in the ICM and it

prevents ICM transformation to TE (Zernicka-Goetz et al., 2009).

Some markers of trophoblast were also identified although technical limitations of

the procedure used to separate ED and Tr meant that many fewer of these were found

than for ED.

In conclusion, results support the idea that the increased survival of embryos

exposed to CSF2 from Day 5-7 of development is the result of increased embryonic

survival before Day 15 and a greater capacity of the embryo to elongate and secrete

IFNT at Day 15. The reduction in embryonic and fetal loss after Day 30-35 caused by

CSF2 is probably not a direct reflection of altered gene expression at Day 15.


121










used for qPCR.


used for qPCR.


Table 4-1. Primers
Accession #
NM_001077997

NM_001030318

NM_001035055

NM_174056

NM_001034221

NM_001025344

NM_001046076

BE664075

XM_609225

NM_176646

XM_001788143

NM_001034034.1


Product size
128 bp


Melting temp
77C


Name Sequence
BMPER F 5'-AGA GGA CTC CTA GTC CAA CAC TCT-3'
R 5'-GGA AAT GAG AGC AAG CAT GTA GAC C-3'
CAST F 5'-AGA GGT CTA TGT GTT CCG TGC AGT-3'
R 5'-ACA GGC TTT CCG TCT TCT GGA TCT-3'
CLDN11 F 5'-CAT TCT GTG AGC TGT CTT GAA GTG-3'
R 5'-ATC AGT GTT TGC ACC CGT AAA GCC-3'
FGF2 F 5'-TCT CTC GGG AAA CTG CTG ACT TGT-3'
R 5'-CCC ACT GTT TCA CTC ATA CAG AAT TT-3'
GATA5 F 5'-TCA CAT TGT AAT CAT CGT GGA CCC G-3'
R 5'-CAG AAC AAG GAA GGC TCT TTA CTG CC-3'
GDF3 F 5'-CTC CAT GCT CTA CCA GGA CAA TGA-3'
R 5'-ACC CAC ACC CAC ACT CAT CAA CTA-3'
GJC1 F 5'-AGA GAA CGG GAA ACA CAG CGT TC-3'
R 5'-CTG GAA GAC ACA AAT GTA AAG TTC TGC AAC-3'
IGFL1 F 5'-AGG CAC TCT GTG AAT TCT GCA ACC-3'
R 5'-TCT TGC CAC CTT TGG AAG TGG AGA-3'
NODAL F 5'-GAA GAC CAA GCC CTT GAG TAT GCT A-3'
R 5'-GCA CCC ACA TTC CTC CAC AAT CAT-3'
SERPINA5 F 5'-GGG ATT GTA CTG TCC TGT GGG TTA-3'
R 5'-TAT TCG CGT CAG GCC TCC ATT CTT-3'
TMEFF1 F 5'-AAT AGA GGA CGA CGA CAG AAG CA-3'
R 5'-AAG TCA CCA GTT CAA ACC ATT CTG G-3'
GAPDH F 5'-ACC CAG AAG ACT GTG GAT GG-3'
R5'-CAA CAG ACA CGT TGG GAG TG-3'


136 bp

81 bp

110 bp

80 bp

83 bp

80 bp

91 bp

91 bp

80 bp

80 bp

177 bp


122


86C

75C

74C

77C

78C

77C

79C

78C

80C

77C

88C









Table 4-2. Estimates of effect of CSF2 on embryonic survival at Day 15 after expected
ovulation.
Control CSF2 P-value

Number of cows receiving embryos 20 15

Number of cows (percentage) with 16/20 14/15 N.S.
CL on Day 15 (80%) (93%)

Number (percentage) of cows with 7/20(35%) 10/15 0.07
a recovered embryo, of all cows (66%)

Number (percentage) of cows with 7/16 10/14 N.S.
a recovered embryo, of cows with (44%) (71%)
aCL

Number (percentage) pregnant, of 8/20(40%) 10/15 N.S.
all cows (67%)

Number (percentage) pregnant, of 8/16 10/14 N.S.
cows with a CL (50%) (71%)

a Cows were considered pregnant if there was a detectable embryo or antiviral
activity in the flushing


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Table 4-3. Canonical pathways containing a significant number of genes differentially expressed between embryonic disc
and trophoblast.


Pathway"


P-value


Genesa


Human Embryonic Stem Cell Pluripotency


Factors Promoting Cardiogenesis in
Vertebrates
Coagulation System

Wnt/3-catenin Signaling

Role of Oct4 in Mammalian Embryonic
Stem Cell Pluripotency
Basal Cell Carcinoma Signaling
Role of Osteoblasts, Osteoclasts and
Chondrocytes in Rheumatoid Arthritis

Hepatic Fibrosis / Hepatic Stellate Cell
Activation
Axonal Guidance Signaling




Role of Macrophages, Fibroblasts and
Endothelial Cells in Rheumatoid Arthritis


0.000001


0.000032

0.000105

0.000141

0.000380

0.000741
0.001738


0.001778

0.001778




0.004365


RAC2, NODAL, FGF2, FGFR1, SMAD6, FZD1, LEFTY2,
TCF7, SOX2, NANOG, SMO, PDGFRA, BMP6, WNT11,
POU5F1, FZD7
NODAL, CER1, SMO, FZD1, DKK1, BMP6, TCF7,
WNT11, PRKD1, FZD7, PRKCB
FIO, PROS1, SERPINA5, PROC, A2M, SERPINF2,
SERPIND1
RAC2, GJA1, SFRP2, FZD1, SOX2, CDH2, DKK3, SMO,
DVL3, PPP2R2C, SOX8, SFRP1, DKK1, WNT11, FZD7
SOX2, TDH, NANOG, SPP1, PHC3, FBXO15, POU5F1

SMO, DVL3, FZD1, BMP6, GLII, TCF7, WNT11, FZD7
RAC2, SPP1, SFRP2, SMAD6, FZD1, TCF7, DKK3,
NGFR, SMO, SFRP1, DKK1, BMP6, WNT11, TNFRSF11B,
FZD7
COL1A2, IGFBP4, CD40, FGF2, NGFR, FGFR1,
PDGFRA, EDNRA, MMP2, A2M, TNFRSF11B
DPYSL2, PRKACB, FYN, RAC2, PLXNC1, NRP2, EPHB2,
DPYSL5, FZD1, ROBOI, SDC2, CXCL12, NGFR, SMO,
RASSF5, BMP6, GLII, WNT11, PRKD1, FZD7, PRKCB,
UNC5C
RAC2, SFRP2, FGF2, FZD1, TCF7, ROR2, DKK3,
CXCL12, NGFR, SMO, ATF4, SFRP1, DKK1, WNT11,
PRKD1, TNFRSF11B, FZD7, PRSS35, PRKCB









Table 4-3. Continued
Pathwayb P-value Genesa


Pantothenate and CoA Biosynthesis
Ovarian Cancer Signaling

Role of NANOG in Mammalian Embryonic
Stem Cell Pluripotency
LPS/IL-1 Mediated Inhibition of RXR
Function

Leukocyte Extravasation Signaling

RAR Activation
Glycerolipid Metabolism


Prolactin Signaling
FXR/RXR Activation
LXR/RXR Activation
CTLA4 Signaling in Cytotoxic T
Lymphocytes
PXR/RXR Activation
G-Protein Coupled Receptor Signaling

HER-2 Signaling in Breast Cancer


0.004677
0.004786


DPYSL2, DPYS, CILP2, ENPP2
PRKACB, RAC2, GJA1, SMO, EDNRA, MMP2, FZD1,
TCF7, WNT11, FZD7


0.005888 SOX2, RAC2, NANOG, SMO, FZD1, BMP6, WNT11,
FZD7, POU5F1
0.006607 CYP2C9, APOC4, APOC2, FM05, FABP2, ALDHIA1,
UST, NGFR, FABP1, FABP7, ACSL1, MGST3,
TNFRSF11B
0.010965 CLDN10, RAC2, CLDN11, CLDN5, MMP25, CXCL12,
MMP2, RASSF5, ITGAL, PRKD1, MMP19, PRKCB
0.012023 PRKACB, RAC2, ALDHIA1, CRABP2, ADCY3,
0.014454 SMAD6, NCOR1, CRABP1, PRKD1, ADH4, PRKCB
ADH6, PNLIPRP2, LIPA, ALDHIA1, LPL, DGAT2,
APOC2, ADH4
0.016982 FYN, PRLR, PDK1, TCF7, PRKD1, PRKCB
0.017378 RAC2, APOB, PCK2, APOC2, HNF4A, FOXA3, MTTP
0.018197 APOC4, NGFR, LPL, NCOR1, APOC2, TNFRSF11B
0.018197 FYN, CD3G, RAC2, LCK, APIS2, PPP2R2C, CD3D

0.020417 PRKACB, RAC2, ALDHIA1, CYP2C9, PCK2, HNF4A
0.021380 GPR161, PRKACB, FYN, RAC2, RASGRP1, RGS10,
ADCY3, ATF4, EDNRA, DRD2, PDE4D, PRKCB
0.026303 RAC2, NRG1, MMP2, PARD6G, PRKD1, PRKCB


125









Table 4-3. Continued
Pathwayb P-value Genesa
Maturity Onset Diabetes of Young (MODY) 0.027542 FABP2, FABP1, HNF4A
Signaling
TGF-3 Signaling 0.029512 ZNF423, NODAL, INHA, GSC, SMAD6, HNF4A
Acute Phase Response Signaling 0.032359 RAC2, TF, NGFR, APOA2, CRABP2, A2M, SERPINF2,
CRABP1, TNFRSF11B, SERPIND1
Calcium-induced T Lymphocyte Apoptosis 0.033113 CD3G, LCK, CD3D, PRKD1, PRKCB
Virus Entry via Endocytic Pathways 0.046774 FYN, RAC2, ITGAL, PRKD1, FOLR1, PRKCB
Molecular Mechanisms of Cancer 0.047863 PRKACB, FYN, RAC2, DIRAS3, ADCY3, SMAD6, FZD1,
RASGRP1, SMO, IHH, BMP6, GLI1, PRKD1, BCL2L11,
PRKCB, FZD7
Colorectal Cancer Metastasis Signaling 0.047863 PRKACB, RAC2, MMP25, DIRAS3, ADCY3, SMO, MMP2,
FZD1, TCF7, WNT11, FZD7, MMP19
a Genes symbols in black are overexpressed in ED, genes symbols in red are overexpressed in Tr.
b Pathways are from Ingenuity (www.ingenuity.com)


126









Table 4-4. Genes with the greatest fold change for embryonic disc (ED) compared with
trophoblast (Tr).
Intensity Means
Accession # Name Fold Tr ED
Change
Overexpressed in ED


NM_175817


XR_028016

NM_001076949
NM_001025344
NM_174188

NM_001077921

NM_001105463

NM_001105411

NM_177484
NM_001040502

NM_001083369

NM_001078019

XM_001789128

NM_001076945

XM_612940


Fatty acid binding protein 1
(FABP1)
Transforming growth factor-
Beta IG-H3 (TGFBI)
Septin 3 (SEP3)
Nanog homeobox (NANOG)
Secreted phosphoprotein 2
(SPP2)
Amyloid beta precursor protein
A 2 (APBA2)
SRY (sex determining region
Y)-box 2 (SOX2)
GDNF family receptor alpha
1 (GFRA1)
Transferrin (TF)
Alpha-1 acid glycoprotein
(AGP)
Transmembrane protein 130
(TMEM130)
Solute carrier family 7,
member 3 (SLC7A3)
DAZ interacting protein 1
(DZIP1)
Stearoyl-CoA desaturase 5
(SCD5)
Carboxypeptidase A6
(LOC540749)


119.93 4.90 610.36


95.21

89.88
75.69
68.26

64.85

57.68

56.96

54.89
52.29

50.70

49.32

48.07

46.19

45.16


24.99

7.44
11.51
7.32

7.25

7.59

4.59

31.07
16.37

4.74

13.38

4.38

7.65

4.53


2379.0

669.04
1390.0
499.21

470.18

873.88

261.73

6954.9
1006.4

240.07

660.05

210.50

460.06

238.31


127









Table 4-4. Continued
Intensity Means
Accession # Name Fold Tr ED
Change


Overexpressed in Tr
XM_614458 CAP-GLY domain containing
linker protein 1 (CLIP1)
XM_866711 Nuclear receptor co-
repressor 1 (NCOR1)
XM_614825 Centrosomal protein
(CEP350)
NM_001034342 ATF4 activating transcription
factor 4 (ATF4)
XM_612376 Centromere protein F
(CENPF)
XM 585315 Biorientation of chromosomes in
cell division 1 (BODIL)
NM_001102181 Cromodomain helicase DNA
binding protein 2 (CHD2)
NM_174436 Psrotein kinase, cGMP-
dependent, I (PRKG1)
XM_580572 Dickkopf homolog 1 (DKK1)
NM_174239 Aldehyde dehydrogenase 1,
member Al (ALDHIA1)
XM_581155 Filamin A interacting protein
1 (FILIP1L)
NM_001045901 Growth arrest and DNA-
damage-inducible y
(GADD45G)
XM_606804 RAR-related orphan receptor
B (RORB)
XM_001256996 Ribosomal protein L10a
(LOC790558)
XM_864189 Ribosomal protein L36a
(RPL36A)


-2.53 333.15


-2.57


184.07


-2.59 104.34

-2.60 342.05

-2.64 345.30


-2.71

-2.81

-2.88

-2.92
-3.02


296.27

287.45

73.98

2519.12
457.08


-3.04 440.05

-3.56 2178.12


-4.74

-5.23

-8.26


36.98

81.89

96.67


128


131.55

71.62

40.32

131.44

130.74

109.40

102.12

25.68

887.33
151.46

144.76

611.84


7.79

15.65

11.70









Table 4-5. The 15 most abundant genes overexpressed in embryonic disc or
trophoblast.


Accession #
Embryonic Disc
NM_001034790
NM_001166505
NM_175801
XM_001251618

NM_176646
NM_174580

NM_001098378

NM_205802

NM_001075171
NM_001034231
NM_001097568
NM_174438
NM_001114857

NM_001101149

NM_001035046
Trophectoderm
XM_580572

NM_001045901

XM_581232

NW 877106.1

NM_174239


Name


Intensity Means


Stathmin 1 (STMN1)
Tubulin, alpha la (TUBA1A)
Follistatin (FST)
GL12416-like (TUBA3D)
Serpin peptidase inhibitor, clade A,
member 5 (SERPINA5)
POU class 5 homeobox 1 (POU5F1)
Transmembrane protein 88
(TMEM88)
Lysosomal protein transmembrane 4
beta (LAPTM4B)
Coiled-coil domain containing 109B
(CCDC109B)
Inhibitor of DNA binding 2 (ID2)
Inhibitor of DNA binding 1 (ID1)
Protein S-alpha (PROS1)
Metallothionein 1E (MTIE)
Procollagen-lysine, 2-oxoglutarate 5-
dioxygenase 2 (PLOD2)
Microsomal glutathione S-transferase
3 (MGST3)

Dickkopf homolog 1 (DKK1)
Growth arrest and DNA-damage-
inducible, gamma (GADD45G)
Rho GTPase activating protein 21
(ARHGAP21)
Retrovirus-related Pol polyprotein
from transposon 297 (LOC608201)
Aldehyde dehydrogenase 1 family,
member Al (ALDHIA1)


ED
39876.8
39837.2
29163.8
26547.3

18250.3
17887.4


Fold Change
8984.0 4.4
4416.9 9.0
13194.4 2.4
2820.9 9.4


1213.3
489.3


15.0
29.2


17535.0 7907.4

13950.3 2251.5


12955.7
12754.3
11206.3
9820.1
9815.9


3284.5
4141.6
2432.3
2079.4
393.8


3.9
3.0
4.6
4.7
18.9


9500.8 4296.1


8855.9
ED
887.33


1347.2

2519.12


6.6
Fold Change
-2.92


611.84 2178.12

392.39 841.24


339.91


-3.56

-2.19

-2.31

-3.02


783.89


151.46 457.08


129









Table 4-5. Continued
Accession # Name Intensity Means
Trophectoderm ED Tr Fold Change
Filamin A interacting protein 1-like
XM_581155 (FILIPIL) 144.76 440.05 -3.04
Protein phosphatase 1, regulatory
NM_174824 (inhibitor) subunit 16B (PPPIR16B) 182.95 386.36 -2.11
Centromere protein F, 350/400ka
XM_612376 (CENPF) 130.74 345.30 -2.64
ATF4 activating transcription factor 4
NM_001675.2 (A TF4) 131.44 342.05 -2.60
CAP-GLY domain containing linker
XM_614458 protein 1 (CLIP1) 131.55 333.15 -2.53
Biorientation of chromosomes in cell
XM_585315 division 1-like (BODIL) 109.40 296.27 -2.71
Chromodomain helicase DNA binding
NM_001102181 protein 2 (CHD2) 102.12 287.45 -2.81
SH3 domain containing ring finger 2
NM_001083753 (SH3RF2) 115.48 236.26 -2.05
Tetratricopeptide repeat domain 17
XM_589712 (TTC17) 94.55 219.57 -2.39
NM_001103246 N-acetyltransferase 9 (NA T9) 82.36 202.23 -2.46


130









Table 4-6. Differences in expression of selected genes between embryonic disk and trophoblast as determined by
microarray analysis and qPCR.
Fold Change a


Serpin peptidase inhibitor, clade A

Transmembrane protein with EGF-like and
two follistatin-like domain
a ED/Tr


Symbol Microarray qPCR


Name

BMP binding endothelial regulation

Calpastatin

Claudin 11

Fibroblast growth factor 2

Gata binding protein 5

Growth differentiation factor 3

Gap junction protein, gamma 1

IGF-like family member 1

Nodal


BMPER

CAST

CLDN 11

FGF2

GATA5

GDF3

GJC1

IGFLI

NODAL

SERPINA5

TMEFF I


26.0

-2.1

36.0

8.9

15.4

36.7

11.4

20.0

16.8

15.0

11.1


1389

-2.4

205.0

118.6

61.4

167.7

3.2

39.4

608.9

12.8

11.3


P-value for
qPCR
<0.0001

<0.006

<0.0001

<0.0001

<0.0001

<0.0001

N.S.

<0.0005

<0.0001

<0.05

<0.0001




































Figure 4-1. Separation of a Day 15 concepts into embryonic disc and trophoblast. The
left panel shows an entire Day 15 elongated embryo and the right panel
shows an enlargement of the same embryo to visualize the embryonic disc.
The embryo was bisected along the red lines.


132











8284356

2500000

2000000

1500000

1000000

500000


Control


Control


CSF2


CSF2


Figure 4-2. Individual values of antiviral activity in uterine flushings (top) and length of
recovered embryos (bottom). Triangles represent tubular embryos and
circles represent filamentous embryos. In the bottom panel, embryos that
were not recovered intact are represented by open circles. The horizontal
bars represent the mean value for each treatment. Embryo length was not
significantly affected by treatment. Antiviral activity was also not affected by
treatment. However, when considering only those cows in which detectable
antiviral activity was present, there was a tendency for antiviral activity to be
greater for cows receiving CSF2 treated embryos (P=0.07).


133


-0-




A




*

I I


0
0
-0
00
*

O 0
























0 I0 |I



u0 .,
J -





Figure 4-3. Hierarchical Cluster of the transcriptomes of samples of embryonic disc (ED)
and trophoblast (Tr) for control and CSF2 treated embryos. ED samples form
a separate cluster from Tr but there was no distinct clusters based on
treatment.






























134









CHAPTER 5
GENERAL DISCUSSION

Technological advances in manipulation of mammalian embryos outside the

maternal environment have provided opportunities to study preimplantation embryo

development and to optimize genetic selection and fertility. In vitro embryo production

(IVP) has great potential to improve fertility and enhance breeding schemes in beef and

dairy production systems. Nonetheless, transfer of IVP embryos has not met its

potential because these embryos are altered in terms of ultrastructure (Rizos et al.

2002), metabolism (Khurana and Niemann 2000) and gene expression (Lonergan et al.

2006). These differences are associated with reduced embryo survival rates following

transfer (Farin and Farin 1995; Drost et al. 1999; Hasler 2000), increased pregnancy

loss (Hasler 2000; Block et al. 2003) and increased proportion of calves with congenital

malformations and neonatal abnormalities (Farin et al. 2006).

One approach to improve post-transfer survival of IVP embryos is to modify culture

medium with growth factors to more closely mimic the microenvironment found in vivo.

Modification of embryo culture conditions can significantly impact the competence for

development of the resulting blastocyst. Many attempts have been made to mimic

aspects of the uterine environment in culture and decrease alterations in embryo

morphology and physiology. Several growth factors and cytokines have been tested for

their actions on bovine embryonic development in vitro, including epidermal growth

factor (Sagirkaya et al. 2007), interleukin-1 (Paula-Lopes et al. 1998), IGF-1 (Block et al.

2003; Jousan and Hansen 2004; Lima et al. 2006) and CSF2 (de Moraes and Hansen

1997a). Only IGF-1 has been tested for improvement of post-transfer survival of bovine

IVP embryos. In this case, IGF-1 treated embryos had increased survival rates after


135









transfer when transfers were performed in heat-stressed cows but not when performed

in cows not subject to heat stress (Block et al. 2003; Block and Hansen 2007).

In this dissertation, we have identified CSF2 as another maternally-derived

cytokine that can alter embryonic development in a way that increases the likelihood

that a transferred embryo will continue to term. The actions of CSF2 on embryonic

development that promote survival include changes in gene expression at Day 6 of

development that block apoptosis and could conceivably result in alterations in

gastrulation. In addition, CSF2 tended to increase embryonic survival between transfer

and Day 15 and to increase the competence of the Day 15 embryo to exerts its

antiluteolytic actions on the endometrium (Figure 5-1).

In Chapter 2, the ability of CSF2 to increase blastocyst development and post-

transfer survival of bovine IVP embryos, as well as cell number, cell allocation and

apoptosis were analyzed. The results indicate that addition of CSF2 to the culture

medium at Day 5 after insemination increases blastocyst development, pregnancy rate

at Day 35 and decreases pregnancy loss thereafter. On the other hand, addition of

CSF2 at Day 1 increased blastocyst development at a lesser extent and did not have an

effect on pregnancy rate. The differential effect of CSF2 may be dependent on stage of

development of the embryo. Early addition of CSF2 could cause internalization and

degradation of the ligand-receptor complex before the embryo has overcome the EGA

process and is not ready to respond to the beneficial effects of CSF2.

Another effect of CSF2 that may be related to increased embryonic survival is a

preferential increase in the number of cells in the ICM. The number of cells in the ICM

correlates strongly with viability after transfer in mouse embryos (Lane and Gardner


136









1997). The ICM/TE ratio between bovine embryos produced in vitro differs considerably

from embryos generated in vivo (Iwasaki et al. 1990; Crosier et al. 2001). Our results

show that CSF2 can minimize the detrimental effects of in vitro culture by increasing the

ICM/TE ratio.

Another particularity of this study is that embryos were produced using X-sorted

semen. It is known that the female embryo undergoing the transition from the morula to

blastocyst stage does not tolerate glucose as well as the male embryos (Gutierrez-Adan

et al. 2000). In addition, male embryos develop faster in culture than female embryos

(Gutierrez-Adan et al. 2000; Kimura et al. 2005). The gender difference in growth rate

and sensitivity to glucose is probably due to differential expression of the X-

chromosomes. In the female embryo, both X-chromosomes are active at the cleavage-

stage (Mak et al. 2004) and the gene for glucose 6-phosphate dehydrogenase, an

enzyme that controls the entry of glucose into the pentose-phospate pathway, is located

on the X-chromosome. Another gene, hypoxanthine phosphoribosyl transferase 1, which

encodes an enzyme involved in controlling the amount of oxygen radicals, is also on the

X-chromosome (Goldammer et al. 2003). The excess in free radical and the poor

tolerance to glucose could retard the development of female embryos. Since the

primary action of CSF2 is to promote cellular survival, there is a possibility that the

effects of CSF2 in this study were amplified by the fact that at least 85% of the embryos

were female. To further examine the changes in the embryo caused by CSF2 and the

consequences of these changes into adulthood, a follow up study to investigate the

reproduction and health status of the heifers born from the CSF2 treated embryos would

be applicable.


137









Addition of CSF2 to mouse culture medium partially alleviates the long-term

adverse consequences for postnatal growth caused by IVP. Adult mice that originated

from embryos cultured in medium without any cytokine have increased body weight,

increased central fat and increased fat relative to total body mass compared with mice

that originated from embryos cultured in the presence of CSF2 or embryos developed in

vivo (Sjoblom et al. 2005). When pregnant, female mice generated from in vitro culture

have a larger placenta, which decreases the placenta/fetus weight ratio, compared with

mothers that were developed from embryos produced in vivo or cultured in vitro in the

presence of CSF2 (Sjoblom et al. 2005).

Thus far, two molecules, IGF1 and CSF2 have been reported to improve the post-

transfer survival of bovine embryos, with IGF1 only being effective during the hot

months of the year. This peculiarity of IGF1 was not specifically tested for CSF2;

however the increased pregnancy rates found with addition of CSF2 at Day 5 after

insemination were from transfers done in both the fall and winter seasons. While the

transfer of embryos that were treated with CSF2 at Day 1 after insemination (when

pregnancy rates were not increased) were done only during the summer season. The

combination of IGF1 and CSF2 plus other growth factors and cytokines (IGF2, FGF2,

TGFB and LIF) have been tested for embryo development and cell number. The

addition of a single cytokine as well as the combination of all cytokines improved

development and increased blastocyst cell number compared with medium alone (Neira

et al. 2010). However, this study was not able to clarify whether the combination of

multiple cytokines is better than the addition of a single cytokine since all the possible

combinations were not compared at simultaneously. It is possible that the production of


138









bovine embryos in the presence of both IGF1 and CSF2 could compensate for the

inability of IGFlto improve embryo survival during the winter and therefore result in

greater post-transfer survival irrespective of season. IGF1 acts as a mitogenic and

cellular survival factor, but it fails to surmount the negative effects of cryopreservation

on embryos (Velazquez et al. 2009). While CSF2 can also act as a mitogenic factor, its

central role is cell survival (Trapnell et al. 2009), including being able to protect one cell

mouse embryos from freezing damage and decrease the apoptotic index to zero (Desai

et al. 2007).

It is unlikely that the absence of CSF2 would cause absolute infertility in the cow.

Given that the CSF2 receptor shares the 3c activator subunit with IL3 and IL5, it is likely

that in the absence of CSF2, the receptors would be activated by one or both of these

two cytokines (Hansen et al. 2008). Moreover, CSF2 deficient mice (CSF2 -/-) can

produce viable puppies (Robertson et al. 1999). On the other hand, in human and mice,

excessive production of CSF2 has been reported to cause multiple pathologies, mostly

related to autoimmune diseases (Hansen et al. 2008). An increased immune response,

besides increasing the chances of diffuse inflammation, could also result in less

tolerance for the fetus.

In Chapter 3, the transcriptome of the CSF2 embryo was analyzed. Embryos

produced in vitro have altered gene expression patterns when compared with embryos

produced in vivo. In vitro culture of bovine embryos results in increased abundance of

the transcripts for heat shock protein 70.1, copper/zinc-superoxide dismutase, glucose

transporters-3 and -4) (Lazzari et al. 2002), altered levels of expression of X-linked

genes (King 2008) and differential expression of IGF family genes (Bertolini et al. 2002;


139









Sagirkaya et al. 2006; Moore et al. 2007). Furthermore, blastocysts produced in vitro

have higher expression of BCL2-associated X protein, a pro-apoptotic protein, and

arcosine oxidase, an oxidative enzyme and decreased expression of mitochondrial

manganese-superoxide dismutase, an important antioxidant defense in cells exposed to

oxygen (Rizos et al. 2002). The effects from embryo culture in vitro persist into fetal life

being presented as fetal abnormalities and large offspring syndrome. These deformities

could be consequence of alterations in the transcripts at the preimplantation stage or an

epigenetic effect as IVP embryos have decreased expression of transcripts for enzymes

involved in the de novo methylation process (Smith et al. 2009).

The results of Chapter 3 indicate that addition of CSF2 to embryo culture altered

the expression of 3 major groups of genes (development and differentiation process,

cell communication and apoptosis). The genes involved in the developmental process

and differentiation were functioning to inhibit neurogenesis and stimulate mesoderm or

muscle formation, and pluripotency. In cell communication process the signaling system

that was most representative was the WNT system, analysis of the data indicates

inhibition of p-catenin dependent WNT signaling. The apoptosis signaling pathway was

also inhibited by CSF2. The biological effect of CSF2 on apoptosis genes was

appropriately proved by submitting the embryos to heat shock and analyzing the

percentageage of apoptotic blastomers by a well known assay. However, the differential

expression of genes promoting mesoderm and delaying neural tissue development

should be further studied.

The pre-gastrulation period is when the earliest structural decisions are being

made; alterations of the genome during gastrulation may cause damage that will only


140









manifested in subsequent cell generations (Rutledge 1997). Certain changes in early

development can also induce mid to late gestation anatomic malformations and fetal

lethality (Rutledge 1997).

To further investigate the effects of CSF2 in the gastrulation process, a

imunohistochemistry study of embryos recovered at different stages of development,

such as Day 15 (when both mesoderm and neural tissue are beginning to form) and

Day 21 (when gastrulation and neurulation are completed and somites are starting to

form), could allow for properly visualization of the structures controlled by the referred

genes. The use of antibodies that recognizes muscle specific a-actin isomers from

cardiac, smooth and skeletal muscle or y-actin from non-smooth muscle, cytokeratin for

epithelial tissue and anti-neurofilament for neural tissue could reveal the preferential

development of one tissue over the other. If the changes in these early stages are

confirmed, a histomorphological study could be performed after the organs are formed

(Day 45) to define if the damage persisted and if that would be one of the reasons for

the pregnancy losses that happen after Day 35. The development and morphology of

the placenta would also be of interest in this study.

The effects of CSF2 to inhibit WNT signaling pathway could be examined in a

comparative study with control embryos, CSF2 treated embryos and embryos submitted

to WNT inhibitors.

In Chapter 4, CSF2 treated embryos were tested for characteristics that could

make them more likely to establish and maintain pregnancy. CSF2 treated embryos

were greater in size, secrete more IFNT and have a higher recovery rate. However,

there was no significant effect of treatment when expression of the embryonic disc and


141









trophectoderm dissected from elongated control and CSF2 embryos were. The lack of

difference may be due to the fact that only filamentous embryos were selected in both

groups. Perhaps if we had analyzed all the embryos recovered this bias could have

been avoided. Another possibility is that the development of the placenta is facilitated by

CSF2, as seen by a longer trophectoderm, and such an effect could improve

implantation and possibly decrease embryonic loss.

The last study also resulted in the identification of over 500 genes preferentially

expressed in the embryonic disc. Many of these have been described earlier as being

ICM or trophectoderm markers in mouse and human embryos. Identification of new

markers of ICM and TE could be used to facilitate development of the bovine embryonic

stem cell, and advance the understanding of the embryonic developmental process.

Lastly, the study of different DNA methylation patterns may provide more insight

into the actions of CSF2 to promote embryo development and survival. The knowledge

attained by these studies could be pertinent for the development of more efficient

systems in the production of embryos in vitro, which in turn will result in enhanced

pregnancy success following embryo transfer in cattle. Understanding the mechanisms

that regulate early bovine embryonic development may prove useful to study infertility in

other species, including human.


142










CSF2


> ,Apoptosis
Differential gene
expression:
tMesoderm markers
Neural cell
development







Day 15
C, Gene Expression -
normal
Fetal Programming --






Figure 5-1. Summary of effects CSF2 on embryo development and post-transfer
survival. Treatment of embryos with CSF2 from Days 5-7 after insemination
increases development of oocytes to blastocyst, increases the number of
cells in the inner cell mass, decreases apoptosis, increases expression of
genes regulating mesoderm formation and epithelial mesenchymal transition
and decrease expression of genes regulating neural cell development. CSF2
treatment increases recovery of embryos on Day 15, length, IFNT secretion
and expression of IFNT and KRT18 mRNA. Recipients that receive an
embryo treated with CSF2 have increased pregnancy rates at Days 30-35,
decreased pregnancy losses after Days 30-35 and increased calving rate.


143









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BIOGRAPHICAL SKETCH

Barbara Loureiro was born in 1980 in Linhares, Espirito Santo, Brazil, to Maria

Jose and Carlos Alberto Barbosa Loureiro. The elder of two children, Barbara lived her

whole life in Sao Mateus, until she graduated from high school in 1997. Following

graduation from high school, she entered the School of Veterinary Medicine at

Universidade Federal Rural de Pernambuco, Brazil. There she was awarded the

research scholarship "Brazilian Scientific Initiation Program" sponsored by the National

Research Program (PIBIC/CNPq). During the three years of the scholarship she

developed research in the Bovine Clinic and acquired good experience in scientific

work. During her time in college, she was also interested in the educational process

and she served as president of the Veterinary Students Association. She helped

develop the academic program for the School of Veterinary Medicine and represented

the students as a board member at the Educational Council of the School of Veterinary

Medicine and at the Administrative Council of the Veterinary Department. She received

her degree in Veterinary Medicine in July of 2003.

Concomitant with Veterinary School, she received education in the Special

Education Program focused on teaching Agricultural Science, begning in 2002. There

she had the opportunity to teach at the Federal Agricultural Technical School of

Pernambuco in the Animal Science program and develop extension projects in

Environmental Health. She received her Teaching Specialist degree in May of 2004.

Immedialty after graduation from Veterinary School she entered a Master of

Veterinary Science program at Universidade Federal Rural de Pernambuco. One year

into her master's program she received the opportunity to do an intership with Dr. P.J.

Hansen at the University of Florida. Her research was focused on apoptosis in the


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preimplantation embryo. After conclusion of her master's degree, she was awarded a

CAPES/Fulbright scholarship to persue a Doctor of Philosophy Degree in the Animal

Mollecular and Cellular Biology Graduate Program and continue her work with Dr.

Hansen. In 2010 she received the Sigma Xi Graduate Research Award. After

completing the requirements for the doctoral degree, Barbara will continue her career as

a research scientist back in Brazil.


173





PAGE 1

1 REGULATION OF THE PREIMPLANTATION B O V I N E EMBRYO BY COLONY STIMULATING FACTOR 2 By B RBARA LOUREIRO 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 2010

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2 2010 Brbara Loureiro

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3 To my husband, parents, family and friends

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4 ACKNOWLEDGMENTS First I thank my advisor, Dr. Peter J. Hansen, for the opportunity to join his group. I truly appreciate his patience and support. He always challenged me to do my best and above all he believed I could do it. His love for science and respect for his students is something that I will take for the rest of my life. Also, I thank my committee me mbers Dr. Nasser Chegini, Dr. Jos Eduardo Portela Santos and Dr. Alan Ealy for their insight and knowledge. Moreover, I am grateful for their accessibility and willingness to help, as well as their encouragement and support during the completion of this d issertation. I would like to thank my old lab mates Dr. Dean Jousan, Dr. Luiz Augusto de Castro e Paula, Dr. Jeremy Block, Dr. Maria Beatriz Padua, Dr. Katherine Elizabeth Hendricks, Dr. Lilian Oliveira, Moises Franco, Adriane Bell ( in memoriam ) and Amber Brad. I am grateful to them for helping me adapt to Gainesville, with the techniques in the lab and fo remost for their friendship. I a lso thank my present lab mates Aline and Luciano Bonilla, Justin Fear and Sarah Fiel d s for their help and assistance. Tha nks are extended to Dr. James Moss and Dr. Silvia Carambula for the great scientific discussions and not so scientific conversations. I thank the management and personnel at Central Packing Co. in Center Hill, FL for providing the ovaries used in most of the experiments of this dissertation and William Rembert for his assistance in collecting the ovaries and for always being so friendly Special thanks go to the management and personnel at North Florida Holsteins (Bell, FL), the University of Florida Dairy Research Unit (Hague, FL) and Brooksco Dairy (Quitman, GA).

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5 I am also very grateful to the faculty, staff and students of the Department of Animal Sciences and the Animal Molecular and Cell Biology Program for all of their support and friendship. I than k CAPES and Fulbright for the financial suport, assintance with documents and constant help. Special l y the staff from Brazil S i lvio dos Santos Salles, Sandra Lopes, Giselle Melo and Glayna Braga. My very special thanks go to my friends in Brazil Lemia, Al ine, Luciana, Eruska, Erissa and Virginia for cheering for me since the beginning of this journey. Also, I could not forget the friends that supported me in every aspect Mr. Helio Alencar and family, Mr. Reginaldo Barros ( in memoriam ) and family and all fr iends from Recife. Finally, I want to thank my husband Maur cio, for his love, support, help with the experiments and endless patience at these final moments. I also thank my mother and father in law, Odilon and Penha, my sister in law Tatiana and her fami ly, and my brother in law Willian for their involvement and support. This accomplishment would not have happened without the encouragement, support and love of my parents Maria Jos and Carlos Alber to Barbosa Loureiro, my sister mili, my grandmother Marg arida, my uncle Jose Maria, my a unties Cau, Bam and Te and my cousins Giu, Gabri and Lidi. Even from far way, they lived every moment of this journey with me and I am forever grateful to them.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 16 Introduction ................................ ................................ ................................ ............. 16 Embryonic Development in the Bovine ................................ ................................ ... 18 Time Course of Early Development ................................ ................................ .. 18 Changes in Gene Expression During Cleavage Stages ................................ ... 1 9 Changes in Metabolism of the Early Embryo ................................ .................... 20 Developmental Changes in Apoptosis ................................ .............................. 22 Epigenetic Modifications ................................ ................................ ................... 24 Compaction and Blastocyst Formation ................................ ............................. 25 Hatching and Elongation ................................ ................................ .................. 29 Maintenance of the Corpus Luteum ................................ ................................ 30 Attachment to the Endometrium ................................ ................................ ....... 31 Placentomes ................................ ................................ ................................ ..... 32 Alterations in Embry onic Development In vitro ................................ ....................... 32 Growth Factors and Cytokines as Uterine Regulators of Embryonic Development ................................ ................................ ................................ ....... 35 Insulin like Growth Factor 1 ................................ ................................ .............. 37 Interleukin 1 ................................ ................................ ................................ ...... 38 Fibroblast Growth Factor ................................ ................................ .................. 39 Tumor Necrosis Factor ................................ ................................ ..................... 40 Colony Stimulating Factor 2 ................................ ................................ .................... 41 Biology and Signaling ................................ ................................ ....................... 41 CSF2 Secretion in the Uterine Tract ................................ ................................ 43 Actions of CSF 2 on Embryo Development and Survival ................................ .. 44 CSF2 and Interferon tau Secretion ................................ ................................ ... 46 Goals of the Current Investigation ................................ ................................ .......... 46 2 COLONY STIMULATING F ACTOR IMPROVES DEVEL OPMENT AND POST TRANSFER SURVIVAL OF BOVINE EMBRYOS PRODU CED IN VITRO ............. 49 Introduction ................................ ................................ ................................ ............. 49 Materials and Methods ................................ ................................ ............................ 51 Materials ................................ ................................ ................................ ........... 51

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7 Effects of CSF2 on Embryo Development and Blastocyst Properties .............. 52 Production of embryos ................................ ................................ ............... 52 Interactions between oxygen concentration and presence of CSF2 .......... 53 Cell number and differentiation of blastocysts ................................ ............ 54 Apoptotic blastomeres ................................ ................................ ............... 55 Effects of CSF2 and IGF1 on Pregnancy and Calving Success after Transfer to Recipients ................................ ................................ ................... 55 Production of Holstein embryos using X sorted semen ............................. 55 Animals ................................ ................................ ................................ ...... 57 Synchronization and timed embryo transfer ................................ ............... 57 Effect of CSF2 and IGF1 added at Day 1 of culture on development and post transfer survival of bovine embryos ................................ ................ 58 Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of culture on development and post transfer survival of bovine embryos ... 58 Statistical Analysis ................................ ................................ ............................ 59 Results ................................ ................................ ................................ .................... 60 Effects of CSF2 on Embryo Development ................................ ........................ 60 Effects of CSF2 on Blastocyst Total Cell Number, Cell Differentiation and Apoptosis ................................ ................................ ................................ ...... 61 Effects of CSF2 and IGF1 on Pregnancy and Calving Success after Transfer to Recipients ................................ ................................ ................... 61 Discussion ................................ ................................ ................................ .............. 62 3 COLONY STIMULATING F ACTOR 2 CAUSES CHANG ES IN THE TRANSCRIPTOME OF THE BOVINE PREIMPLANTATI ON EMBRYO INCLUDING ALTERATION S IN EXPRESSION OF D EVELOPMENTAL AND APOPTOSIS GENES ................................ ................................ .............................. 72 Introduction ................................ ................................ ................................ ............. 72 Materials and Methods ................................ ................................ ............................ 73 In vitro Production of Embryos ................................ ................................ ......... 73 RNA Purification and Processing ................................ ................................ ..... 74 Microarray Hybridization ................................ ................................ ................... 75 Analysis of Microarray Data ................................ ................................ .............. 76 Quantitative Real Time PCR ................................ ................................ ............ 77 Regulation of Apoptosis by CSF2 ................................ ................................ ..... 78 Results ................................ ................................ ................................ .................... 79 Transcriptomal Profile ................................ ................................ ...................... 79 Biological Process Ontologies Affected by CSF2 ................................ ............. 80 Genes Involved in Cellular Development and Differentiation ............................ 81 Genes Involved in Signal Transduction and Cell Communication .................... 82 Genes Involved in WNT Signaling ................................ ................................ .... 82 Genes Involved in Ap optosis Signaling Pathway ................................ .............. 83 Quantitative Real Time PCR ................................ ................................ ............ 83 Actions of CSF2 to Block Heat Shock Induced Apoptosis ................................ 84 Discussion ................................ ................................ ................................ .............. 84

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8 4 CONSEQUENCES OF EMBRYONIC EXPOSURE TO CSF2 FROM DAY 5 TO 7 AFTER INSEMINATION ON TROPHOBLAST ELONGATION, INTERFERON TAU SECRETION AND GENE EXPRESSION IN THE EMBRYONIC DISC AND TROPHECTODERM ................................ ................................ ............................. 103 Introduction ................................ ................................ ................................ ........... 103 Materials and Methods ................................ ................................ .......................... 104 In vitro Production of Embryos ................................ ................................ ....... 104 Transfer Into Recipients ................................ ................................ ................. 105 Embryo Recovery and Evaluation ................................ ................................ .. 106 Antiviral Assay ................................ ................................ ................................ 107 Analysis of the Transcriptome of Trophectoderm and Embryonic Disc .......... 108 Microarray Hybridization ................................ ................................ ................. 109 Analysis of Microarray Data ................................ ................................ ............ 109 Quan titative Real Time PCR ................................ ................................ .......... 110 Statistical Analysis ................................ ................................ .......................... 111 Results ................................ ................................ ................................ .................. 112 Embryo Survival After Transfer ................................ ................................ ...... 112 Embryonic Growth and Development ................................ ............................. 113 Antiviral Activity in Uterine Flushings ................................ .............................. 113 Changes in the Transcriptome of Embryonic Disc and Trophectoderm ......... 113 Characteristics of Genes Differentially Expressed Between Embryonic Disc and Trophoblast ................................ ................................ .......................... 114 Identification of Likely Candidate Genes for Use as Embryonic Disc Markers 115 Validation of Microarray Results Using qPCR ................................ ................ 116 Discussion ................................ ................................ ................................ ............ 117 GENERAL DISCUSSION ................................ ................................ ............................ 135 LIST OF REFERENCES ................................ ................................ ............................. 144 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 172

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9 LIST OF TABLES Table page 2 1 Effect of CSF2 on total cell number, inner cell mass (ICM), trophectoderm (TE) and ICM/TE ratio of blastocysts at Day 7 after insemination ...................... 68 2 2 Effect of CSF2 on total cell number and TUNEL positive blastomeres in blastocysts at Day 7 after insemination ................................ .............................. 68 2 3 Effect of CSF2 and IGF1 added at Day 1 of culture on embryonic development at Day 7, pregnancy risk at Day 30 35 (based on ultrasonography), calving r ate and pregnancy loss among recipient cattle that received embryos that were cultured in 5% O 2. ................................ .................. 69 2 4 Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of culture on embryonic development at Day 7, pregnancy risk at Day 30 35 (based on ultrasonography), calving rate and pregnancy loss among recipient cattle that received embryos that were cultured in 5% O 2. ................................ .................. 70 3 1 Primers and Probes used on qPCR. ................................ ................................ ... 92 3 2 Gene ontologies in the biological process category that were regulated by CSF2. ................................ ................................ ................................ ................. 95 3 3 Differentially regulated genes involved in WNT signaling. ................................ .. 98 3 4 Differentially regulated genes involved in apoptosis. ................................ .......... 99 4 1 Primers used for qPCR. ................................ ................................ .................... 122 4 2 Estimates of effect of CSF2 on embryonic survival at Day 15 after expected ovulation. ................................ ................................ ................................ .......... 123 4 3 Cano nical pathways containing a significant number of genes differentially expressed between embryonic disc and trophoblast. ................................ ....... 124 4 4 Ge nes with the greatest fold change for embryonic Disc (ED) compared with trophoblast (Tr). ................................ ................................ ................................ 127 4 5 The 15 most abundant genes overexpressed in embryonic disc or trophoblast. ................................ ................................ ................................ ....... 129 4 6 Differences in expression of selected genes between embryonic disk and trophoblast as determined by microarray analysis and qPCR. ......................... 131

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10 LIST OF FIGURES Figure page 1 1 M echanisms by which CSF2 regulates cellular survival, differentiation, functions and activation ................................ ................................ .................. 48 2 1 Percentage age of oocytes that developed to the blastocyst stage at Day 7 (Panel A ) and 8 after insemination (Panel B ) ................................ .................... 71 3 1 Genes expressed in control and CSF2 treated em bryos at Day 6 of development. ................................ ................................ ................................ .... 100 3 2 Validation of microarray results using quantitative PCR. ................................ 101 3 3 Regulation of heat shock induced apoptosis in Day 6 bovine embryos by CSF. ................................ ................................ ................................ ................. 102 4 1 Separation of a Day 15 conceptus into e mbryonic disc and trophoblast. ........ 132 4 2 Individual values of antiviral activity in uterine flushings (top) and length of recovered embryos (bottom). ................................ ................................ ........... 133 4 3 Hierarchical Cluster of the transcriptomes of samples of embryonic disc (ED) and trophoblast (Tr) for control an d CSF2 treated embryos.. ........................... 134 5 1 Summary of effects of CSF2 on embryo development and pos t transfer survival. ................................ ................................ ................................ ............ 143

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11 LIST OF ABBREVIATION S AQP Aquaporins ATP Adenosine triphosphate Beta common BNC Binucleated cell CCCP Carbonyl cyanide 3 chloro phenylhydrazone CpG Cytosine guanine dinucleotide DNA Deoxyribonucleic acid EAG Embryonic genome activation ED Embryonic disc ES Embryonic stem FSH Follicle stimulating hormone GnRH Gonadotropin releasing hormone GTP Guanosine triphosphate H hour ICM Inner cell mass IFNT Interferon tau IVP In vitro produced JAK Janus kinase KSOM Potassium s implex optimized medium Min minute mRNA Messenger RNA mtDNA Mitochondrial DNA NADPH Nicotinamide adenine dinucleotide phosphate NKT Natural Killer T

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12 PGF PI3K P hosphatidylinositol 3 kinase PKC Protein kinase C RNA Ribonucleic acid RNAse Ribonuclease STAT Signal transduction and activation of transcription Sec Second SOF Synthetic oviduct fluid T E Trophectoderm Tr Trophoblast TUNEL Terminal deoxynucleotidyl transferase mediated dUTP Vs Versu s

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13 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 REGULATION OF THE PREIMPLANTATION BOVINE EMBRYO BY COLONY STIMULATING FACTOR 2 By B rbara Loureiro August 2010 Chair: Peter J. Hansen Major: Animal Molecular and Cell Biology Colony stimulating factor 2 (CSF2) is a multifunction al cytokine originally recognized as a hematopoietic factor but now known to be expressed in several reproductive tract tissues including the bovine oviduct and endometrium Colony stimulating factor 2 may be an important intracellular regulator of endometri al, oviductal and embryonic function s during early pregnancy in the cow and other species. Addition of CSF2 to culture medium improved the proportion of in vitro produced embryos developing to the blastocyst stage in cow, mouse, human and pig and increased post transfer embryonic survival in mice. A series of experiments was conducted to understand the role of CSF2 in preimplantation embryonic development including its effects on blastocyst formation, the embryonic transcriptome, and embryonic survival. The first experiment was conduct ed to test whether addition of CSF2 to culture medium could enhance development and post transfer survival of in vitro produced bovine embryos. Treatment of embryos with CSF2 at Day 1 after insemination increased the percentage age of embryos that became transferable morulae or blastocysts at Day 7 but had no significant effect on pregnancy rate at Day 30 35 or calving rate. When CSF2 was added at Day 5 after insemination there was increase in

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14 the percentageage of embryos that b ecame transferable morulae or blastocysts at Day 7, an increase in the percentageage of recipient cows pregnant at Day 30 35, an increase in calving rate and a decrease in pregnancy loss after Days 30 35. Furthermore, blastocysts formed after treatment with C SF2 had an increase in the number of cells in the inner cell mass. T he second experiment was conducted to analyze the transcriptome of CSF2 treated embryos t o identify genes involved in mediating CSF2 effects on development to the blastoctcyst stage and surviv al after transfer. The Agilent bovine microarray platform was used. E mbryos were treated with CSF2 at Day 5 and selected at Day 6 after insemination A total of 160 genes were differentially expressed with 67 being higher in CSF2 treated embryos and 93 being lower. Analysis identified 13 biological process ontologies that were grouped into three major groups. The first group included genes functionally involved in developmental process es and differentiation Actions of CSF2 would tend to inhibit genes involved in neurogenesis and stimulate genes involved in mesoderm or muscle formation. The second group were genes involved in cell communication with the most characteristic effect caused by CSF2 being inhibition of catenin dependent WNT signalin g. The third group were genes involved in apoptosis signaling which was inhibited by CSF2. The antiapoptotic actions of CSF2 were confirmed in another experiment in which CSF2 decreased the percentageage of blastomer e s in Day 6 embryos becoming apoptotic after h eat shock. The third study evaluate d possible mechanisms by which CSF2 acts during Day 5 to 7 of development to improve embryonic and fetal survival. Embryos were treated with CSF2 or served as controls and were transferred to recipient cows at Day 7 after

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15 ovulation. Embryos were recovered at Day 15 and embryonic disc and trophoblast analyzed for gene expression by microarray analysis. Results suggest that higher pregnancy rates at Day 30 35 represent increased embryonic survival before Day 15 an d a greater capacity of the embryo to elongate and secrete interferon tau ( IFNT ) at Day 15. This conclusion is based on greater recovery of embryos from cows receiving CSF2 treated embryos at Day 15 (P<0.07), a tendency for CSF2 treated embryos to be longe r than control embryos, and greater antiviral activity (a measure of IFNT bioactivity) in uterine flushings of cows receiving CSF2 treated embryos (P<0.07, when considering those cows with detectable antiviral activity). Analysis of gene expression in fil amentous embryos indicate d no difference in transcription among this subset of embryos that survived to Day 15 and elongated successfully. Therefore, the reduction in embryonic and fetal loss after Day 30 35 caused by CSF2 is probably not a direct reflect ion of altered gene expression at Day 15. Taken together these results indicate that CSF2 can regulate embryonic development, increase embryonic survival after transfer and decrease pregnancy loss. The ability of CSF2 to improve development is probably due to a set of effects that include an increase in the number of cells in the inner cell mass, a decrease in cell death and differentially regulation of the embryo transcriptome. Furthermore, before implantation CSF2 embryos tend to be longer and secret more IFNT The increased pregnancy rates observed after Day 35 of pregnancy may be a combined result of the factors mentioned above. Moreover, CSF2 treatme nt can be inflicting epigenetic changes that persist latter in development or even morphological changes that are consequence of the differentially gene expression early before implantation.

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16 CHAPTER 1 LITERATURE REVIEW Introduction The maternal microenvironment of the oviduct and uterus is a major determinant of the health and well being of the newly formed offspring during embryonic, fetal, neonatal and adult life. During development, the needs of the embryo constantly changes and, to cope with those changing requirements, the reproductive tract undergoes specific physiological and biochemical modific ations throughout gestation ( Buhi 2002; Spencer et al. 2007; Hugentobler et al. 2010) The importance of the uterine environment for pregnancy has been shown in several experiments. By comparing the probability that twin embryos would survive pregnancy in embryo transfer studies, McMillam ( 1998) estimated that only about 50 70% of recipients are capable of maintaining a transferred embryo. In other embryo transfer experiments with cows, pregnancy rates were affected by a variety of physiological states of the recipient animal incl uding heat stress (Chebel et al. 2008) circulating concentrations of urea (Tillard et al. 2007) treatment with somatotropin (Moreira et al. 2001) milk yield (Y niz et al. 2008) and asynchrony between recipient and embryo (Kubisch et al. 2004) Evidence that the uterine environment can affect adult life comes from many species including the sheep, in which a reduction in vitamin B12, folate and methionine content of the maternal diet around the time of conception caused high blood pressure, increased adiposity, insulin resistance and altered immune function in adult offspring (Sinclair et al. 2007) Males suffered greater effects than females. Long term effects in adult sheep are probably manifestation of epigenetic modifications of the genome.

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17 The importance of the uterine environment for embryonic development means that in vitro systems for embryo production can be compromised unless critical features of the repro ductive tract environment are replicated in vitro In beef and dairy production systems, the in vitro produced (IVP) embryo is important for increasing genetic selection and as a required technical procedure for production of transgenic animals (Hansen and Block 2004) Embryo transfer can also be used to improve fertility in heat stressed females (Block e t al. 2003; Block and Hansen 2007) Embryo transfer is effective in this regard because embryos are transferred to the recipient at Day 7 of pregnancy when the embryo is resistant to heat stress (Ealy et al. 1993; Ealy et al. 1995) E arly e mbryonic development is not absolutely dependent upon regulatory molecules present in the reproductive tract because embry os produced in vitro in medium without growth factors can give rise to live calves after transfer to recipients (Block and Hansen 2007) However, culture conditions are suboptimal and result in alte rations at the morphological (Fischer Brown et al. 2002; Fischer Brown et al. 2004) ultrastructural (Rizos et al. 2002a) physiological (Ushijima et al. 1999) and transcriptional levels (Bertolini et al. 2002; Sagirkaya et al. 2006; Smith et al. 2009) compared with embryos produced in vivo Using microarray analysis, 200 genes were found to be differentially expressed between embryos produced in vivo and in vitro (Smith et al. 2009) In addition, several abnormalities have been reported in calves resulting from IVP embryos (Farin and Farin 1995; Lazzari et al. 2002; Miles et al. 2005) Thus, one or more components of the maternal environment are necessary for optimal embryonic development and birth of a healthy calf.

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18 There are strong lines of evidence to implicate colony stimulating factor 2 (CSF2), otherwise called g ranulocyte macrophage colony stimulati ng factor, as a physiologically important regulator of early embryonic development. This cytokine is expressed in several rep roductive tract tissues including the oviduct and endometrium of the cow (de Moraes et al. 1999) and human (Zhao and Chegini 1994; Chegini et al. 1999) Addition of CSF2 to culture medium improved the proportion of in vitro embryos d eveloping to the blastocyst stage in cow (de Moraes and Hansen 1997 a ) mouse (Robertson et al. 2001) human (Sj blom et al. 1999) and pig (Cui et al. 2004) and increased post transfer embryonic survival in mice (Sj blom et al. 2005) The literature review here presented will focus on the events that take place during early embryonic development in the cow, unless stated otherwise, and the effects of CSF2 on preimplantation embryonic developmen t. Embryonic Development in the Bovine Time Course of Early Development After ovulation, the bovine oocyte is picked up by the cilia covered fimbria and guided through the infundibulum and ampulla of the oviduct (Oxenreider and Day 1965) It is in the ampullary isthmic junction of the oviduct that the oocyte is fertilized (Bazer et al. 2009) Fusion of the two gametes and release of the sec ond polar body represents the completion of meiosis (Marteil et al. 2009) The first cleavage of the zygote occurs around 23 31 hours after insemination ( Maddox Hyttel et al. 1988) The developing embryo leaves the oviduct and moves into the uterus approximately at Day 5 of pregnancy when it is about 16 cells (Betteridge et al. 1988) Until hatching at Day 7 8, the embryo is surrounded by a protein coat called the zona pellucida (composed of three glycoproteins; ZP1 ZP3) that is important for

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19 prevention of polyspermy, to keep the blastomeres together and to conserve the microen viroment of the peritvitelline space (Litscher et al. 2009; Van Soom et al. 2010) After hatching, the blastocyst is transformed to an ovoid form until elongat ion of the trophoblast is initiated between Days 12 and 14. Also around this period, gastrulation and specification of the germ layers occur in the embryonic disc (ED). By Day 24 the conceptus can fill the entire length of both uterine horns (Blomberg et al. 2008) Implantation starts on Day 20, when the trophectoderm (TE) adhere s to the endometrial luminal epithelium of the mother (Blomberg et al. 2008; Bazer et al. 2009) Changes in Gene Expression During Cleavage Stages During early development, the embryonic genome is inactive and the embryo relies on maternal messenger ribonucleic acid (mRNA) for pro tein synthesis (Thelie et al. 2009) The recruitment mechanisms by which dormant RNA is either targeted for translation or decay are still largely uncharacterized. The current model involves lengthe ning of the poly(A) tail, which triggers binding of the poly(A) binding protein and binding of translation initiation factors (Memili and First 2000; Groisman et al. 2002) RNA concentration is highest in the germinal vesicle stage oocyte and from then until the 8 cell stage, RNA is gradually depleted (Gilbert et al. 2009; Vall e et al. 2009) Evidence in the mouse suggests that this decline is important for activation of the embryonic genome (Li et al. 2010) Depletion of maternal argonaute 2 ( Ago2 ) which encodes a catalytic RNA hydrolase (RNase), disrupts gene expression and the 2 cell embryo fails to become a blastocyst (Li et al. 2010) In the bovine, embryonic genome activation (EGA) occu rs at the 8 to 16 cell stage (Memili and First 2000) through an unknown mechanism.

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20 Changes in Metabolism of the Early Embryo The type of substrate metabolized by the embryo changes with development At early stages, the fuel for ATP formation by the mitochondria is provided by pyruvate, while the uptake of glucose is low. As the embryo develops to the compact morula and blastocyst stage, ATP synthesis increases and glucose starts to contribute to th e citric acid cycle through conversion to lactate and then pyruvate (Thompson 2000) Glucose can also generate ribose required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosp hate (Thompson 2000) Furthermore, glucose, pyruvate and lactate production and/or consumption can be different between the two cell types of the Day 8 blastocyst. While inner cell mass (ICM) cells consume more glucose than pyruvate, the converse is true for TE cells (Gopichandran and Leese 2003) Lactate production is higher for TE than for ICM cells (Gopichandran and Leese 2003) The number and activity of mitochondria also change as the embryo develops. A primordial oocyte contain s as few as 10 mitochondrial DNA (mtDNA) copies wh ereas a fully grown oocyte can contain more than 100.000 copies of mtDNA, with one or two copies of mtDNA per organelle (Ferreira et al. 2009; Chiaratti et al. 2010) At this stage the mitochondria is immature and d oes not present any activity, therefore the energy i s supplied through the granulosa cells (Tarazona et al. 2006) Initially present near the periphery of the cell, oocyte mitochondria become more d ispersed at the germinal vesicle breakdown stage, presumably to better serve the cell (Ferreira et al. 2009; Marteil et al. 2009) At this point the levels of activity have highly increased and the mitochondria are capable of generating the necessary ATP (Tarazona et al. 2006) The bovine embryo does not gain the capacity for replenishing the mtDNA until the morula and blastocyst stage s when there is increased expression of nuclear respiratory factor 1

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21 ( NRF1 ) and transcription factor A, mitochondria ( TFAM ) that are regulators of mtDNA replication and transcription (Chiaratti et al. 2010) Embryos with non competent mitochondria stop development before the EGA (Tarazona et al. 2006) High concentrations of glucose are toxic to bovine embryos, especially female embryos undergoing the transition from the morula to blastocyst stage (Guti rrez Ad n et al. 2000) This fact along, wit h the possibility that male embryos develop faster in culture than female embryos (Guti rrez Ad n et al. 2000; Kimura et al. 2005) could explain the skewing o f the sex ratio towards males often seen in calves from IVP embryos (Block and Hansen 2007; Camargo et al. 2010) The gender difference in growth rate and sen sitivity to glucose could be due to differential gene expression either because of differences in activity o f sex or autosomal chromosomes. Both X chromosomes are active in the cleavage stage female embryo (Mak et al. 2004) Unbalanced expression of X linked genes can increase the activity of enzymes involved in energy metabolism and detoxification of oxygen radicals (P rez Crespo et al. 2005) For example, the gene for glucose 6 phosphate dehydrogenase ( G6PDH ) an enzyme that controls the entry of glucose into the pentose phospate pathway, is located on the X chromosome. With both X chromosomes active in the female embryo there is more pentos e phospate pathway activity which leads to poor tolerated imbalance in carbohydrate metabolism (Guti rrez Ad n et al. 2000) Hypoxanthine phosphoribosyl transferase 1, an enzyme involved in controll ing the amount of oxygen radicals, is also on the X chromosome (Goldammer et al. 2003) Free radical actions involve not only cellular damage but also cellular growth (Rieger 1992)

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22 The retarded development in female embryos could be caused by a decrease in oxygen radical levels due to increased activity of the enzyme. By the time the embryo reaches the blastocyst stage, 88% of the genes (193 genes) in the X chromosome are upregulated in the female embryo comparing to the male embryo. However, only 10% of these genes had a 2 fold increase and in fact, 70% of the genes had a fold increase lower than 1.6 comparing to the male embryo (Bermejo Alvarez et al. 2010) These suggest that at the blastocyst stage the X chromosome is starting to be inactivated. Glutathione is the most important cellular antioxidant in the cytosol. There is a large decline in its concentration after fertilization because it is consumed as part of the chromatin decondensation process (Lim et al. 1996; Lub erda 2005) However, de novo synthesis of glutathione increases at the 16 cell stage (around EGA), and reach es its maximum peak at the hatched blastocyst stage (Lim et al. 1996) Development al Changes in Apoptosis Apoptosis is a developmentally regulated process that plays an important role in the survival of the preimplantation embryo. Although the maturing oocyte can undergo apoptosis (Roth and Hansen 2004 a ; Roth and Hansen 2004 b ; Roth and Hansen 2005) the capacity for apoptosis is lost at the 2 cell stage and does not become reacquired until sometim e between the 8 and 16 cell stages (Paula Lopes and Hansen 2002 a ; Gj rret et al. 2005) As the embryo undergoes further development, there is little cha nge in the degree of apoptosis (Loureiro et al. 2007) The lack of apoptosis at the two cell stage caused is in part by a block in activation of caspases (Brad et al. 2007; de Castro e Paula and Hansen 2008 a ) which in turn reflects increased resistance of the mitochondria to depolarization (Brad et al. 2007) A second block to the pathway exists

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23 at the level of the nucleus since addition of carbonyl c yanide 3 chloro phenylhydrazone a mitochondria depolarization agent, did not cause increase in DNA fragmentation even though group II caspases were activated (Brad et al. 2007; Carambula et al. 2009) The sensitivity of embryonic DNA to fragmentation coincides with its levels of methylation. DNA is highly methylated at the 2 cell stage and then becomes progressively more demethylated as development progresses until by the 8 16 cell stage the embryo has low methylation and transcription is activated (Dean et al. 2001; Park et al. 2007) The interaction between apoptosis and methylation was shown when sensitivi ty of 2 cell embryos to carbonyl cyanide 3 chloro phenylhydrazone could be induced with 5 aza 20 deoxycytidine, a DNA methylation inhibitor (Carambula et al. 2009) In the bovine embryo, the consequ ences of apoptosis are dependent upon stage of development. Induction of apoptosis is a major cause for the reduced oocyte competence for fertilization and development caused by heat shock (Roth and Hansen 2004b) Cell stressors like arsenic (Krininger et al. 2002) heat shock (Loureiro et al. 2007) and ceramide (de Castro e Paula and Hansen 2008b) induce apoptosis through the mitochondrial pathway that causes mitochondria depolarization and activation of caspases. It is likely that massive activation of apoptosis by these stress es compromises development. However, when the stress is less severe so that the increase in apoptosis is limited, the apoptotic process itself may be beneficial to embryo survival. At Day 4 of development, inhibition of apoptosis with a caspase inhibitor exacerbated deleterious effects of heat shock on development to the blastocyst stage (Paula Lopes and Hansen 2002b)

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24 Epigenetic Modifications Cytosine methylation at the cytosine guanine dinucleotide (CpG) islands in the genomic DNA is necessary for regulation of chromatin configuration and normal gene expression (Geiman and Muegge 2010) In general, hypermethylated DNA is transcriptionally inactive wh ereas hypomethylated DNA is highly transcribed (Corry et al. 2009) Conservation of methylation in only one parental allele is a form of epigenetic regulation known as imprinting (Wilkins 2006; Tveden Nyborg et al. 2008) This monoallelic expression can be tissue specific. One example of tis sue specific imprinting is the X chromosome, in which the paternally derived copy is inactivated in extraembryonic tissue while its expression is random in fetal and adult somatic tissues (Wilkins 2 006) The degree of methylation change with development. In the bovine zygote, the male pronucleus is partially demethylated at 20 hours after fertilization so that paternal levels of reactivity to antibody to 5 methyl cytosine is 51% of that of maternal DNA (Park et al. 2007) Methylation of the paternal chromosomes increases to levels similar to the female by 28 hours after fertilization when embryos are at about the 2 cell stage (Park et al. 2007) Embryonic genome activation occurs at the 8 to16 cell stage (Gilbert et al. 2009) Up to this stage, embryos are highly methylated (Dean et al. 2001) and c oincident with embryonic genome activation, methylation declines (Dean et al. 2001) De novo methylation after the 16 cell stage is not identical for all the nuclei. By the blastocyst stage, the ICM contains highly methylated nuclei and the TE lower amounts of methylated DNA (Dean et al. 2001) The degree of methy lation influences cell potential for differentiation (Western et al. 2010)

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25 Compaction and Blastocyst Formation Around the 32 cell stage, at Day 5 of pregnancy, the bovine embryo forms a solid mass known as the morula that then undergoes a process called compaction (Betteridge et al. 1988) Prior to compaction, the blastomeres are spherical and lack specialized intercellular junctions. During compaction, cells become flattened against one another, thus maximizing intercellular contact and obscuring intercellular boundaries (Sheth et al. 2000; Johns on and McConnell 2004) This process is mediated primarily by activation of e cadherin, a Ca 2+ dependent adhesion molecule. For full adhesive function and cytoskeletal anchorage, e cadherin forms a core adhesion complex with a protein known as catenin (Niessen and Gottardi 2008) This type of junction is responsible for generating contact dependent growth and polarity signals with apical and basolateral domains in all blastomeres (Johnson and McConnell 2004) Blastomere initiation of polarity seems to be mediated by the Par complex protein (Par 3 and 6), atypical protein kinase C (aPKC) and caudal cell division cycle homolog (S. cerevisiae; cd (Eckert and Fleming 2008) Inhibition of aPKC and Par 3 causes failure of asymmetric cleavage latter in development (Plusa et al. 2005) Another important junctional complex for epithelial differentiation is the tight junction. Formed by claudin, ocludin and zona ocludins proteins (ZO 1 and 2), this junction tightly connects opposing cell membranes, creating a barrier that is virtually impermeable to fluid diffusion through the intercellular space (Tsukita et al. 2001) In mouse embryos, after compaction ZO 1 binds to rab guanosine triphosphate hydrolase (GTPase) and Par 3 6/aPKC to the e cahdehirin catenin complex. When cells start to differentiate, the peripheral membrane proteins cingulin and ZO 2 assemble to the

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26 complex which in turn results in loss of Par 3 6/aPKC. Finally, duri ng blastocoel formation, ZO complex. At this point the embryo generates an impermeable seal between TE cells and the blastocoel cavity (Sheth et al. 2000) Cavitation, in which the fluid filled blastocoel is formed, involves polarized transport of ions and water (Watson et a l. 2004) Vectorial transport of Na + and Cl ions through the TE into the blastocoel generates an osmotic gradient that drives fluid across the epithelium (Kawagishi et al. 2004) In the mouse embry o, it is a carrier mediated process that involves several types of ion transporters, including a Na + channel, Na + / H + exchangers and ATPase Na + /K + transporting, alpha 1 ( Atp1a1 ). The Atp1a1 are localized in the basolateral membrane region of the TE (Madan et al. 2007) Blastocoel expansion is significantly retarded in the absence of extraembryonic Na + in the presence of inhibitors of Na + channels or silencing RNA (siRNA) for Atp1a1 (Kidder 2002; Madan et al. 2007) Also present in the TE membrane are water channels known as aquaporins (AQP). They allow rapid water flow across the membrane in the direction of the osmotic gradient (Liu and Wintour 2005) Murine preimplantation embryos express mRNA for multiple AQP throughout preimplantation development (Liu and Wintour 2005) AQP 3 mRNA increases at the morula to blastocyst transition and AQP 8 protein is first detected in the cell margins at the morula stage (Barcroft et al. 2003) Both A QP 3 and 8 are found in the basolateral membrane of the TE while AQP 9 is predominantly observed in the apical membrane domain of the TE (Liu and Wintour 2005) AQP 8 is highly selective for water molecules while AQP 3 and 9 are less selective, allowing the passage of small solutes (van Os et al. 2000)

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27 Following compaction and cavitation, the bovine blastocyst at Days 6 7 starts to differentiate into ICM cells, which retain a pluripotent phenotype (Pant and Keefer 2009) and TE cells which will become the outer layer of the placenta (Hamilton 1946; Betteridge et al. 1988) TE cells are the first to differentiate and have the characteristics of an epithelium (Mar ikawa and Alarc n 2009) The ICM cells go through a second lineage segregation becoming the epiblast, which will give rise to the fetus itself, and primitive endoderm that becomes the parietal and visceral endoderm, which latter contributes do the yolk sac (Blomberg et al. 2008) Around the third week of development gastrulation, neurulation and formation of the somites will take place in the epiblast (Maddox Hyttel et al. 2003) In the bovine, it is not known how the blastomere decides to become a TE cell or ICM cell. There are two theories to explain TE and ICM differentiation in the mouse embryo. According to the inside out theory, position determines cell fate. At the morula stage, cell to cell contact increases and some cells become enclosed by surrounding cells while other cells in the outside layer are in contact with the external environment for part of their surface. Asymmetric cell c ontact induces epithelial differentiation into TE whilst symmetric contact of the enclosed ICM inhibits differentiation (Eckert and Fleming 2008; Marikawa and Alarc n 2009) An alternative theory suggests that the cell decides its fate and the location in which it will reside, rather than the location deciding what fate the cell will have (Yamanaka et al. 2006) Several studies have corroborated the inside outside model. For example, imunosurgically isolated ICM have the potential to form TE in vivo (Rossant and Lis 1979) and tight junction and a b lastocoel in vitro (Eckert et al. 2005)

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28 A few genes have been identified as being responsible for formation of the ICM and TE in human and mouse. The first transcription factor to appear in the la te morula ICM is Sex determining region Y box2 ( Sox2 ) (Guo et al. 2010) It is a key factor for maintenance of pluripotency and for reprogramming of differentiated cells into induced stem cells (Takahashi and Yamanaka 2006) Pou domain class 5 transcription factor 1 ( Pou5f1 or Oct4 ) is a transcription factor necessary for the maintenance of the pluripotency in the ICM and it prevents ICM tra nsformation to TE (Zernicka Goetz et al. 2009) Another stem cell marker present in the ICM is Nanog homeobox ( Nanog ) (Marikawa and Alarc n 2009) Caudal type homeobox 2 ( Cdx2 ) is specifically expressed in TE and its presence is necessary to repress Pou5f1 expression in TE. Loss of Cdx2 results in failure to downregulate Pou5f1 and Nanog in outer cells of the blastocyst and subsequent death of those cells (Strumpf et al. 2005) Nodal is found to be expressed in the ICM and primitive endoderm of the blastocyst and also latter in the epiblast and visceral epithelium (Mesnard et al. 2006) In the bovine, the mechanism of early segregation and differentiation is different in some respects from the mouse. POU5F1 is expressed in both ICM and TE of in vitro and in vivo produced blastocys ts until Day 10 of development (Eijk et al. 1999; Kirchhof et al. 2000; Mesnard et al. 2006) whereas CDX2 has a weak expression (Degrelle et al. 2005) NANOG mRNA and protein are also found in both ICM and TE of bovine blastocysts and elongated embryos, but with greater expression in the ICM and ED (Degrelle et al. 2005; Mu oz et al. 2008) A microarray experiment that analyzed the transcriptome of ICM and TE of human blastocysts has identified new marker ge nes to complement the existing markers for

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29 ICM/TE such as POU5F1 and CDX2 Pathway analysis of the microarray data identified signaling pathways related to integrin mediated cell adhesion and overexpression of several NA+/K+ ATPases in TE (Adjaye et al. 2005) reflecting their role in controlling permeabilization and fluid transport across the epithelium. Keratin 18 ( KRT18 ), a cytoeskeletal protein, was predominantly expressed in the TE cells and imu nocytochemistry detected its expression only in the cell to cell contacts of the TE (Goossens et al. 2007) Pathways involved in cell cycle were also differentially expressed. Genes that activate th e WNT signaling pathway were shown to be overexpressed in the ICM while genes encoding Casein kinase 1 alpha ( CSNK1A ) and disheveled activator of morphogenesis 1 ( DAAM1 ), which are agonists of the WNT pathway, were both over expressed in the TE (Adjaye et al. 2005) Hatching and Elongation The blastocyst becomes fully expanded when it reaches about 160 to180 cells (Van Soom et al. 1997) Ther eafter, the blastocyst hatches from the zona pellucida by a combination of cell growth and volume increase in the blastocoel (Van Soom et al. 1997; Houghton et al. 2003) The hatching process in the bovine embryo is apparently a mechanical process more than an enzymatic one (Fl chon and Renard 1978; Massip and Mulnard 1980; Massip et al. 1982) Once the blastocyst has hatched, the ICM forms a protuberance called the ED, Day 12. By Day 12, the layer of TE cells covering the ED is degraded via apoptosis and the disc cells are exposed to the maternal milieu (Williams and Biggers 1990; Guillomot et al. 2004) Elongation begins b etween Days 12 and 14 (Betteridge et al. 1988; Vejlsted et al. 2006) and is concomitant with gastrulation (Hue et al. 2001) The development of the

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30 trophoblast provides a large placental surface area that is better able to initiate the maternal conceptus cross talk and exchange essential nutrients for survival of the conceptus (Spencer and Bazer 2004) As part of the process of elongation, the conceptus undergoes shape changes going from spherical to ovoid, then tubular, and finally to the elongated stage. As a result, the conceptus increases in siz e more than 1000 fold by Day 24 of gestation so that it can extend the entire length of both uterine horns (Maddox Hyttel et al. 2003) Elongation is accomplished by an increase in cell number accom panied by an increase in protein synthesis (Thomson 1998; Degrelle et al. 2005) Elongation of bovine embryos appears to be in part determined by uterine signa ls, given that extended in vitro culture beyond the blastocyst stages results in formation of TE outgrowths and attachment of the embryo to the bottom of the culture dish rather than elongation (Ale xopoulos et al. 2005) However, in vitro produced blastocysts elongate when transferred to recipients (Block et al. 2007) One candidate as a uterine elongation factor is insulin like growth factor binding protein 1 ( IGFBP1 ). In the bovine endometrium, IGFBP1 mRNA increases in amount by Day 16 of pregnancy and is significantly different in pregnant versus non pregnant animals (Simmons et al. 2009) In vitro IGFBP1 stimulates migration and mediated attachment of ovine TE cells while had no effect on cell proliferation (Simmons et al. 2009) Maintenance of the Corpus Luteum One of the roles of the elongated conceptus is to produce the pregnancy recognition signal, interferon tau ( IFNT ), which blocks luteal regression caused by progesterone (Thatcher et al. 2001; Spencer et al. 2007) IFNT is secreted by the mononuclear cells of the primitive extra embryonic trophoblast a few Days prior to when

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31 the conceptus attac hes to the uterine wall (Helmer et al. 1987) The peak production of IFNT occurs on Days 16 to 17 of pregnancy (Thatcher et al. 2001) The w ay IFNT prevents development of the luteolytic mechanism has been well documented in sheep. superficial ductal glandular epithelia (Spencer and Bazer 2004) This action prevents the induction of oxytocin receptor transcription by estrogen and, therefore, oxytocin induced luteolytic pulses of PGF 2 (Asselin et al. 1997; Spencer et al. 1998; Pru et al. 2001; Spencer and Bazer 2004) In the cow the mechanism must be a little different, since there i s no change in estrogen receptor mRNA in the endometrium from Day 16 pregnant cows and cyclic cows but there is a decrease in oxytocin receptor mRNA (Robinson et al. 1999) Therefore, in the cow, pregnancy may alter the oxytocin receptor regardless of the estrogen one. Between Days 8 and 17 of pregnancy is when the conceptus ordinarily inhibits pulsatile PGF 2 secretion but is also when at least 40% of total embryonic losse s occur (Thatcher et al. 2001) Attachment to the Endometrium The ruminant conceptus does not actually implant in the uterus. Rather placentation occurs because of apposition and interdigitation wi th limited invasion of trophoblast cells into the endometrial epithelium. Placentation starts at about Day 20 of pregnancy (Cha vatte Palmer and Guillomot 2007 ) Cell contact is initiated in the regi on of the ED and extends towards the ends of the conceptus (Assis Neto et al. 2009a) In sheep and cow embryos, the trophoblast attaches to the caruncular epithelium mainly and to the intercaruncula r mucosa to a lesser extent. In these areas, transitory villi grow on the trophoblast and invade the uterine glands. This process ensures an anchorage of

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32 the conceptus in the uterine cavity and localized absorption sites of the glandular secretions (Chavatte Palmer and Guillomot 20 07 ) The limited invasion of the maternal tissue is caused by trophoblast cells that migrate into the endometrial epithelium and fuse with uterine epithelial cells to form binucleated cells (BNC). The BNC represent 20% of the trophoblast and produce placental lactogen and a group of inative aspartyl proteinases called pregnancy associated glycoproteins that are delivered to the maternal compartment (Szenci et al. 1998; Klisch et al. 1999) Fusion of the BNC with uterine epithelial cells forms syncytial plaques (Schlafke and Enders 1975) Due to the presence of uterine syncytium, the ruminant placenta is classified as synepitheliochorial. Placentomes The definitive placenta is differentiated into two regions cotyledons a nd intercotyledonary tissue. Placentomes are formed by interdigitation of cotyledons with corresponding structures on the endometrium called caruncles. Their primary role is nutrient and gas exchange between the fetus and the mother (Schlafer et al. 2000; Enders and Carter 2004) Cotyledons can be observed macroscopically after Day 37 of gestation and 80 to 120 cotyledons eventually form (Schlafer et al. 2000; Assis Neto et al. 2009) By the second trimester of gestation, the number of viable luteal cells decrease and the cotyledons are responsible for producing the p rogesterone necessary for maintenance of pregnancy (Shemesh 1990; Izhar et al. 1992) Alterations in Embryonic Development In vitro Pregnancy risk in recipients of IVP embryos are generally not superior to pregnancy risk following artificial insemination (Rodrigues et al. 2004; Sartori et a l. 2006) and are less than following the transfer of in vivo derived embryos (Hasler 2000)

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33 Furthermore, in vitro produced ( IVP ) embryos that survive the embryonic period are more likely to be lost later on. While pregnancy loss after the first two months of gestation for AI and superovulated embryos is generally around 5 to 14% (Santos et al. 2004; Demetrio et al. 2007; Jousan et al. 2007) pregnancy loss after Day 40 of gestation for IVP embryos ranged from 12 to 24% (Hasler 2000; Block et al. 2003; Demetrio et al. 2007) A major problem with IVP embryos compared with embryos produced in vivo is poor survival to cryopreservation (Enright et al. 2000; Rizos et al. 2002) The high sensitivity of IVP embryos to chilling and freezing can be related to a higher accumulation of cytoplasmic lipid droplets (Ushijima et al. 1999) In addition, blastocysts cultured in synthetic oviduct fluid medium (SOF) had lower expression of connexin 43, a gap junction protein that is essential for the transport of cryoprotectants and fluids during freezin g and thawing, when compared with embryos developed in coculture or in vivo (Rizos et al. 2002a) The period of embryo development, rather than the period of maturation or fertilization, is the most critical one for perturbations resulting in reduced capacity of the blastocyst for cryopreservation. Survival rates after cryopreservation of blastocysts t hat were produced by maturation and fertilization in vivo but cultured in vitro were 0% compared with 70% for blastocysts that were matured, fertilized and cultured in vivo (Rizos et al. 2002a) A s imilar conclusion that the period of embryonic development is crucial for high competence for cryosurvival was acquired when cryopreservation rates of embryos produced by in vitro maturation, fertilization and embryo culture (0%) were compared with cryopre servation rates of embryos that were matured and fertilized in

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34 vitro but cultured in the ewe oviduct (63%) (Enright et al. 2000; Rizos et al. 2010) Transcript abundance of 5 genes, elongation factor 1 gamma ( EEF1G ) guaninenucleotide binding protein ( GNB3 ) forkhead transcription factor ( FOXP3 ) represssor of estrogen receptor activity and high mobility group protein 2 ( ESR1 ) were significantly lower for blasto cysts cultured in SOF than for blastocysts produced in vivo or produced in vitro and allowed to develop in the ewe oviduct (Corcoran et al. 2007) Abnormalities for calves resulting from IVP embryos have included increased calf birth weight (Lazzari et al. 2002) altered organ development (Farin and Farin 1995) and alterations in placentome number and placental structure (Miles et al. 2005) Those abnormalities can result in an increase in the cases of dystocia and cesarean section as well as perinatal mortality (van Wagtendonk de Leeuw et al. 2000) The low pregnancy risk and increased fetal loss that are characteristic of transfers with IVP embryos are probably connected to a variety of cellular and molecular deviations during early embryonic development. Morphological evaluation of Day 14 embryos revealed that IVP embryos hav e high incidence of no detectable ED (Bertolini et al. 2002; Fischer Brown et al. 2002; Fischer Brown et al. 2004) In v itro culture of bovine embryos in the presence of high concentrations of serum or bovine serum albumin resulted in increased number of cells in Day 7 blastocysts, size of blastocysts on Day 12, and the relative abundance of the transcripts for heat shock p rotein 70.1 ( HSP70.1) copper/zinc superoxide dismutase, glucose transporters 3 and 4 ( SLC2A3 and SLC2A4 ), fiblroblast growth factor 2 (basic) ( FGF2 ), and insulin like growth factor 1 receptor ( IGF1R) when compared with in vivo derived embryos (Lazzari et al. 2002) Other studies show IVP embryos with alterations in the level of expression of X linked

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35 genes, increased chomosomally abnormal cells (King 2008) and differential expression of IGF family genes (Bertolini et al. 2002; Sagirkaya et al. 2006; Moore et al. 2007) The expression of BCL2 associated X protein ( BAX) an apoptosis related gene, was higher in blastocysts produced in vitro using SOF than for those developed in coculture or in vivo (Rizos et al. 2002a) Oxidative stress genes are also differentially expressed, with mitochondrial manganese superoxide dismutase, an important antioxidant defense in cells exposed to oxygen, strongly expressed in blastocyts developed in vivo when compared with IVP embryos while sarcosine oxidase, an oxidative enzyme, highly expressed on IVP embryos when compared with its in vivo counterparts (Rizos et al. 2002a) Microarray techniques have been used to i nvestigate differences in the transcriptome between IVP and in vivo embryos (Smith et al. 2009) In vivo produced stimul lyase activity while genes in the G protein coupled receptor signaling pathway were significantly lower for in vivo embryos. However, none of the genes differentially regulated in this study matched the imprinted genes thoug ht to be responsible for large offspring syndrome. Transcripts for enzymes involved in the de novo methylation process were downregulate in the IVP embryos compared with the in vivo derived embryos; this could lead to aberrant methylation and subsequent fe tal abnormalities (Smith et al. 2009) Growth Factors and Cytokines as Uterine Regulators of Embryonic Development Among the molecules secreted by the reproductive tract that can regulate embryonic development are various growth factors and cytokines. Originally described as protein molecules that promote cell proliferation and inhibit apoptosis, growth factors

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36 are now known to play roles in endocrine, paracrine, and autocrine regulation of a wide v ariety of cell functions. Historically, cytokines are associated with hematopoietic and immune cells; however, they are now known to be secreted by a wide variety of cells and tissues including endocrinologically responsive tissues within the reproductive tract. While some cytokines can be growth factors, like CSF1 and CSF2, others have an inhibitory effect on cell growth, and they can target cells to undergo apoptosis and cell death. The embryo itself expresses receptors for an array of growth factors and PDGFR ) and IGF1R and IGF2R are found at the oocyte stage and throughout embryo development (Yoshida et al. 1998; Wang et al. 2008 a ) Messenger RNAs for FGF2 receptor ( FGF2R) are present in all stages of oocyte maturation and embryonic development up to the 2 cell stage, and again at the blastocyst stage (Yoshida et al. 1998) Transcripts for FGFR1c, FGFR2b, FGFR3c and FGFR4 are found on in vitro produced blastocysts and the in vivo elongated Day 17 conceptus (Cooke et al. 2009) Epidermal growth factor receptor ( EGFR ) mRNA and protein have been shown to be present in spherical embryos on Day 13 and elongated embryos on Day 16 (Kliem et a l. 1998) Expression of mRNA and protein of the CSF2 receptor alpha subunit ( CSF2R ) blastocyst stage (Sj blom et al. 2002) The list of cytokines and growth factors present in the uterus is large and their physiological roles are still being resolved. The purpose of this section of the literature review is to provide examples of specific growth factors and cytok ines implicated in

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37 regulation of embryonic development in the cow and to illustrate the specific roles they may play. Insulin like Growth Factor 1 The main source of insulin like growth factor 1 (IGF1) is the liver, which secretes IGF1 in response to grow th hormone (Scharf et al. 1996) However, it is not assured that changes in circulating IGF1 cause changes in uterine IGF1 concentrations since IGF1 concentrations in uterine fluid did not change in parallel with plasma concentrations of IGF1 (Bilby et al. 2004) The uterus, oviduct and the embryo also produce IGF1 (Velazquez et al. 2008 ) although their contribution to the total IGF1 pool in uterine and oviductal fluid is unknown. Addition of IGF1 to culture medium increases the proportion of IVP bovine embryos that develop to the blastocyst stage (Block et al. 2003; Lima et al. 2006) In addition, IGF1 increased blastocyst total cell number (Makarevich and Markkula 2002; Sirisathien et al. 2003) and altered the relative abundance of developmentally important mRNA transcripts at the blastocyst stage including increases in IGFBP1 2 3 5 (Prelle et al. 2001; Block et al. 2007) desmocollin 2, ATP1A1 (Block et al. 2007) and SLC2 genes (Oro peza et al. 2004) and decreases in expression of the gene for heat shock protein 70 ( HSP70 ) (Block et al. 2007) and IGF1R (Enright et al. 2000; Prelle et al. 2001; Block et al. 2007) In addition, IGF1 protected embryos from heat shock, allowing increased development and reduced apoptosis (Jousan and Hansen 2004; Jousan et al. 2007; Jousan et al. 2008) and protected bovine embryos from the anti developmental actions of the prooxida nt menadione (Moss et al. 2009) Furthermore, IGF1 treated embryos showed increased pregnancy rates after they were transferred to recipients exposed to

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38 heat stress (Block et al. 2003; Block and Hansen 2007) Thus, IGF1 may function in pregnancy to increase embryonic development and protect embryos from specific stresses capable of disrupting development Interleukin 1 Interleukin1, beta (IL1B) is a polypeptide found in uterine flushes at least from Days 11, 14 and 17 of cyclic cows (Davidson et al. 1995) There is evidence that the source of this ILB1 is the luminal and glandular epithelium and stroma of the endometrium (Paula Lopes et al. 1999) On the other hand ILB1 could not be detected in pregnant cows on Days 25 and 30 of gestation (Davidson et al. 1995) When endometrium collected from pregnant cows was cultured with ILB1 it increased the secretion of PGE 2 and PGF from epithelial cells (Betts and Hansen 1992; Davidson et al. 1995) and from stromal cells (Davidson et al. 1995) ILB1 treatment also decreased DNA synthesis in stromal cells but had no effect on epithelial cells (Davidson et al. 1995) The presence of ILB1 in the uterus indicates that this cytokine might have a role in early embryonic development in cattl e. Addition of ILB1 to cultured bovine embryos increased the percentage age of embryos becoming a blastocyst but only if ILB1 was added at the first Day of culture and if the embryos were cultured in high density (25 30 embryos/drop) (Paula Lopes et al. 1998) The fact that effects are seen at high embryo density may mean that ILB1 acts to stimulate some embryo derived growth factor that in turn increases embryonic development. The importance of ILB1 for pregnancy was demonstrated in mouse (Sim n et al. 1998) Injections of ILB1 receptor antagonist around the preimplantation period significantly decreased the number of implantation sites in this species. When flushed at

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39 Day 8, 10 times more blastocysts were found in the uterine flush of the ILB1 receptor antagonist treated mice. Even though all the blastocysts appeared to be morphologically normal, they were delayed in development comparing to th e non injected group, were still surrounded by the zona pellucida and were not able to implant (Sim n et al. 1998) Fibroblast Growth Factor Fibroblast growth factors (FGFs) are represented by more than 22 FGF genes and 4 FGFR genes with different temporal and spatial patterns of expression during development (Itoh and Ornitz 2004; Itoh and Ornitz 2008; Gotoh 2009) Specifics FGFs, i.e. FGF2 are required for self renewal and maintenance of pluripotency activity in mouse and human embryos and embryonic stem (ES) cells (Gotoh 2009) Interestingly, FGF4 is necessary for cellular differentiation in mouse and human ES cells (Kunath et al. 2007) but is needed to maintain multipotency and self renewal in troph oblast stem cells (Guzman Ayala et al. 2004) In the cow, FGF2 has been identified within the endometrium and in the uterine lumen at Days 17 18 of the estrous cycle in pregnant and non pregnant fe males (Michael et al. 2006) Moreover, in the ewe flush, FGF2 concentrations in uterine flushings increase around Days 12 13 after estrus (O c n Grove et al. 2008) In bovine embryos, the best characterized effect of FGF2 is on TE growth and IFNT secretion. Supplementation of a TE bovine cell line (CT 1) with FGF2 increases cell proliferation and IFNT secretion (Michael et al. 2006) In addition, treatment of IVP blastocysts with FGF2 increased the expression of IFNT mRNA but had no effect on blastocyst cell number. Other FGFs, i.e. FGF1 and FGF10, have also increased the

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40 steady state amounts of mRNA for IFNT in CT 1 cells as well as IFNT biological activity (Cooke et al. 2009) Tumor Necrosis Factor Tumor necrosis factor (TNF) is a multifunctional cy tokine that first identified as a regulator of immunological and inflammatory responses in several tissues, including the reproductive tract (Hunt et al. 1996) It is produced by macrophages and ovi ductal epithelial cells in the mouse (Hunt 1993) human (Morales et al. 2006) and bovine (Wijayagunawardane and Miyamoto 2004) and by blastocysts in mouse and human (Hunt et al. 1996) In the bovine endometrium, TNF can increase PGF secretion and lead to luteolysis (Murakami 2001; Skarzynski et al. 2005; Siemieniuch et al. 2009) Exposure of mouse blastocysts to TNF decreased cellular proliferat ion and increased the percentage age of blastomeres that were apoptotic (Pampfer et al. 1997) Furthermore, there was a decrease in the number of cells in the ICM and an increase in reabsorpti on rate when these blastocysts were transferred to recipients (Wuu et al. 1999) In bovine, TNF did not affect development of embryos to the blastocyst stage (Soto et al. 2003) but it did increased the percentage age of apoptotic blastomeres in embryos exposed to the cytokine at Days 4, 5 and 6 after insemination (Loureiro et al. 2007) It is also possible that TNF is associated with mastitis, as elevated concentrations of TNF are found in the animals after infections in the mammary gland or infusion of lipopolysaccharide (Hansen et al. 2004) Cows that present mastitis have a reduction in fertility as there is an increase in the Days to first service, Days open and service per conception (Barker et al. 1998; Schrick et al. 2001) In mice, embryonic losses

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41 associated with diabetes have been related to excessive production of TNF in the uterus (Pampfer 2001) Colony Stimulating Factor 2 Biology and Signaling CSF2 is a polypeptide growth factor of 124 amino acids and with a molecular weight of 14,138 (Metcalf 1985) Most adult organs synt hesize detectable amounts of CSF2 (Burgess and Metcalf 1980) ; however increased secretion requires stimulation of cytokines, antigens, microbial products or inflammatory agents (Conti and Gessani 2008) In the human serum, concentrations of CSF2 range from 20 to 100 pg/ml (Conti and Gessani 2008) The main role of CSF2 is to promote su rvival and activation of neutrophils, eosinophils and macrophages, as well as dendritic cell maturation and differentiation of alveolar macrophages and invariant natural killer T cells ( i NKT) (Barreda et al. 2004; Conti and Gessani 2008; Hercus et al. 2009) It is thought to promote the necessary communication between the hematopoietic cells and local tissues in the event o f inflammation (Hercus et al. 2009) as well as to enhance proinflamatory cytokine production (Brissette et al. 1995) ( Figure 1 1 ). CSF2 def icient mice present deficient alveolar macrophage maturation, which leads to the development of abnormal lungs (Stanley et al. 1994) and compromised i NKT cellular differentiation (Bezbradica et al. 2006) Administration of exogenous CSF2 corrected these defects (Bezbradica et al. 2006) Furtheremore, CSF2 injection in m ice caused an increase in the levels of circulatory neutrophils and cycling peritoneal macrophages (Hamilton 2002) and administration of CSF2 specific antibody caused a decrease in inflammation in t he skin (Sch n et al. 2000) Other inflammatory reactions

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42 are promoted through activation of adhesion events and increased cell survival through inhibition of apoptosis (Hamilton 2002) Many or all cells in the stem and progenitor compartments exhibit receptors for CSF2 and are responsive to stimulation by this molecule (Metcalf et al. 1980) CSF2 receptors are expressed at very low levels (100 1000 per cell) and comprise a cytokine associated proteins important for the signaling downstream of the receptor (Quelle et al. 1994; Guthridge et al. 1998; Carr et al. 2001; Mirza et al. 2010) reaction causing dimerization of both subunits and receptor activation (Carr et al. 2001; Mirza et al. 2010) Upon binding to CSF2, the CSF2 receptor complex is a high order (Hansen et al. 2008) ( Figure 1 1 ). CSF2 and CSF2 receptors can be rapidly consumed by internalization of the complex followed by ligand endocytosis, lysosomal degradation and direct proteosomal (Barreda et al. 2004) One prominent interaction partner of CSF2 is Janus kinase 2 (JAK2), a tyrosine si tes for Src homology 2 domains of other proteins such as members of the signal transducer and activator of transcription (STAT) family. JAK2 can also phosphorylate STAT proteins themselves. In parallel, additional signaling pathways can be activated,

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43 such as the Ras Raf extracellular signal regulated kinase (Ras Raf ERK) pathway, which is important for triggering the cell cycle and is activated by binding of the adapter (Degroot 1998; Barreda et al. 2004; Choi et al. 2007) While activation of JAK2/STAT is mainly responsible for CSF2 induced cell proliferation, phosphatidylinositol subunit has a role in regulation of apoptosis and cell survival (Degroot 1998; Dhar Mascareno et al. 2005) One difference between these pathways in response to CSF2 is the concentration necessary for activation. For example, the apoptosis inhibition reaction occurs at significantly lower concentrations (100 fold) than those required for stimulation of cell proliferation (Barreda et al. 2004) ( Figure 1 1 ). cell membranes of mouse and human preimplantation embryos (Robertson et al. 2001; Sj blom et al. 2002) CSF2 Secretion in the Uterine Tract CSF2 is expressed at the protein and mRNA level in endometrial epithelial cells of humans and mice (Chegini et al. 1999; R obertson et al. 2001) in human fallopian tube (Zhao and Chegini 1994) and in human first trimester decidua (Segerer et al. 2009) In the co w, CSF2 has been localized in the oviductal epithelium (greater in ampulla) (de Moraes et al. 1999) endometrium (mostly in the luminal epithelium and the apical portions of the glands) (de Moraes et al. 1999; Emond et al. 2004) and in the myometrium after Day 30 of pregnancy (Emond et al. 2004) O n Days 14 17 after estrus, concentrations of CSF2 in uterine flushing in cattle tended to be higher in pregnant cows than cyclic cows (de Moraes et al. 1999) For

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44 cyclic cows, concentrations of CSF2 tended to be lowest during estrus compared with Days 7, 13 16 and 18 of the cycle (de Moraes et al. 1999) This pattern in the cow is unlike the mouse where CSF2 concentrations in the uterus peak during estrus (Robertson et al. 1996) Moreover, CSF2 release in the transforming growth factor ( TGF B ) (Tremellen et al. 1998) Expression declines at implantation under the (Robertson et al. 1996) but bioactive CSF2 can be detected in placental and decidual tissues for the duration of pregnancy (Crainie et al. 1990) In pregnant bitches, CSF2 mRNA was found at the onset of implantation/placen tation (Beceriklisoy et al. 2009) Actions of CSF2 on Embryo Development and Survival The use of knock out models in mice have indicated that CSF2 is important for normal embryonic development. Des pite a regular number of implantation sites, offspring from CSF2 null mice are 25% of normal size, have a 4.5 fold increase in mortality rate during the first three weeks of life and a fetal growth retardation that persists through adulthood (Seymour et al. 1997; Robertson et al. 1999) Total cell number of blastocysts from CSF2 / mice was reduced by 14 18% (Robertson et al. 2001) Cell number of embryos from CSF2 / mice was increased 24% when cultured with exogenous CSF2 (Robertson et al. 2001) Mitogenic actions of CSF2 were also demonstrated in a study in which mouse embryos were cultured in media supplemented with CSF2. The cytokine increased total cell numbers and ICM cell numbers of CF1 mice blastocysts (more sensitive strain), but only i n the absence of human serum albumin (Karagenc et al. 2005) There was no effect of CSF2 on TE cell numbers.

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45 Another line of evidence for beneficial effects of CSF2 on preimplantation embryonic deve lopment is the improvement in the proportion of cultured embryos that develop to the blastocyst stage in vitro in the cow (de Moraes and Hansen 1997 a ) human (Sj blom et al. 1999) mouse (Robertson et al. 2001) and pig (Cui et al. 2004) Bovine embryos seem to be more responsive to CSF2 when it is added to culture at Day 5 after insemination than when added within 24 hours of insemination When added at Day 5, CSF2 increased the proportion of oocytes becoming blastocyst s (de Moraes and Hansen 1997 a ) When added after insemination CSF2 had no effect on the proportion of embryos developing past the 8 cell stage (de Moraes and Hansen 1997 a ) The late effect of CSF2 coi ncides with the time that the embryo enters the uterus (Betteridge et al. 1988) Murine embryos cultured in CSF2 have a higher rate of metabolic activity and requirements. CSF2 elicited a 50% increase in the uptake of the non metabolizable glucose analogue, 3 O methyl glucose, in mouse blastocysts (Robertson et al. 2001) One possible reason for effects of CSF2 on the proportion of embryos becoming blastocysts may be the antiapoptotic effects of the cytokine. Culture of mouse embryos with CSF2 decreased the number of apoptotic b lastomeres by 50% and increased the number of viable ICM cells by 30% as a result of both antiapoptotic and proliferative effects (Sj blom et al. 2002) In another study, CSF2 protected one cell mou se embryos from freezing damage and decreased the apoptotic index to zero (Desai et al. 2007) The means by which CSF2 protects against apoptosis in embryos is not clear. In neurons, polymorphonucl ear neutrophils and HeLa cells, CSF2 can activate the PI3K Akt pathway which decreases apoptosis by induction of BCL 2 and BCL2L1 (previous

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46 known as BcL XL) (A ntignani and Youle 2007; Sch bitz et al. 2008) Using microarray analysis, CSF2 was recently shown to decrease expression of stress response genes and apoptosis genes in mouse blastocysts (Chin et a l. 2009) In vivo CSF2 null mutant mice had elevated expression of heat shock protein 1 ( Hsph1 ) (Chin et al. 2009) Sj blom et al. (2005) found that survival of mouse embryos after transfer into re cipie nt females was greater when embryos were cultured with CSF2 than when embryos were cultured without the cytokine (90% vs 76%). Along with this finding, there was evidence that CSF2 treatment during the preimplantation period affected placental develop ment after transfer. Compared with values for fetuses produced in vivo fetal placental weight ratio decreased by 8% for fetuses from CSF2 treated embryos versus 11% for fetuses from embryos cultured in medium alone. CSF2 and Interferon tau Secretion One w ay in which CSF2 could improve post transfer embryonic survival in cattle would be through increased secretion of IFNT by elongated conceptuses. Imakawa et al. (1993 and 1997) and Rooke et al. (2005) demonstrated that CSF2 stim ulates production of IFNT by sheep trophoblast in vitro Similar results were found when a cell line derived from bovine trophoblast cells (CT 1 cells) was treated with porcine CSF2 (Michael et al. 2006) On the other hand, de Moraes et al. (1997 b ) found no beneficial effect of bovine CSF2 on IFNT secretio n by bovine blastocysts at Day 7 8 after insemination Goals of the Current Investigation The aim of this dissertation is to understand the role of CSF2 in preimplantation embryonic development including its effects on blastocyst formation, the embryonic transcriptome, and long term embryonic survival. Based on the literature review, it is

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47 likely that CSF2 can act as a regulator of preimplantation embryonic development, enha nce embryo competence for survival, induce genes that promote proliferation, inhibit apoptosis and promote critical functions necessary for post transfer survival. To accomplish our goal we pursued the following research objectives: Research objective 1: to determine the effects of CSF2 on blastocyst yield and capactity for survival after transfer into recipients, and evaluate properties of the blastocyst formed after CSF2 treatment. Research objective 2: to determine the transcriptome of CSF2 treated embr yos. Research objective 3: test whether treatment of embryos with CSF2 increases post transfer growth and IFNT secretion and alters the transcriptome of the ICM and TE. Results from the proposed studies will add information regarding the biology of CSF2 during preimplantation embryonic development. The first objective establishes that CSF2 is an important regulator of embryonic development and post transfer survival. The second objective seeks to identify genes and pathways through which CSF2 regulat es embryo development and survival. The third objectives evaluates whether exposure to CSF2 during the preimplantation period leads to changes in gene expression later in pregnancy that could enhance subsequent embryonic and fetal development.

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48 Figu re 1 1 Mechanisms by which CSF2 regulates cellular survival, differentiation, functions and activation. CSF2 initiates signaling by first binding to the CSF2 enhancing activates PI3K and Akt signaling resulting in cell survival without proliferation. At high concentrations, CSF2 activates STAT or Shc dependent pathways stimulating cell survival, cellular activation and proliferation. Pulmonary CSF2 regulates the expression of numerous genes enabling multiple immune and non immune functions. CSF2 is also known as GM CSF. Figure reproduced with permi ssion from Current Opinion in Immunology 2009, 21:514 521.

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49 CHAPTER 2 COLONY STIMULATING F ACTOR IMPROVES DEVEL OPMENT AND POST TRANSFER SURVIVAL OF BOVINE EMBRYOS PRODU CED IN VITRO Introduction The embryo executes its developmental program in the female reproductive tract in an environment largely dictated by the mother. An inadequate maternal environment can lead to reduced embryonic survival (Hansen 2007; Leroy et al. 2008; Robinson et al. 2008) and epigenetic changes that persist into adulthood (Sinclair et al. 2007) The maternal environment affects embryonic development by providing to the embryo an array of nutrients and regulatory molecules and by expression of cell adhesion molecules that facilitate eventual attachment and placentation (Spencer et al. 2008) Among the regulatory factors shown to affect preimplantation development in the cow, for example, are insulin like growth factor 1 (Block 2007) i nterleukin 1, beta (Paula Lopes et al. 1998) activin (Park et al. 2008) and granulocyte macrophage colony stimulating facto r (CSF2) (de Moraes and Hansen 1997 a ) There are strong lines of evidence to implicate CSF2 as a physiologically important regulator of early embryonic development. The cytokine is expressed in lumi nal epithelium and other tissues of the oviduct and endometrium (Zhao and Chegini 1994; Chegini et al. 1999; de Moraes et al. 1999; Emond et al. 2004) In mice, production of CSF2 fluctuates during the estrous cycle, peaking during estrus (Robertson et al. 1996) Expression declines at implantation under the inhibitory (Robertson et al. 1996) Release of CSF2 into the uterine ma, including TGFB (Tremellen et al. 1998) In cattle, amounts of immunoreactive CSF2 in the endometrium of cyclic cows tends to be low during estrus and high from Days 13 to 17 after estrus before declining

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50 as estrus approaches (de Moraes et al. 1999; Emond et al. 2004) Immunoreactive CSF2 in the endometrial luminal epithelium increases in response to t he maternal recognition of pregnancy signal interferon tau ( IFNT ) so that amounts of immunoreactive CSF2 are higher in pregnant cows at Day 16 and 18 after estrus compared with cyclic cows (Emond et al. 2004) Addition of CSF2 to culture medium improved the proportion of in vitro embryos developing to the blastocyst stage in the cow (de Moraes and Hansen 1997 a ) human (Sj blom et al. 1999) and pig (Cui et al. 2004) and increased post transfer embryonic survival in mice (Sj blom et al. 2005) Total cell numbers of blastocysts from CSF2 / mice were significantly reduced by 14 18% compared with wild type controls (Robertson et al. 2001) Despite a regular number of i mplantation sites, offspring from CSF2 / mice were 25% of normal size, exhibited fetal growth retardation that persists through adulthood, and experienced increased mortality during the first three weeks of life (Seymour et al. 1997; Robertson et al. 1999) The bovine preimplantation embryo is a good model for studying maternal regulation of embryonic development. Development to the blastocyst stage is not ab solutely dependent upon regulatory molecules present in the reproductive tract because embryos that give rise to live calves after transfer to recipient can be produced in culture in growth factor free media (Block and Hansen 2007) However, embryos produced by in vitro oocyte maturation, fertilization and culture have aberrant biochemical and molecular properties compared with embryos produced in vivo (Lazzari et al. 2002; Rizos et al. 2002) Deviation in embryonic function associated with production in vitro is due in part to an inadequate environment during the

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51 preimplantation period. This is so becaus e the gene expression and cryotolerance of bovine embryos produced by in vitro fertilization was enhanced when embryos were cultured in vivo in the sheep oviduct after insemination (Rizos et al. 2002; Rizos et al. 2002) Here we tested the possible role of CSF2 as one of the regulatory molecules that mediate maternal effects during the preimplantation period on survival through the embryonic and fetal periods. The approach was to determine whether addition of CSF2 to culture medium enhances the development and post transfer survival of bovine embryos produced in vitro As a positive control, some embryos were treated with in sulin like growth factor 1 (IGF 1), which to date, is the only growth factor shown to improve survival of bovine embryos transferred to recipients. This molecule protects embryos from the effects of elevated temperature (Jousan and Hansen 2004; Jousan et al. 2008) and pregnancy and calving rates of cows exposed to heat stress were improved when embryos used for transfer were treated with IGF1 (Block et al. 2003; Block and Hansen 2007) However, no improvement in post transfer survival was seen when cows were not heat stressed (Block and Han sen 2007) The current studies were conducted in both hot and cool seasons. Materials and Methods Materials The media HEPES Tyrodes Lactate (HEPES TL), in vitro fertilization (IVF) TL, and Sperm TL were purchased from Caisson (Sugar City, ID) and used to prepare HEPES Tyrodes albumin lactate pyruvate (TALP) and IVF TALP as previously described (29). Oocyte collection medium (OCM) was Tissue Culture Medium 199 (TCM 199) with

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52 bov ine steer serum (Pel Freez, Rogers, AR) containing 2 U/ml heparin, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocyte maturation medium (OMM) was TCM 10% (v/v) bovi ne steer serum, 2 g/ml estradiol 17 20 g/ml bovine follicle stimulating hormone (Folltropin V; Belleville, ON), 22 g/ml sodium pyruvate, 50 g/ml gentamicin sulfate, and 1 mM glutamine. Percoll was from Amersham Pharmacia Biotech (Uppsala, Sweden). Potassium simplex optimized medium (KSOM) that contained 1 mg/ml bovine serum albumin (BSA) was obtained from Caisson. Essentially fatty acid free (EFAF) BSA was from Sigma (St. Louis, MO). On the Day of use, KSOM was modified for bovine embryos to pro duce KSOM BE2 as described elsewhere (Soto et al. 2003) Prostaglandin F 2 (PGF) was Lutalyse from Pfizer (New York, NY, USA) and gonadotrophin releasing hormone (GnRH) was Cystorelin from Merial (Duluth, GA, USA). Dithiothreitol (DTT) was from Sigma Aldrich (St. Louis MO, USA). Recombinant bovine CSF2 was donated by CIBA GEIGY (Basle, Switzerland). Recombinant human modified IGF1 (E3R) was obtained from Upstate Biotech (Lake Placid, NY, USA). Eff ects of CSF2 on Embryo Development and Blastocyst Properties Production of embryos Embryo production was performed as previously described (Soto et al. 2003) Cumulus oocyte complexes (COCs) were obtained by slicing 2 to 10 mm follicles on the surface of ovaries (a mixture of beef and dairy cattle) obtained from Central Beef Packing Co. (Center Hill, FL). Those COCs with at least one complete layer of compact

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53 cumulus cells were washed two times i n OCM, placed in groups of 10 in 50 l microdrops of OMM overlaid with mineral oil and matured for 20 22 h at 38.5 C in a humidified atmosphere of 5% (v/v) CO 2 in humidified air. Matured COCs were washed once in HEPES TALP and transferred in groups of 50 to 4 well plates containing 425 l of IVF TALP and 20 l of PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 M epinephrine in 0.9% [w/v] NaCl) per well and fertilized with ~ 1 x 10 6 Percoll purified spermatozoa from a pool of frozen thawed semen from three bulls. After 8 10 h at 38.5 C in a humidified atmosphere of 5% (v/v) CO 2 in humidified air, putative zygotes were removed from fertilization wells, denuded of cumulus cells by vortex in HEPES TALP, and placed in groups of 30 in 50 l microdrops of KSOM BE2. Putative zygotes were cultured at 38.5 C in a humidified atmosphere of 5% CO 2 or 5% CO 2 5% O 2 and 90% N 2 Embryos received treatment at Day 0 (i.e., immediately after insemination ) or Day 5 after insemination according to the specific experimental design. Interactions between oxygen concentration and presence of CSF2 This experiment was designed to determine the effects of CSF2 on embryonic development at culture cond itions of high oxygen [5% (v/v) CO 2 in air] or low oxygen (5% CO 2 5% O 2 and 90% N 2 v/v) and when added at Day 0 or 5 after insemination. After removal from fertilization drops, embryos were washed, placed in microdrops of KSOM BE2 medium and cultured in high O 2 or low O 2 If treatment was Day 0 after insemination embryos were placed in a 50 l KSOM BE2 drop 10 ng/ml CSF2. When treatment was added at Day 5, embryos were placed in a 45 l KSOM BE2 drop and 5 l of KSOM BE2 10 ng/ml CSF2 were added to the drop at Day 5 after insemination

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54 The concentration was chosen based on results from other experiments that addition of CSF2 at this concentration would increase blastocyst yield (de Moraes and Han sen 1997 a ) Cleavage rate was assessed at Day 3 after insemination and the percentageage of oocytes that became blastocysts was assessed at Days 7 and 8 after insemination. The experiment was replicated 11 times using 2673 oocytes. Cell number and diffe rentiation of blastocysts The experiment was designed to test whether CSF2 would increase cell number of bovine blastocysts and alter cell allocation between the trophectoderm (TE) and inner cell mass (ICM). After insemination embryos were cultured in 45 l microdrops of KSOM BE2 at 38.5C in a humidified atmosphere 5% CO 2 5% O 2 and 90% N 2 At Day 5 after insemination, 5 l of CSF2 (to create a final concentration of 10 ng/ml) or 5 l of vehicle (KSOM BE2) were added to the drops. Zona intact blastocysts were removed from culture at Day 7, washed two times in 50 l microdrops of 10 mM KPO 4 pH 7.4 containing 0.9% (w/v) NaCl and 1 mg/ml PVP (PBS PVP) by transferring the embryos from microdrop to microdrop. To label TE cells, embryos were placed in 100 l PBS PVP containing 0.5% (v/v) Triton X 100 and 100 g/ml propidium iodine for 30 s at 37C. Embryos were immediately washed in PBS PVP. Embryos were then incubated in 50 l drops of PBS PVP containing 4% (w/v) paraformaldehyde and 10 g/ml Hoechst 33258 fo r 15 min at room temperature to fix the embryos and stain ICM. Embryos were washed in PBS PVP and mounted on slides using Prolong Gold Antifade (Invitrogen, Eugene, Or, USA) and coverslips placed on the slides. Labeling of propidium iodine and Hoechst was observed using a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Gttingen, Germany). Each embryo was analyzed for the number of

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55 ICM (blue nuclei) and TE (pink nuclei), and total cell number (blue + pink nuclei) with a DAPI filter using a 20x objectiv e. Digital images were acquired using AxioVision software (Zeiss) and a high resolution black and white Zeiss AxioCam MRm digital camera. T he experiment was replicated 3 times using 18 36 embryos/group. Apoptotic blastomeres This experiment was designed to test the hypothesis that CSF2 decreases the percentage age of blastomeres that are positive for the Terminal deoxynucleotidyl transferase d UTP nick end labeling ( TUNEL) reaction. Embryos were cultured and treated with CSF2 as described above for analysis of TE and ICM. On Day 7 after insemination blastocysts, expanded blastocysts and hatching blastocysts were removed from culture and washed two times in 50 l micr odrops of PBS PVP. Embryos were fixed in a 50 l microdrop of 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature, washed twice in PBS PVP, and stored in 600 l of PBS PVP at 4 C until analysis for TUNEL labeling as described previously (Jousan and Hansen 2004; Jousan et al. 2008) The experiment was replicated 3 times using 31 58 embryos/group. Effects of CSF2 and IGF1 on Pregnancy and Calving Succ ess after Transfer to Recipients Production of Holstein embryos using X sorted semen Embryo production was performed as described above with slight modifications. Holstein COCs were purchased from one of three suppliers (Evergen, Storrs, CT, USA; Bomed, M adison, WI, USA; Trans Ova, Sioux City, IA, USA) and shipped overnight in a portable incubator at 38.5C in maturation medium or collected from slaughter house ovaries. After 20 24 hours, COCs were removed from maturation medium, washed one

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56 time in Hepes T ALP and transferred in groups of 30 to fertilization drops covered with TALP and then 3 l PHE and 20 l sperm purified by Percoll gradient were added to this. The final concentration of sperm was 1x10 6 per ml. Fo r each replicate, two to four straws of X sorted semen from one Holstein bull (Sexing Technologies Inc., Navasota TX) were used depending on the number of oocytes that had to be fertilized. In total 4 different bulls were used throughout the experiment. Af ter 20 22 h at 38.5C and 5% (v/v) CO 2 presumptive zygotes were removed from fertilization drops, vortexed in 30 l Hepes TALP for 5 min in a microcentrifuge tube and washed two times to remove cumulus cells and associated spermatozoa. Putative zygotes (2 5 30/drop) were placed in 45 l (treatment at Day 5) or 50 l (treatment at Day 0) of KSOM BE2 overlaid with mineral oil. Embryos were cultured at 38.5C in a humidified atmosphere of 5% CO 2 5% O 2 and 90% N 2 (v/v). Treatments were added at either Day 0 (i.e., immediately after insemination ) or Day 5 after insemination as described for each experiment. The proportion of oocytes that cleaved was recorded at Day 3 after insemination Morula, blastocyst and expanded blastocyst stage embryos classified as Grade 1 (Robertson and Nelson 1998) were harvested on Day 7 (Farm 1 and 2) and Day 8 (Farm 3) and loaded into 0.25 ml straws in HEPES TALP supplemented with 10% fetal calf serum and 50 M DTT. Straws containing selected embryos were then placed horizontall y into a portable incubator (Cryologic, Mulgrave, Vic, Australia) at 38.5C and transported to the respective farm.

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57 Animals The experiment was conducted at 3 locations: Farm 1 ( Brooksco Dairy, Quitman, ty of Florida Dairy Research Unit, Holstein cows between 76 and 154 Days in milk (mean = 84) were used as recipients from June 29 December, 12, 2007. Cows were housed in a free stall barn equipped with fans and a sprinkler system. Overall, 15 replicates were completed with 8 30 recipients per replicate. At Farm 2, a total of 100 primiparous and multiparous lactating Holstein cows between 75 and 376 Days in milk (mean = 161) were used as recipients from December 7, 2007 February 1, 2008. Recipients were housed in a free stall barn equipped with fans and sprinklers. Overall, 6 replicates were co mpleted with 12 26 recipients per replicate. A single replicate was performed at farm 3 on December 23, 2007 using a total of 21 multiparous lactating Holstein cows between 168 and 468 Days in milk (mean = 267). Recipients were housed in a free stall barn equipped with fans and sprinklers. Synchronization and timed embryo transfer Each week, eligible cows were organized into a group (i.e. replicate) and ovulation synchronized for embryo transfer. The timed ovulation protocol was the Ovsynch 56 procedure (Brusveen et al. 2008) Day 0 was considered the Day of expected ovulation. Hormon al treatments consisted of 100 g GnRH, i.m. on Day 10; 25 mg PGF i.m. on Day 3; and 100 g of GnRH i.m. at 56 h ours after PGF For first service cows only, the timed ovulation protocol was preceded by a Presynch protocol (two injections of 25 mg PGF i.m. 14 Days apart), with the last injection 14 Days before initiation of the

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58 timed ovulation protocol. Cows w ere diagnosed for the presence of corpus luteum (CL) at Day 7 after anticipated ovulation using an Aloka 500 ultrasound equipped with a 5 MHz linear array transducer. Cows diagnosed with a CL received epidural anesthesia [5 ml of 2% (w/v) lidocaine] and a single embryo transferred to the uterine horn ipsilateral to the ovary via the transcervical route. Effect of CSF2 and IGF1 added at Day 1 of culture on development and post transfer survival of bovine embryos The experiment was conducted from June 29 through August 31, 2007 at Farm 1 in 7 replicates. At Day 1 after insemination presumptive zygotes were randomly distributed to 50 l KSOM BE2 drops with one of three treatments: 1) KSOM BE2 alone, 2) 10 ng/ml CSF2 or 3) 100 ng/ml IGF1 Cleavage rate was assessed at Day 3 after insemination. At Day 7 after insemination, Grade 1 morula, blastocyst and expanded blastocyst stage embryos, considered transferable embryos, were harvested and transferred to lactating dairy cows synchronized using the PreSynch/Ov synch protocol. Pregnancy was diagnosed between Days 30 35 of gestation using ultrasonography and the incidence of calving recorded. Transfers were performed for 51 55 cows/treatment. Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of cult ure on development and post transfer survival of bovine embryos The experiment was conducted from September 7, 2007 through February 1, 2008 at Farms 1 3 in 15 replicates. In 8 replicates, presumptive zygotes were randomly assigned to either 50 l KSOM BE2 (control) 2) 50 l KSOM BE2 + 100 ng/ml IGF1 added at Day 1 after insemination or 3) 50 l KSOM BE2 + 10 ng/ml CSF2 added at Day 5 after insemination. Embryos assigned to the CSF2 group were placed in 45 l KSOM BE2 drop at Day 1 after insemination At Da y 5 after insemination an aliquot of 5 l KSOM BE2 + 100 ng/ml CSF2 was added to the drops to achieve a final

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59 concentration of 10 ng/ml CSF2. The remaining 7 replicates used similar treatments except the control treatment was changed so that embryos were placed in 45 l drops of KSOM BE2 at Day 1 after insemination and 5 l KSOM BE2 were added to the drops at Day 5 after insemination. Because there was no statistical difference between the two types of control embryos, control data were pooled. Cleavage r ate was assessed at Day 3 after insemination. At Day 7 after insemination, Grade 1 morula, blastocyst and expanded blastocyst stage embryos were harvested and transferred to lactating dairy cows synchronized using the PreSynch/Ovsynch 56 or Ovsynch 56 prot ocol. Pregnancy was diagnosed between Days 30 35 of gestation using ultrasonography and the incidence of calving recorded. Transfers were performed for 44 107 cows/treatment. Statistical Analysis Data on the percentageage of oocytes that cleaved and that became blastocysts and transferable embryos, the percentage age of cells that were TUNEL positive and the ICM/TE ratio were analyzed by least squares analysis of variance using the General Linear Models procedure of SAS (SAS for Windows, Version 9.0, Cary, NC). Data were transformed by arcsin transformation before analysis. The mathematical model included main effects and all interactions. Replicate was considered as a random effect and other main effects were considered fixed. Tests of significance were ma de using error terms determined by calculation of expected mean squares. All values reported are least squares means SEM. Probability values are based on analysis of arcsin transformed data while least squares means are from analysis of untransformed da ta. Data regarding the percentageage of cows that became pregnant after transfer were analyzed by logistic regression using the LOGISTIC procedure of SAS. Treatment

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60 effects were separated into individual degree of freedom comparisons using orthogonal contrasts. T hree sets of contrasts were compared. The first, testing the hypothesis that both CSF2 and IGF1 would increase pregnancy and calving rate, were control vs IGF1 and CSF2 and IGF1 vs CSF2 The second, testing the hypothesis that only CSF2 woul d increase pregnancy and calving rate, were control and IGF1 vs CSF2 and control vs IGF1 An additional contrast was also made to compare control to CSF2. Pregnancy loss was analyzed for the first and second experiment and for all the losses in both expe 2 analysis. Results Effects of CSF2 on Embryo Development The experiment evaluated whether addition of CSF2 at Day 0 or 5 after insemination would increase the percentage age of oocytes that became blastocysts. Embryos were cultured in either h igh oxygen (air) or low oxygen (5%, v/v). Overall, there was no effect of CSF2 on cleavage rate at Day 3 after insemination, with averages varying from 73 80%. There was a tendency ( P=0.09 ) for CSF2 to increase the percentage of oocytes that became blastoc ysts on Day 7 after insemination ( Figure 2 1 Panel A). Development was higher in low oxygen (P<0.0001) but there was no interaction between Day of treatment or oxygen concentration. On Day 8 after insemination ( Figure 2 1 Panel B), CSF2 increased ( P =0.05 ) bl astocyst development development was higher (P<0.0001) in low oxygen and there was a treatment x Day x oxygen interaction (P=0.06). The observed 3 way interaction between treatment, Day and oxygen is due to the fact that the difference in response to addition of CSF2 at Day 0 vs Day 5 depended upon oxygen concentration. For embryos cultured in low oxygen, CSF2 increased blastocyst development to a greater extent when added at Day 5 rather

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61 than at Day 1. For embryos in high oxygen, CSF2 increased blast ocyst development to a greater extent when added at Day 1. Effects of CSF2 on Blastocyst Total Cell Number, Cell Differentiation and Apoptosis As shown in Table 2 1 blastocysts formed in the presence of CSF2 had a tendency for an increased (P=0.066) nu mber of ICM cells and greater ICM/TE ratio (P< 0.02) when compared with control embryos. There was no significant effect of CSF2 on total cell number ( Table 2 3 ) or on the percentage of blastomeres labeled as TUNEL positive ( Table 2 4 ). Effects of CSF2 an d IGF1 on Pregnancy and Calving Success after Transfer to Recipients In the first experiment, treatments were added at Day 1 of culture and recipients were used in the summer during heat stress ( Table 2 3 ). There was a significant increase in the percenta ge age of cleaved embryos that became transferable morulae or blastocyst on Day 7 after insemination when embryos were treated with CSF2 (control vs CSF2, P<0.01; control and IGF vs CSF2, P<0.02; control vs IGF and CSF2, P<0.02; Table 2 3 ). Treatment did not have a significant effect on pregnancy rate at Day 30 35 of gestation or on calving rate although, numerically, pregnancy and calving rates were higher in the IGF1 group than for the other two groups. Pregnancy loss between Day 30 35 and term was signi ficantly lower for recipients that received a CSF2 treated embryo compared with control embryos (CSF2 vs control, P <0.05; CSF2 and IGF1 vs control, P <0.05; Table 2 3 ) Of the calves born, 85% were female. The second experiment was conducted largely during the cool season. As for the first experiment, IGF1 was given to embryos at Day 1 after insemination. In contrast, CSF2 was added at Day 5 of culture. Treatment with CSF2 was modified because of

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62 data indicating greater effects on development when added at Day 5 (see Figure 2 1 ) and the failure of CSF2 treatment at Day 1 to affect pregnancy rate ( Table 2 3 ). There was a significant increase in the percentage age of cleaved embryos that became transferable morulae or blastocyst on Day 7 after insemination w hen embryos were treated with CSF2 at Day 5 of culture (CSF2 vs control, P<0.01; CSF2 vs IGF, P<0.01; CSF2 and IGF vs control, P<0.02; CSF2 vs control and IGF, P<0.02; Table 2 4 ). Moreover, pregnancy rate (CSF2 vs control and IGF1 P<0.05; CSF2 vs IGF, P< 0.06) and calving rate (CSF2 vs control, P<0.05; CSF2 vs control and IGF, P<0.05) were higher for cows receiving embryos treated with CSF2 than embryos receiving control or IGF1 treated embryos. There was no effect of treatment on pregnancy loss for any of the treatments but again pregnancy loss was numerically lower for cows receiving embryos treated with IGF1 or CSF2 than for cows receiving control embryos. Of the calves born, 85% were female. When data from the two transfer experiments were pooled, the d ifference in pregnancy loss between cows receiving control embryos (9/40; 22.5%) and cows receiving either an embryo treated with either IGF1 (3/36; 8.3%) or CSF2 (8/98; 8.1%) was significant (P<0.025). Discussion Experiments reported here implicate CSF2 a s an important regulator of preimplantation development. Treatment of preimplantation bovine embryos with CSF2 increased the proportion of embryos that became blastocysts, in creased the cell number in the ICM and improved the survivability of embryos afte r transfer to recipients. The effect of CSF2 on post transfer survival involved both an increase in the proportion of embryos that established pregnancy by Day 30 35 (when treatment was from Day 5

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63 7 after insemination) and a reduction in the proportion of embryos at Day 30 35 which were lost before completion of gestation (when treatment was from Day 1 7 or 5 7 after insemination). The fact that treatment with CSF2 during such a narrow window of development (from Day 1 7 or Day 5 7) altered embryonic funct ion much later in pregnancy (after pregnancy diagnosis at Day 30 35) suggests that CSF2 is exerting epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development. The likel ihood that actions of CSF2 during the preimplantation period on survival after Day 30 35 represent modifications of the epigenome implies that CSF2 may be one of the molecules through which changes in maternal physiology lead to alterations in fetal progra mming. An example of the importance of maternal function for conceptus development is induction of fetal overgrowth in sheep by transfer of embryos into an advanced uterine environment (Wilmut et al. 1981) or premature elevation of progesterone concentrations (Kleemann et al. 1994) Bovine embryos produced in vitro which are not exposed to most of the regulatory molecu les produced by the reproductive tract, are associated with an array of fetal abnormalities including increased rates of abortion, fetal overgrowth, and altered metabolism (Farin and Farin 1995; Lazzari et al. 2002; Miles et al. 2005) Maternal effects on conceptus development involve epigenetic alterations in DNA methylation patterns, as has been demonstrated for sheep exp osed to nutritional stress (Sinclair et al. 2007) and for embryos produced in vitro in cattle (Suzuki et al. 2009) Treatment with CSF2 increased the proportion of embryos becoming blastocysts regardless of whether it was added immediately after insemination or at Day 5 after

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64 insemination (a time when embryos were at the morula stage of development). Similar results wer e seen in an earlier experiment with bovine embryos (de Moraes and Hansen 1997 a ) Thus, the action of CSF2 to increase the ability of embryos to advance to the blastocyst stage of development involv es actions on the embryo during the transition from the morula to blastocyst stage of development. Perhaps, CSF2 is mitogenic and increases the number of embryos that have cell numbers sufficient for blastocoele formation. Another possibility, that CSF2 increases cell number by blocking apoptosis, is less likely. Although culture of human embryos with CSF2 decreased the number of apoptotic blastomeres by 50% (Sj blom et al. 2002) no effect of CSF 2 on apoptosis was observed in the present experiment. Alternatively, CSF2 could increase one or more of the molecules involved in blastocyst formation. The observation that blastocysts from CSF2 treated embryos had proportionally more ICM cells relative to TE indicates the capacity of CSF2 to affect blastocyst differentiation. In the human (Sj blom et al. 2002) and mouse (Karagenc et al. 20 05) CSF2 treatment in culture resulted in more cells in the ICM of blastocysts. The magnitude of the effect of CSF2 on blastocyst yield depended on the timing of exposure and the oxygen concentration used for culture. For embryos cultured in low oxygen, the increase in blastocyst yield caused by CSF2 was greater when the cytokine wa s added at Day 5 than at Day 0. The opposite was true when embryos were cultured in high oxygen. Differences in response between Day 0 and Day 5 may reflect degradation of CS F2 during culture or down regulation of CSF2 receptors. The importance of oxygen as a determinant of the effect of timing of CSF2 precludes simple explanations, however.

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65 The improvement in survival of embryos after transfer to recipients also varied with timing of CSF2 exposure. In the initial experiment, in which embryos were exposed to CSF2 from Day 1 7 after insemination, there was no effect of CSF2 on pregnancy risk at Day 30 35 of gestation. In the second experiment, however, when CSF2 treatment was from Day 5 7 after insemination, pregnancy risk at Day 30 35 was greater for cows receiving embryos treated with CSF2 than for cows receiving control embryos. Caution must be taken when interpreting these results. The first experiment was done during heat stress, a factor that can compromise embryonic survival (Hansen 2007) and involved relatively small numbers of transfers so that real treatment effe cts may not have been observed. What is clear is that, CSF2 reduced the loss of pregnancies occurring after Day 30 35 of gestation regardless of whether CSF2 was administered from Day 1 7 or 5 7 after insemination. In the mouse, as well, treatment of embryos with CSF2 in culture enhance d fetal and postnatal growth (Sj blom et al. 2005) To our knowledge, CSF2 represents the only regulatory molecule shown to affect post placentation events when acting during the preimplantation per iod. As mentioned above, the effect of CSF2 to improve survivability of the conceptus after Day 30 35 of gestation is likely the result of alterations in the conceptus epigenome. Actions of CSF2 to affect pregnancy risk at Day 30 35 could involve changes in embryonic development related to functions important for establishment of pregnancy. One action of CSF2 that might enhance survival after transfer is the increase in number of ICM cells in the blastocyst. In the mouse, CSF2 increased the number of ICM c ells (Karagenc et al. 2005) and post transfer survival of embryos (Sj blom et al. 2005) In the mouse, the number of cells in the ICM correl ates with

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66 viability after transfer (Lane and Gardner 1997) The ICM/TE ratio for bovine embryos produced in vitro differs considerably from embryos generated in vivo (Iwasaki et al. 1990; Crosier et al. 2001; Knijn et al. 2003) Moreover, a high proportion of embryos produced in vitro have no detectable embryonic dis c by Day 14 of gestation (Fischer Brown et al. 2004; Block et al. 2007) At Day 16 of gestation, embryonic discs were reported to be smaller for embryos produced in vitro than embryos produced in vivo (Bertolini et al. 2002) Perhaps, CSF2 minimizes the detrimental effects of c ulture on embryonic disc development or survival by increasing the ICM/TE ratio. Another way in which CSF2 might improve post transfer embryonic survival at Day 30 35 of gestation would be through increased secretion of the anti luteolytic pregnancy recog n ition signal IFNT by elongated embryos. At least 40% of total embryonic losses have been estimated to occur between Days 8 and 17 of pregnancy (Thatcher et al. 1995) when the conceptus ordinarily i nhibits pulsatile PGF 2 secretion. Treatment with CSF2 has been reported to increase IFNT production by elongated sheep embryos (Imakawa et al. 1993; Imakawa et al. 1997; Rooke et al. 2005) and a cell line derived from bovine trophoblast cells (Michael et al. 2006) On the other hand, de Moraes and Hans en (1997 ) found no beneficial effect of bovine CSF2 on IFNT secretion by bovine blastocysts at Day 7 8 after insemination CSF2 could also affect expression of other genes during the preimp lantation period. The production of embryos in vitro alters the expression of several developmentally important genes (Bertolini et al. 2002; Rizos et al. 2002; Sagirkaya et al. 2007; McHughes et al. 2009) and such alterations in gene expression can persist at least through Day 25 of gestation (Moore et al. 2007)

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67 Treatment of embryos with IGF1 represents a positive control. This treatment has been reported to increase embryo survival when transfers to lactating cows are performed during p eriods of heat stress but to be without effect on embryonic survival when heat stress is not present (Block et al. 2003; Block and Hansen 2007) Similar result s were obtained in the present study. In the first embryo transfer study, where cows were exposed to heat stress, pregnancy risk at Day 35 was highest for cows receiving embryos treated with IGF1 Treatment effects were not significant but the lack of sig nificance may represent the small number of animals used for the study. In the second embryo transfer study, conducted mostly outside the time of year when heat stress is present, there was no improvement in pregnancy risk compared with the controls. Int erestingly, like CSF2, IGF1 reduced pregnancy loss af ter Day 30 35 in both studies. Thus, this growth factor may also cause changes in preimplantation development that result in changes in post placentation development conducive for conceptus survival. A practical outcome of this study is that CSF2 may prove useful as an additive to culture media for in vitro production of bovine embryos for commercial embryo transfer programs. There is a compelling need for such treatments. Pregnancy rates follow ing transfer of embryos produced in vitro is lower than following transfer of in vivo derived embryos (Farin and Farin 1995) In addition, pregnancy loss after ~ Day 40 of gestation is higher for emb ryos produced in vitro than for embryos produced in vivo IVP embryos that survive the fetal period are more likeable to be lost later on (Farin and Farin 1995)

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68 Table 2 1 Effect of CSF2 on total ce ll number, inner cell mass (ICM), trophectoderm (TE) and ICM/TE ratio of blastocysts at Day 7 after insemination Control CSF2 P value Total cell number ICM 154 9.02 41.9 4.1 170 10.8 69.7 4.6 NS 0.066 TE 112 5.6 106 6.2 NS Ratio ICM/TE 0.42 0.023 0.66 0.026 0.02 Values are expressed as the mean SEM Replicates= 3; N= 18/36 embryos/group Table 2 2. Effect of CSF2 on total cell number and TUNEL positive blastomeres in blastocysts at Day 7 after insemination Control CSF2 P value Total cell number Percentage of apoptosis 146 5.3 10.4 1 154 5.5 8.7 1 NS NS Values are expressed as the mean SEM Replicates= 3; N= 31/58 embryos/group

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69 Table 2 3 Effect of CSF2 and IGF1 added at Day 1 of culture on embryonic development at Day 7, pregnancy risk at Day 30 35 (based on ultrasonography), calving rate and pregnancy loss among recipient cattle that received embryos that were cultured in 5% O 2 Transferable embryo yield a ( percentage %) Pregnancy Risk Calving Rate Pregnancy Loss Control (C) 17% 2 18/52 = 35% 14/52 = 27% 4/18 = 22% IGF1 (I) 18% 2 24/55 = 43% 22/55 = 40% 2/24 = 8% CSF2 ( Day 1; G1) 25% 2 18/51 = 35% 18/51 = 35% 0/18 = 0% Orthogonal Contrasts SET 1: C vs I and G1 <0.02 NS NS <0.05 SET 1: I vs G1 < 0.06 NS NS NS SET 2: C and I vs G1 <0.02 NS NS NS SET 2: C vs I NS NS NS NS SET 3: C vs G1 <0.01 NS NS <0.05 SET 3: C and G1 vs I NS NS NS NS a Transferable embryo yield: Grade 1 or 2 morulae and blastocysts at Day 7.

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70 Table 2 4 Effect of CSF2 added at Day 5 of culture and IGF1 added at Day 1 of culture on embryonic development at Day 7, pregnancy risk at Day 30 35 (based on ultrasonography), calving rate and pregnancy loss among recipient cattle that received embryos that were cultured in 5% O 2 Transferable embryo yield a ( percentage %) Pregnancy risk Calving rate Pregnancy loss Control (C) 10% 1 27/79 = 34% 17/74 = 23% 5/22 = 22% IGF1 (I) 14% 2 12/44 = 27% 11/44 = 25% 1/12 = 8% CSF2 ( Day 5; G5) 14% 1 47/107 = 43% 39/104 = 37% 5/44 = 11% Orthogonal Contrasts SET 1: C and I vs G5 <0.02 <0.05 <0.05 NS SET 1: C vs I NS NS NS NS SET 2: C vs I and G5 <0.02 NS NS NS SET 2: I vs G5 <0.05 <0.06 NS NS SET 3: C vs G5 <0.01 NS <0.05 NS SET 3: C and G1 vs I NS NS NS NS a Transferable embryo yield: Grade 1 or 2 morulae or blastocysts at Day 7.

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71 Figure 2 1 Percentage age of oocytes that developed to the blastocyst stage at Day 7 (Panel A ) and 8 after insemination (Panel B ). There was a tendency for an increase in t he percentage age of blastocysts at Day 7 (treatment, P=0.09; treatment x Day x oxygen= NS) and a significant increase in blastocyst development at Day 8 (treatment, P=0.05; treatment x Day x oxygen= 0.06) when embryos were treated with CSF2. There was an effect of oxygen on the percentage age of oocytes that became blastocysts at Days 7 and 8 on both Days of treatment (P<0.0001)

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72 CHAPTER 3 COLONY STIMULATING F ACTOR 2 CAUSES CHANG ES IN THE TRANSCRIPTOME OF THE BOVINE PREIMP LANTATION EMBRYO INC LUDING ALTERATIONS I N EXPRESSION OF DEVELO PMENTAL AND APOPTOSI S GENES Introduction Colony stimulating factor 2 (CSF2) is an important local regulator of embryonic function during early pregnancy in several mammalian species. It is expressed in the oviduct (Zhao and Chegini 1994; de Moraes et al. 1999) endometrium (Chegini et al. 1999; de Moraes et al. 1999; Robertson et al. 2001; Emond et al. 2004) and decidua (Segerer et al. 2009) Addition of CSF2 to culture medium improved the proportion of cultured embryos developing to the blastocyst stage in the cow (de Moraes and Hansen 1997 a ; Chapter 2 ) ; human (Sj blom et al. 1999) mouse (Robertson et al. 2001) and pig (Cui et al. 2004) and also causes a preferential increase in the number of cells in the inner cell mass (ICM) in the human ( Sj blom et al. 2002 ) mouse (Karagenc et al. 2005) and cow ( Chapter 2) In the mouse blastocyst, CSF2 increased expression of genes involved in glucose transport (Robertson et al. 2001 ) and decreased incidence of apoptosis (Sj blom et al. 2002; Desai et al. 2007) A recent report using the cow as a model indicates that changes in blastocyst function caused by CSF2 are sufficient to increase the competence of the embryo for sustained development through the embryonic and fetal periods of pregnancy. In particular, addition of CSF2 to culture medium at Day 5 after insemination increas ed post transfer survival of bovine in vitro produced (IVP) embryos by increasing pregnancy rate at Day 30 35 of gestation and decreasing loss of pregnancies after Day 30 35 ( Chapter 2 ). The actions of CSF2 to enhance competence of the embryo for post bla stocyst development could involve changes in the embryonic transcriptome that

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73 alter important functions for establishment of pregnancy and for differentiation and growth of the conceptus. The objective of the present study was to determine changes in the t ranscriptome of the bovine embryo caused by CSF2 and thereby identify gene pathways and ontologies that are involved in actions of C S F2 on formation of the blastocyst and post transfer pregnancy success. Materials and Methods In vitro Production of E mbryo s Embryo in vitro production (IVP) was pe rformed as previously described (Chapter 2) with modifications described below. Cumulus oocyte complexes (COCs) from ovaries from a mixture of beef and dairy cattle were collected in Tissue Culture Medium 199 (TCM supplemented with 2% (v/v) bovine steer serum (Pel Freez, Rogers, AR) containing 2 U/ml heparin, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocytes were allowed to matur e for 20 22 h in groups of 10 in 50 l microdrops of TCM bovine steer serum, 2 g/ml estradiol 17 20 g/ml bovine follicle stimulating hormone (Folltropin V; Belleville, ON, Canada), 22 g/ml sodium pyruvate, 50 g/ml gentamicin sulfate, and 1 mM glutamine. Matured oocytes were then washed in HEPES TALP (Parrish et al. 1986 ; Caisson, Sugar City ID, USA) and transferred in groups of 50 to four well plates containing 600 L of IVF TALP supplemented with 25 L PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 M epinephrine in 0.9% [w/v] NaCl), and fertilized with 30 L Percoll purified spermatozoa (~ 1x10 6 sperm cells). Sperm were prepared from a pool

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74 of frozen thawed semen from three different bulls; a different set of bulls was generally used for each replicate). Fertilization proceeded for 18 20 h at 38.5 C in a humidified atmosphere of 5% (v/v) CO 2 in humidified air. Putative zygotes were removed from fertilization plates, denuded of cumulus cells by vortexing in HEPES TALP, and placed in groups of 30 in 45 l microdrops of KSOM BE2 (Soto et al. 2003) Embryos were cultured at 38.5 C in a humidified atmosphere of 5% CO 2 or 5% CO 2 5% O 2 and 90% N 2 (v/v). Cleavage rate was assessed at Day 3 after insemination. At Day 5 after insemination, 5 l of KSOM BE2 or 5 l of KSOM BE2 containing 100 ng/ml C SF2 (a gift from Novartis, Basle Switzerland) were added to each drop to achieve a final CSF2 concentration of 0 or 10 ng/ml. The concentration of CSF2 was one that increased blastocyst yield and post transfer pregnancy rates when added at Day 5 after inse mination (de Moraes and Hansen 1997 a ; Chapter 2 ) On Day 6, 24 hours after treatment, morulae and early blastocysts were selected and washed three times in 50 l microdrops of 10 mM PO 4 buffer, pH 7.4 containing 0.9% (w/v) NaCl and 1 mg/ml polyvinylpyrrolidone (PBS PVP) by transferring the embryos from microdrop to microdrop. Embryos were frozen at 80C in PBS PVP in groups of 50. A total of 4 groups of embryos from each treatment were prepared in a total of 6 replicates of in vitro production. RNA Purification and P rocessing Total cellular RNA was extracted from embryos with the RNeasy Plus Micro kit (Qiagen RNA was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was determined by Agilent 2100 Bioanalyzer

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75 with RNA 6000 Nano LabChip kit (Agilent Technologies, Santa Clara CA, USA). Only samples that showed RNA integrity > 7 were used for the microarray hybridization and quantitative PCR analysis. Extracted RNA was stored at 80 C until microarray analysis. Microarray Hybridization The effect of CSF2 on gene expression was assessed using the Bos taurus Two Color Microarray Chip from Agilent v1 (Agilent Technologies ). This array contains 21,475 unique 60 mer probes, representing approximately 19,500 distinct bovine genes arranged on a slide as 4 arrays in a 4 x 44K format. The probes were developed by cluster ing more than 450,000 mRNA and EST sequences of the bovine genome (btau 2.1). A total of four separate samples of RNA from CSF2 treated embryos and four separate samples of RNA from control embryos were subjected to microarray analysis. All microarray prot ocols were carried out by Mogene LLC (St. Louis MO, USA), an Agilent Certified Service Provider. Prior to labeling, samples of RNA (1.5 3.0 pg/ l) were concentrated to 5 l using a Savant SpeedVac (Therno Scientific) at low heat and amplified into ss cDNA using the NuGEN WT Ovation Pico RNA amplification System (NuGen Technologies, Inc, San Carlos, CA, USA) following manufacturer's instructions. Amplified ss cDNA was purified using Zymo Spin IIC columns (Zymo Research, Orange, California, USA) and stored at 20 o C overnight. Product yield and purity were determined by the Nanodrop 1000 spectrophotometer assuming that 1 absorbance unit at 260 nm of ss cDNA is equal to 33 g/ml. All 260/230 values were greater than 2. Aliquots containing 2 g of ss cDNA were l abeled with the Agilent Genomic DNA Enzymatic Labeling kit to incorporate cyanine 3 or 5 labeled CTP. For half the

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76 replicates, ss cDNA from control embryos was labeled with Cy3 and ss cDNA from CSF2 treated embryos was labeled with Cy5. For the other rep licates, control embryos were labeled with Cy 5 and CSF2 treated embryos with Cy3. Hybridizations were set up using 2 g of each sample (Cy3 and Cy5 components) for a total of 4 g per array. Volumes were brought to 44 l with Diethylpyrocarbonate (DEPC) w ater and then 11 l of Agilent 10x GE Blocking Agent was added. The mixtures were incubated at 98 o C for 3 minutes and then cooled to room temperature for 5 minutes before adding 55 l of Agilent 2x Hi RPM Hybridization buffer. Tubes were flash spinned on a microfuge and lightly vortexed before loading 100 l of the hybridization mixture onto each array. Hybridization was carried out for 17 h at 65C and 10 rpm in a SureHyb gasket slide (Agilent). Washing and scanning procedures were carried out using standa rd Agilent guidelines for gene expression microarray processing. At the end of hybridization, microarray slides were sequentially washed using standard Agilent guidelines for gene expression microarray processing. Microarray slides were scanned immediately using an Agilent G2505B scanner. Analysis of Microarray Data Images were extracted and pre processed using the Agilent Feature Extraction Software v 9.5 with default analysis parameters for the initial extraction, signal the generated data. The software produces background adjustment and normalizations for the dye of individual genes. The intensity of each spot was summarized as the median pixel intensity. All the generated values were then transformed to log 2 The Lowess method was used for intensity normalization within each array. JMP Genomics 3.1 for SAS 9.1.3 software

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77 (SAS Inst., Inc., Cary, NC) was used for data global normalization and identification of differentially expressed genes. The PROC ANOVA procedure was used for simultaneous comparisons and the quantile method for intensity normalization. The model included replicate and treatment. Replicate (array) was considered random and treatment was considered fixed. Correction for false discovery rate was performe d by the Benjamini and Hochberg method (Benjamin i and Hochegerg 1995) Only genes with median pixel intensity of at least 2.8 were considered to be expressed. To increase the reliability of the results, only genes with a 1.5 fold difference and false disco 0.01 were considered differentially expressed. David Bioinformatics Database ( http://david.abcc.ncifcrf.gov/ ; Huang et al. 2009) was used to categorize the ge nes into different ontologies. The Ingenuity pathway analysis software ( http://www.ingenuity.com ) was used to determine differentially regulated pathways and to modify existing canonical pathways to better fit the findings from the literature. The significance of the association between the list of genes and the canonical pathway was measured by the ratio of the number of molecules from the data set that mapped to the pathway divided by th e total number of molecules in the pathway and a p association between the genes and the pathway is explained by chance alone. To more completely fit differentially expressed genes into pathways, analyses of each gene based on available literature was used to modify certain canonical pathways to include additional genes. Quantitative Real Time PCR Quantitative Real Time PCR analysis (qPCR) of 16 differentially expressed genes

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78 housekeeping gene (GAPDH) for use as an internal control. Specific primers ( Table 3 1 ) were designed using Integrated DNA Technologies software (http://idtdna.com). The qPCR analys is was carried out by Mogene LLC with the Applied Biosystems Taqman Gene Expression Mastermix (Foster City, CA, USA). Each reaction consisted each primer and probe being 200 nM. All probes had a 5' 6 Fam label and a 3' IA Black Quencher. Following incubation at 95C for 10 min, 40 cycles of denaturation (95C for 15 s) and annealing/synthesis (60C for 1 min) were completed. Each RNA sample was analyzed in triplicate. Res ponses were quantified based on the threshold cycle (CT). All CT responses from genes of interest were normalized to the housekeeping T T for each sample was calculated by T of CSF2 treated group fro m the control. Fold change of genes in the CSF2 treated group was determined by solving for 2 relative to the controls. Treatment effects were analyzed by the median scores procedure of SAS (SAS for Windows, Version 9.0, Cary, NC, USA). Regulation of A poptosis by CSF2 This experiment was designed to verify whether CSF2 could regulate apoptosis by testing whether treatment of embryos with CSF2 reduced the induction of apoptosis caused by heat shock. The experimental design involved a 2 x 2 factorial arrangement of treatments (control vs heat shock and 0 or 10 ng/ml CSF2). Putative z ygotes were randomly distributed in 45 l microdrops of KSOM BE2 at 38.5C in a humidified atmosphere of 5% CO 2 5% O 2 and 90% (v/v) N 2 At Day 5 after insemination, 5 l of vehicle (KSOM BE2) or CSF2 (to create a final concentration of 10 ng/ml) were added to the drops. On Day 6 (24 h after treatment), morulae and blastocysts were selected,

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79 placed in previously conditioned microdrops of the same treatment and then cultured at either 38.5C or 42C for 15 h in an atmosphere of 5% CO 2 Embryos were then returned to an environment of 38.5C in a humidified atmosphere of 5% CO 2 5% O 2 and 90% (v/v) N 2 for 9 h back Embryos were then removed from culture and washed three times in 50 l microdrops of PBS PVP. Embryos were fixed in a 50 l microdrop of 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature, washed twice in PBS PVP, and stored in 600 l of PBS PVP at 4 C until analysis for terminal deo xynucleotidyl transferase dUTP nick end labeling (TUNEL) using the in situ Cell Death Detection Kit TMR red (Roche Diagnostics, Indianapolis, USA) to determined apoptotic nuclei as described previously (Loureiro et al. 2007) The experiment was replicated 2 times using 65 81 embryos/group. Results Transcriptomal P rofile As shown in a Venn diagram ( Figure 3 1 A), there were a total of 17884 genes that were expressed by embryos from both treatments (con trol and CSF2), 276 genes expressed exclusively expressed in CSF2 treated embryos and 282 genes present only in control embryos (intensity cutoff for expression = 2.8). Hierarchical analysis ( Figure 3. 1B) of microarray results indicated that the four CSF2 s amples formed a distinct cluster from the four control samples. Differentially expressed genes were determined as those genes where there were at least a 1.5 false discovery rate o f 0.01). A total of 214 genes met these criteria and 160 of these could be annotated (67 genes upregulated by CSF2 and 93 genes downregulated).

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80 The five most upregulated genes in terms of fold change were calcium channel, voltage dependent, alpha 1G subun it ( CACNA1G ; 9.2 fold increase), stearoyl coenzyme A desaturase ( SCD ; 6.5 fold increase), Kruppel like factor 8 ( KLF8 ; 5.8 fold increase), secreted frizzled related protein 4 ( SFRP4 ; 4.0 fold increase), and alcohol sulfotransferase ( SULT2A1 ; 3.2 fold incre ase). The five most downregulated genes, in terms of fold change were olf actomedin 4 ( OLFM4 ; 4.2 fold decrease), wingless type 16 ( WNT16 ; 3.8 fold decrease), neural precursor cell expressed, developmentally down regulated 4 ( NEDD4 ; 3.8 fold decrease), MAP kinase activating death domain ( MADD ; 3.7 fold decrease), and coiled coil domain containing 103 ( CCDC103 ; 2.9 fold decrease). Biological Process Ontologies A ffected by CSF2 Analysis using the David software categorized the differentially expressed ge nes into 13 biological process ontologies ( Table 3 2 ). These ontologies could be grouped into four functional groups. One was for genes involved in development and differentiation. There were 42 differentially expressed genes in the developmental process ontology (26% of the differentially expressed annotated genes), 32 genes in the multicellular organ development ontology, 26 genes in the system development ontology, 26 genes in the anatomical structure development ontology, 23 genes in the cellular deve lopmental process cell differentiation ontology and 5 genes in the pattern specification process. The second was for differentially expressed genes involved in signal transduction and cell communication. There were 45 differentially expressed genes in the cell communication ontology (28% of the differentially expressed genes), 44 differentially expressed genes in the signal transduction ontology, and 25 genes in the cell surface receptor linked signal transduction ontology. The third was for genes

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81 involved in apoptosis with 9 differentially expressed genes in the regulation of apoptosis ontology and 6 genes in the induction of programmed cell death ontology. The fourth group was for genes involved in cell adhesion with 13 differentially expressed genes in t he biological adhesion cell adhesion ontology. There was no pattern for CSF2 to regulate cell adhesion and migration genes in a way that would consistently promote or inhibit cell adhesion or migration. Genes Involved in Cellular Development and Differen tiation Many of the differentially regulated genes identified by DAVID as being involved in developmental processes were involved in neurogenesis, mesoderm or muscle formation, and pluripotency. A total of 13 genes identified as being involved in neurogene sis were differentially regulated by CSF2, with 4 upregulated genes ( MAB21L2 3.0 fold increase; HUWE 2.5 fold increase; NOTCH2, 2.2 fold increase RTN4 1.6 fold increase) and 8 downregulated genes ( DTX3 2.2 fold decrease; HUNK 2.1 fold decrease; CELSR 1.6 fold decrease; SEMA4 1.6 fold decrease; ARSA 1.6 fold decrease; GREM1 1.6 fold decrease; GLIS2 1 6 fold decrease; CHURC1 1.5 fold decrease; and RGS12 1.5 fold decrease). A total of 8 differentially regulated genes were identified as being assoc iated with mesenchyme, mesoderm or muscle cells, including regulation of the epithelial mesenchymal transition. Upregulated genes were KLF8 (5.8 fold increase), MYF6 (3.1 fold increase), HOXA5 (2.2 fold increase), NOTCH2 (2.2 fold increase), CD73/NT5E (2. 2 fold increase) and FHL1 (2.1 fold increase) while DTX3 (2.2 fold decrease) and GREM1 (1.7 fold decrease) were downregulated by CSF2. T wo of these genes are involved in hematopoiesis ( KLF8 and NOTCH2 ). In addition, CSF2 upregulated expression of two oth er genes involved in hematopoiesis, CCL23 (2.5 fold increase ) and HOXA5 (2.2 fold increase), and

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82 decreased expression of another gene involved in hematopoiesis ( CXCL12 1.9 fold decrease). Genes Involved in Signal Transduction and Cell Communication Analysis of differentially regulated genes in signal transduction and cell communication ontologies ( Table 3 2 ) indicates a broad range of effects of CSF2 on expression of genes encoding ligands ( TNFSF8, CXCL2, CXCL12 ), receptors ( OR51E1, CCRL1, GIPR, NC OA7, GRID1, OR2T11, ITPR2, PTGER4, GPR143 ), receptor tyrosine kinases ( ROR2, PTPRK, RIPK3 ), other protein kinases ( PTPN22, MARK2, PRKA2B, PTPRK, DAPK1, CSNK2B, MADD ), transcription factors ( KLF8, MYF6 ) and cAMP regulators ( NOTCH2, PDE7B, CREM, PTGER4 ). Gen es Involved in WNT Signaling One signaling system that was represented often in the list of differentially genes regulated in the most consistent manner was the WNT system. As shown in Table 3 3 expression of a total of 10 genes involved in WNT signalin g were affected by CSF2. Of the 6 upregulated genes, four ( SFRP4/FrpHE, NOTCH2, PPP2R3A and PCDH24 ) are inhibitory to catenin dependent signaling. Of the 4 downregulated genes, two activate the catenin dependent signaling pathway ( WNT16 and CSNK2B) a nd all 4 activate at least one catenin independent signaling pathway ( WNT16, CSNK2B, ROR2 and CELSR2). The data set of differentially expressed genes was also examined for 70 genes reported to be upregulated by WNT signaling (Ambrosetti et al. 2008; Segditsas et al. 2008; Chien et al. 2009) Only three genes in this list were found to be differentially

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83 regulated by CSF2. GREM1 (1.7 fold decrease), SEMA4 (1.6 Fold decrease) and MAB21L2 (3.0 fold increase). Genes I nvolved in Apoptosis Signaling P athway Effects of CSF2 on genes involved in apoptosis signaling are shown in Table 3 4 A total of 16 genes we re differentially regulated. Of the 7 upregulated genes, 3 are anti apoptotic ( TNFSF8, PRKAR2B, CD73/NT5E, and PGR ), one ( NOTCH2 ) is usually, but not always, anti apoptotic, and 2 ( CASP7 and RTN4 ) are pro apoptotic. Of the 9 genes downregulated by CSF2, 6 are pro apoptotic ( NOD2, PIK3IP1, RIPK3, MADD, DAPK1, and CREM ) and 3 are anti apoptotic ( RNF7, CXCL12, and PLD2 ). Quantitative Real Time PCR Quantitative real time PCR (qPCR) was used to confirm the findings of the microarray analysis ( Figure 3 2 ). For 1 1 of the 16 genes examined, the fold change caused by CSF2 as determined by qPCR microarray was in the same direction as the fold change as determined by microarray hybridization. The effect of CSF2 as determined by qPCR was significant (P<0.001) for one o f these genes ( SLC16A10 ), approached significance (P=0.09) for 7 genes ( PPP2R3A, PMM2, ANKRD37, NOTCH2, MAB21L2, MRSPS12, and MADD ) and was not significant for three genes ( VGLL2, ECE1, and TNFSF8 ). One gene, WNT16 whose expression was decreased by CSF2 a s determined by microarray hybridization was increased by CSF2 (P<0.001) as determined by qPCR. While not significant, the same trend was apparent for RIPK3 and HK1 Two other genes affected by CSF2 as determined by microarray, had a fold change near 1.0 as determined by qPCR ( MASP2 and CSNK2B).

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84 Actions of CSF2 to Block Heat Shock Induced A poptosis As another validation of the microarray results, an experiment was performed to test whether embryonic function was changed by CSF2 in a way predicted from the microarray results. In particular, since CSF2 altered gene expression in a way that would inhibit apoptosis, it was tested whether culture of embryos with CSF2 beginning at Day 5 after insemination would block induction of apoptosis caused by heat shock initiated at Day 6. As shown in Figure 3 3 E, culture at 42C for 15 h increased the percentage of blastomeres that were TUNEL positive and reduced total cell number (P<0.0001). While CSF2 did not affect the percentage of cells that were TUNEL positive at 38.5C, it blocked the increase in TUNEL labeling caused by heat shock (CSF2, P<0.005). Discussion Colony stimulating factor 2 is an important regulator of preimplantation embryonic development. In the cow, it can increase the proportion of embryos that d evelop to the blastocyst stage, increase the number of cells in the ICM improve the competence of the embryo to establish pregnancy after transfer to a recipient female, and reduce the probability of fetal loss after Day 30 35 of pregnancy (de Moraes and Hansen 1997 a ; Chapter 2 ) As indicated by changes in the embryonic transcriptome described in this paper, these actions of CSF2 involve changes in expression of genes controlling developmenta l and apoptotic processes. Changes in expression of genes controlling development are likely to lead to a blastocyst whose developmental trajectory favors embryonic survival in the embryonic and fetal period. Actions of CSF2 to increase resistance to induc tion of apoptosis could conceivably contribute to growth of the embryo and survival after transfer into a recipient with suboptimal uterine environment.

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85 A large number (42) of the genes whose expression was altered by CSF2 are genes implicated in developme ntal processes. A total of 26% of annotated genes that were differentially expressed were in the developmental process ontology. Thus, it is likely that CSF2 acts on the embryo to control cell fate and differentiation in the blastocyst. Two major actions o f CSF2 on development could be inferred by examination of the developmental genes regulated by CSF2. The first was regulation of genes involved in neurogenesis in a manner that would act to inhibit neural cell formation and differentiation. The second was stimulation of genes involved in formation or differentiation of mesoderm or mesoderm derived cells. That CSF2 acts to inhibit processes involved in neurogenesis is evident from an examination of the 4 genes involved in neurogenesis increased by CSF2 ( M AB21L2, HUWE, NOTCH2 and RTN4 ) and the 9 genes that were downregulated by CSF2 ( DTX3, HUNK CELSR SEMA4, ARSA GREM1 GLIS2, CHURC1 and RGS12 ). Two of the upregulated genes are required for neurogenesis. Use of antisense oligonucleotides to deplete MAB2 1L2 impaired notochord and neural tube differentiation (Wong and Chow 2002) HUWE1 causes ubiquitination of N myc and induces ES cells to differentiate into neural tissue (Zhao et al. 2009) Conversely, RTN4 is a neural stem cell marker that prevents neurite outgrowth (Zheng et al. 2010) and NOTCH signaling can inhibit mitosis of neuronal precursors (le Roux et al. 2003) However, it is not clear whether NOTCH signaling is enhanced in CSF2 treated embryos, since one of the genes do wnregulated by CSF2, DTX3 is a component of the NOTCH signaling pathway (Pampeno et al. 2001) Moreover, NOTCH signaling is involved in a wide array of developmental events and the phenotype caused by activation of NOTCH pathways

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86 depends upon inputs from multiple inputs (Lewis et al. 2009) The other 8 genes involved in neurogenesis that were downregulated by CSF2 are all inv olved in neural c ell function. Two of these genes, the bone morphogenic protein (BMP) inhibitor GREM1 (Wordinger et al. 2008) and the transcription factor CHURC1 participate in neur u lation. Early in development, ec toderm is inhibited from differentiating into neura l cells by the actions of BMP4. Secretion of BMP antagonists relieves this inhibition and contributes to neural induction (Hemmati Brivanlou and Me lton 1997) Neural formation also requires actions of fibroblast growth factors, which act through the transcription factor CHURC1 to enhance actions of BMP antagonists and activate expression of SOX2 (Sheng et al. 2003) Inhibition of GREM1 and CHURC1 expression by CSF2 could, therefore, result in maintenance of the inhibi tion of neural cell formation. The other genes involved in neurogenesis inhibited by CSF2 are the transcription factor G LIS2 (Zhang et al. 2002) RGS12 a component of the signaling system for nerve growth factor (Willard et al. 2007) CELSR a cadherin involved in planar polarity and neural tube formation (Curtin et al. 2003; Zhou et al. 2007 a ) SEMA4 a member of the semaphorin family of axon guidance proteins (Pasterkamp and Giger 2009) HUNK a protein kinase expressed in fetal brain (Gardner et al. 2000) and ARSA a lysos omal enzyme involved in myelin formation (Gieselmann et al. 1991) In contrast to the apparent inhibition of neural formation caused by CSF2, there was evidence that CSF2 regulated gene ex pression in a way that would increase mesoderm formation and differentiation. One gene increased by CSF2 was KLF8, a Krppel like transcription factor that can induce epithelial mesenchymal transition (Wang et al. 2007) The process of epithelial mesenchymal transition occurs at several

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87 developmental events including gastrulation and leads to epithelial cells becoming migratory and differentiating into mesenchymal cells (Baum et al. 2008) CSF2 also caused upregulation of HOXA5 a mesenchymally restricted morphogen involved in pattern formation (Boucherat et al. 2009) MYF6 a member of the basic helix loop helix family of transcription factors that activates genes involved in muscle cell formation (Rescan 2001) CD73/NT5E, an extracellular nucleotidase and mesenchyma l stem cell marker which can induce proliferation, migration, invasion, and adhesion of breast cancer cells (Barry et al. 2001; Babiychuk and Draeger 2006; Zhou et al. 2007 b ; Wang et al. 2008 b ) and FHL1 a four and a half LIM only protein i nvolved in muscle development ( McGrath et al. 2003) (2.1 fold increase). One effect of CSF2 that was inconsistent with promotion of differentiation of mesoderm derivatives was the inhibition of GREM1 expression because this BMP antagonist promotes the epithelial mesenchymal transition i n kidney and limb bud (Michos et al. 2004) The first differentiation event in the embryo after formation of the blastocyst is gastrulation whereby the three germ layers (ectoderm, mesoderm, and en doderm) are formed. In the cow, this event occurs as early at Day 9 of development when mesoderm can be identified (Maddox Hyttel et al. 2003) Neurulation occurs later in development and the neural groove forms betweens Day 14 and 21 (Maddox Hyttel et al. 2003) Taken together, the net result of actions of CSF2 on expression of genes involved in developmental processes could be a blastocyst i n which formation of mesoderm and mesoderm derived cell types is enhanced while differentiation of the ectoderm into the neural plate is delayed. Perhaps, CSF2 favors embryonic survival by ensuring allocation

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88 of a sufficient number of ICM cells towards mes oderm early in development and by inhibiting neurulation until the appropriate stage of development. A total of 10 genes involved in WNT signaling were differentially expressed by CSF2. Given the involvement of WNT in epidermal fate determination, BMP4 re gulation and endoderm formation (Wilson et al. 2001; Hansson et al. 2009) such changes could conceivably contribute to changes in the process of gastrulation. Analysis of the WNT related genes regulated by CSF2 would indicate inhibition of catenin dependent WNT signaling. Four genes inhibito ry to this pathway ( SFRP4/FrpHE, NOTCH2, PPP2R3A and PCDH24 ) were upregulated by CSF2 and two of the genes downregulated by CSF2 (WNT16 and CSNK2B) promote catenin dependent signaling. Actions of proteins involved in WNT signaling are complex, however, and depend upon the array of other WNT signaling proteins present in the cell (Chien et al. 2009) Moreover, all four of the WNT signaling genes that were downregulated by CSF2 are involved in one o r more catenin independent signaling pathways ( WNT16, CSNK2B, ROR2 and CELSR2) and activation of these pathways leads to an inhibition of catenin dependent gene expression (Chien et al. 2009) Expression of only 3 of 70 WNT induced genes were significantly affected by CSF2 and only two of those ( GREM1 and SEMA4 ) experienced the decrease in expression expected if CSF2 decreased catenin dependent gene expression. Further research is required to delineate regulation of WNT signaling by CSF2 in the preimplantation embryo. It has been demonstrated previously that CSF2 can reduce apoptosis in preimplantation mouse embryos (Sj blom 2002) Similar results were obtained in the present experiment with bovine embryos. Expression of three genes ( CD73/NT5E,

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89 PGR, and PRKAR2B ) that inhibit apoptosis were increased by CSF2. The ecto nucleotidase CD73/NT5E blocks TRAIL induced apoptosis, possibly through interaction with death receptor 5 (Mikhailov et al. 2010) Ligand dependent action of PGR leads to inhibition of apoptosis through an unknown mechanism (Friberg et al. 2009) The cAMP dependent kinase PRKAR2B can inhibits apoptosis through a p53 dependent mechanism (Srivastava et al. 1999) CSF2 also increased expression of two genes that can be pro or anti apoptotic, NOTCH2 (Quillard et al. 2009; Yoon et al. 2009) and the TNF receptor superfamily member TNFSF8 (CD30) (Al Shamkhani 2004) Treatment with CSF2 also inhibited expression of 6 pro apoptotic genes ( MADD, RIPK3, PIK3IP, NOD2, DAPK1 and CREM1 ). MADD is a death domain containing adaptor protein, DAPK1 is a death associ ated protein kinase 1 (Martoriati et al. 2005; Okamoto et al. 2009) and NOD2 is a caspase recruitment domain family (Geddes et al. 2009) PIK3IP1 (He et al. 2008) and RIPK3 are kinase proteins that can trigger apoptotic signaling (Declercq et al. 2009) and CREM decreases amounts of the anti apoptotic protein BCL2 (Jaworski et al. 2003; Mioduszewska et al. 2003) ). Consistent with the idea that CSF2 inhibits apoptosis was the finding that induction of apoptosis in Day 6 embryos by heat shock was reduced by treatment with CSF2. Heat shock induces apoptosis in preimplantation embryos through mitochondrial depolarization and activation of group II I caspases such as caspase 9 (Loureiro et al. 2007) It is possible that the increased number of ICM cells and increased ICM/ TE ratio seen in CSF2 treated embryos ( Chapter 2 ) is a result, at least i n part, in inhibition of apoptosis responses because the incidence of apoptosis is greater in ICM than TE

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90 (Knijn et al. 2003; Fouladi Nashta et al. 2005; Pomar et al. 2005) Increased survival after transfer to recipients ( Chapter 2 ) could also be due, in part, to increased resistance of embryos to adverse maternal environments that could induce blastomere apoptosis after transfer. Quantitative PCR was used to confirm the microarray results. While 11 of 16 genes showed fold changes in the same direction as for microarray analysis, differences were significant in one case only and approached signific ance (P=0.09) in 7 other cases. Lack of significance was likely due to part to the small sample siz e and between sample variation. It could also be, for some genes, the microarray and the qPCR probes recognize different splice variants. The most notable discrepancy between PCR and microarray results was for WNT16, where expression was reduced by CSF2 as determined by microarray hybridization and increased by CSF2 as determined by qPCR. This gene was not very abundant and the large fold change differences may reflect sampling error. While the lack of uniform agreement between microarray and qPCR results means that definitive conclusions regarding effects of CSF2 on individual genes is not possible, it is likely that systems and pathways found by microarray hybridization to be regulated by CSF2 refle ct biological actions of CSF2. Indeed, the experiment on induction of apoptosis by heat shock represents a biological confirmation of the results from microarray analysis with respect to regulation of genes involved in apoptosis. Ev en though a small percentage age of genes were confirmed in the qPCR we were able to confirm the regulation of apoptosis by CSF2 evaluating the percentage age of apoptotic blastomers by a well characterized assay.

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91 It is concluded from the present study that CSF2 can regulated embryonic development by altering the expression of genes controlling developmental process and apoptosis. Results indicate that CSF2 act to increase expression of genes regulating epithelial to mesenchymal transition and decrease neura l cell differentiation. Other CSF2 effects include a decrease in genes involved in apoptosis and an increase in genes that regulate cell survival. Perhaps this change in cellular fate and the decrease in cellular death are responsible for the higher pregna ncy rates at Day 35 and the lower embryonic losses seem on recipients that receive a CSF2 treated embryo.

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92 Table 3 1 Primers and Probes used on qPCR. Gene Name Accession Primer/Probe Ankyrin repeat domain 37 ( ANKRD37 ) XM_586036 Forward a GTG GGA AAA GGA AGT GTT GAT G Probe b TGG TCA TGA AGA GGT GAG GAG AAG GT Reverse C CTT GTA GCT GAA CGG TAG ACC Casein kinase 2, beta polypeptide ( CSNK2B ) XM_585826 Forward a GTT TCC CTC ACA TGC TCT TCA Probe b TGG ATC TTG AAA CCG TAA AGC CTG GG Reverse C TCA CTG GGC TCT TGA AGT TG Endothelin converting enzyme 1 ( ECE1 ) NM_181009 Forward a CAT TCT ACA CCC GCT CTT CAC Probe b CCG ATG CCG CCG AAG TTT AAG G Reverse C GTT CCC ATC CTT GTC GTA CTC Hexokinase 1 ( HK1 ) NM_001012668 Forward a CCA AAG TGT AAT GTG TCC TTC C Probe b TGC CGC TGC CGT CTT CAG ATA A Reverse C GAA GAG AGA AGT GCT GGA AGG Mab 21 like 2 ( MAB21L2 ) XM_585738 Forward a TCT CAC CAA TCC CAA AAG CC Probe b AGC AGT CCC CAG CAC CCT ATA GT Reverse C TCT GGA GTT CTC GCA GTT TG MAP kinase activating death domain ( MADD ) XM_867337 Forward a AGT GCA ATA CAG TCC GAG G Probe b AGT ACA AGA CAC CGA TGG CCC AC Reverse C CAA CAC GGA GTA GCA GAT C

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93 Table 3 1 Continued Gene Name Accession Primer/Probe Mannan binding lectin serine peptidase 2 ( MASP2 ) XM_582170 Forward a TGG TTT GTG GGA GGA ATA GTG Probe b AGT ACA AGA CAC CGA TGG CCC AC Reverse C CAA CAC GGA GTA GCA GAT C Phosphomannomutase 2 ( PMM2 ) NM_001035095 Forward a TCT AAC CCA GTC TCC CCT C Probe b CCT CCT GCA AGT TCC TGT GGC T Reverse C GGG ACC AAA GCT GAA CAA TG Notch homolog 2 ( NOTCH2 ) XM_867242 Forward a ACA CAT GTC TGA GCC ACC Probe b FAM/TCT ATG CAT GAA AGA GTC TGC CTC CA Reverse C TTT CCC GGA TGA CCT TCA Protein phosphatase 2, regulatory subunit B, alpha 1 ( PPP2R3A ) XM_599849 Forward a CAA GAT GAC CAG CAC AGT Probe b TGA TCC CAG AAC TCT AAA AGA TGT CCA GC Reverse C GTT AGT GGC TGC GAT TGA Receptor interacting serine threonine kinase 3 ( RIPK3 ) XM_584025 Forward a CTG ACA GAT TTG ATG CAG AAG TG Probe b AAG GGC TTT CTT GGT GTT TAT TCG GC Reverse C Solute carrier family 16, member 10 ( SLC16A10 ) XM_615239 Forward a GCT CGG ATT CAT GTC TAT ACC C Probe b TGA TGT GGC CTT CTA CCT CGC TG Reverse C AAC AGC ACC TCC AAT AAG GG

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94 Table 3 1 Continued Gene Name Accession Primer/Probe Tumor necrosis factor (ligand) superfamily, member 8 ( TNFSF8 ) NM_001025207 Forward a GCA AAA CTG ACC ATC CTG AAT C Probe b CTG CCG GAA CTG AGA CTG ACA ATA AGA C Reverse C CTG ACG GGA AAC AAA AGC TG Vestigial like 1 (Drosophila) ( VGLL1 ) XM_872858 Forward a AGC TCT GGG CAA TGT CAA G Probe b AGT GGC GTT TCT CTG CTC CGT Reverse C GCA AAA GAT ACT TCC GGC TG Wingless type MMTV integration site family, 16 isoform 1 ( WNT16 ) NM_016087 Forward a GAG GTG TGA AAG CAT GAC TG Probe b TGC CGC TGC CGT CTT CAG ATA A Reverse C GAA GAG AGA AGT GCT GGA AGG Mitochondrial ribosomal protein S12 ( MRPS12 ) NM_01077101 Forward a TCT AGA AGT AGC TGG TCT GGG Probe b CTA TGG GTT AAG GGT CCA CTG GGC Reverse C GAT GGC AGT ACA GAG TCT TGT C a Forward=sense (5') primer b Each probe was synthesized with a 5' 6 FAM reporter dye and 3' IA Black quencher c Reverse=antisense (3') primer

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95 Table 3 2 Gene ontologies in the biological process category that were regulated by CSF2 a Ontology Effect of CSF2 Genes GO:0032502: Developmental process Up MAB21, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTCH2, PCDH1, PTPN2, CD2, RTN4, MYF6, FHL1, SFRP4, SLC26, FGD3, TNFSF8 Down CYLC1, CELSR, ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2, PHGDH, GLIS2, GYPC, PTGER, HUNK, CREM, WNT16, BOLL, CHURC1, RIPK3, CENPI, MADD, NOD2, RNF7, GPR14, DAPK1 GO:0007275: Multicellular organism development Up MAB21, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTCH2, PCDH1, PTPN2, CD2, RTN4, MYF6, FHL1, SFRP4 Down CYLC1, CELSR2, ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2, PHGDH, GLIS2, GYPC, PTGER, HUNK, CREM, WNT16, BOLL, CHURC1 GO:0048731: System development Up MAB21L, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTC H2, PCDH1, PTPN2, CD2, RTN4, MYF6, FHL1, SLC26, FGD3 Down ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2, PHGDH, GLIS2, GYPC, PTGER4, GO:0048856: Anatomical structure development Up MAB21L, HOXA5, CASP7, PGR, PDE3B, IBSP, NOTCH2, PCDH1, PTPN2, CD2, RTN4, MYF6, FHL1, SLC26, FGD3 Down ECE2, SEMA4, GREM1, FEZF1, POLL, ARSA, ROR2, PHGDH, GLIS2, GYPC, PTGER4 GO:0007389: Pattern specification process Up HOXA5, NOTCH2, MYF6 Down GREM1, ROR2

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96 Table 3 2 Continued Ontology Effect of CSF2 Genes GO:0048518: Positive regulation of biological process Up CD2, MASP2, MYF6, NOTCH2, PTPN2, RIPK3, RNF7, TNFSF8 Down BOLL, CHURC1, DAPK1, GLIS2, NOD2, STUB1, VAV1 GO:0007165: Signal transduction Up CASP7, CD2, CCRL, FGD3, GIPR, MARK1, NOTCH2, NXPH4, PDE3B, PRKAR, PTPN2, PDE7B, PGR, PYGO1, OR51E,TNFSF8 Down CREM, CELSR2, CXCL2, CXCL1, CSNK2, DTX3, DAPK1, ECE2, FYB, GPR14, HUNK, ITPR2, LRRN2, MADD, MPP2, RIPK3, NOD2, OR2T1, PLD2, STUB1, TBC1D2, VAV1, WNT16, SFRP4, PTGER, RALGP, ROR2, RGS12 GO:0007154: Cell communication Up CASP7, CD2, CCRL1, ECE1, FGD3, GIPR, MARK1, NOTCH2, NXPH4, PDE3B, PRKAR, PTPN2, PDE7B, PGR, PYGO1 SFRP4, TNFSF8, OR51E Down CREM, CELSR2, CXCL2, CXCL1, CSNK2, DTX3, DAPK1, ECE2, FYB, GREM1, GPR14, HUNK, ITPR2, LRRN2, MADD, MPP2, NOD2, OR2T1, PLD2, STUB1, VAV1, WNT16, PTGER, RIPK3, RALGP, ROR2, RGS12 GO:0007166: Cell surface receptor linked signal transduction Up CD2, CCRL1, GIPR, NOTCH2, NXPH4, PTPN2, PYGO1, SFRP4, OR51E Down CELSR2, CXCL 2, CXCL1, CSNK2, DTX3, ECE2, GPR14, MADD, OR2T1, PLD2, STUB1, VAV1, WNT16, PTGER, ROR2, RGS12

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97 Table 3 2 C ontinued Ontology Effect of CSF2 Genes GO:0012502: Induction of programmed cell death Up CD2, TNFSF8, NOTCH2 Down RNF7, DAPK1, RIPK3 GO:0043065: Regulation of apoptosis Up CD2, TNFSF8, NOTCH2, RTN4 Down RNF7, DAPK1, RIPK3, MADD, NOD2 GO:0022610: Biological adhesion cell adhesion Up CLDN2, IBSP, PCDH15, CD2, THBS3 Down CELSR2, LRRN2, VAV1, F5, OLFM4, ROR2, CXCL12, PPFIBP1 GO:0048869: Cellular developmental process cell differentiation Up TNFSF8, SFRP4, CASP7, MYF6, NOTCH2, PCDH1, FHL1, CD2, RTN4, PTPN2 Down CYLC1, CREM, ECE2, NOD2, DAPK1, RIPK3, BOLL, SEMA4, FEZF1, MADD, ROR2, GLIS2, RNF7 a Gene

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98 Table 3 3 Differentially regulated genes involved in WNT signaling. Least squares mean Symbol CSF2 Control Fold Change P value Role in WNT signaling Reference Upregulated SFRP4/FrpHE 13.07 3.26 4.00 0.04 Binds to and inhibits WNT Chien et al. 2009 NOTCH2 84.22 38.10 2.21 0.03 Blocks induction of WNT induced genes Walsh and Andrews, 2003 PDE7B 4.88 2.89 1.69 0.02 Promotes catenin independent and dependent signaling Ahumad a et al. 2002; Li et al. 2002 PYGO1 135.6 82.08 1.65 0.01 Nuclear cofactor for catenin Jessen et al. 2008 PPP2R3A (PP2A) 28.53 18.09 1.58 0.04 Phosphatase that inhibits catenin dependent signaling Creyghton et al. 2005 PCDH24 320.2 211.28 1.52 0.01 Protocadherin that inhibits activation of catenin Ose et al. 2009 Downregulated WNT16 3.76 14.28 3.80 0.01 WNT ligand for catenin dependent and independent pathways Mazier es et al. 2005; Binet et al. 2009 CSNK2B (CK2) 119.2 256.51 2.20 0.02 Enhances activation of catenin dependent transcription; enhances activation of planar cell polarity; inhibits RAC 1 mediated effects of WNT Wang and Jones, 2006; Bryja et al. 2008 ROR2 34.89 63.48 1.80 0.01 WNT receptor or co receptor for planar cell polarity pathway Chien et al. 2009 CELSR2 (Flamingo) 206.1 316.26 1.60 0.01 Cadherins that interact with WNT and activate planar cell polarity signaling Saburi et al. 2005

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99 Table 3 4 Differentially regulated genes involved in apoptosis. Least squares mean Symbol CSF2 Control Fold Change P value Role in apoptosis Upregulated NOTCH2 84.22 38.10 2.21 0.03 anti/pro apoptotic CD73/NT5E 27.11 12.20 2.22 0.03 anti apoptotic PGR 63.08 34.46 1.83 0.05 anti apoptotic TNFSF8 (CD30) 64.96 37.64 1.73 0.03 a nti/pro apoptotic CASP7 82.11 49.07 1.67 0.03 pro apoptotic PRKAR2B 182.85 119.97 1.52 0.04 anti apoptotic RTN4 492.97 311.20 1.58 0.02 pro apoptotic Downregulated MADD 8.18 29.55 3.60 0.03 pro apoptotic RIPK3 43.02 85.65 2.00 0.03 pro apoptotic PIK3IP1 34.58 62.71 1.80 0.01 pro apoptotic CXCL12 75.16 138.34 1.80 0.02 anti apoptotic NOD2 5.49 9.77 1.80 0.05 pro apoptotic DAPK1 20.42 31.88 1.60 0.02 pro apoptotic CREM 53.9 83.1 1.60 0.02 pro apoptotic RNF7 95.80 155.50 1.60 0.03 anti apoptotic PLD2 308 493.9 1.60 0.02 anti apoptotic

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100 Figure 3 1 Genes expressed in control and CSF2 treated embryos at Day 6 of development. Shown in panel A is a Venn diagram of genes expressed in embryos of both treatments (brown), only in control embryos (red) or only in CSF2 treated embryos (green). Intensity cutoff for expression = 2.8. Shown in panel B is the hierarchic al cluster of differentially expressed genes

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101 Figure 3 2 Validation of microarray results using quantitative PCR. Shown are the fold change increases (positive) or decreases (negative) in expression caused by CSF2 as determined by microrarray hybridization (x axis) and qPCR 9y axis). Probability values for CSF2 effects in the PCR analysis are designated by the color of the circle (red, P<0.001; black, P=0.09; open, P>0.10).

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102 Figure 3 3 Regulation of heat shock induced apoptosis in Day 6 bovine embryos by CSF. Panels A D are representative photomicrographs illustrating the frequency of apoptotic nuclei in embryos at 38.5C (A, D) and 42C (B, E). as affected by heat shock at 42C and treatment with colony stimulating factor 2 (CS F2). Detection of apoptosis was by TUNEL analysis using TMR red conjugated dUTP to identify apoptotic nuclei (red) and Hoescht 33342 to identify all nuclei (blue). Note that exposure of embryos to 42C for 15 h (B) increased the frequency of TUNEL positiv e cells compared with embryos cultured at 38.5C (A). In the presence of CSF2, however, there was no difference in TUNEL labeling between embryos cultured at 38.5C (C) or 42C (D). Panel E shows least squares means for the percentage of cells that were ap optotic (top panel) and total cell number (bottom panel). Data represent results from 65 81 embryos per treatment. Heat shock at 42C for 15 h increased the proportion of cells that were TUNEL positive (P<0.0001) and decreased the total cell number (P<0.0 001). Treatment with CSF2 decreased the percentage of cells that were TUNEL positive (P<0.005) but did not affect total cell number.

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103 CHAPTER 4 CONSEQUENCES OF EMBRYONIC EXPOSUR E TO CSF2 FROM DAY 5 TO 7 AFTER INSEMINATION O N TROPHOBLAST ELONGA TION, INTERFE RON TAU SECRETION AND GENE E XPRESSION IN THE EMB RYONIC DISC AND TROPHECTODERM Introduction Colony stimulating factor 2 (CSF2) is an important regulator of e mbryonic development in the cow. It improve s the proportion of cultured embryos that develop to th e blastocyst stage (de Moraes and Hansen 1997 a ; Chapter 2 ) increase s the number of cells in the inner cell mass ( Chapter 2), and decreases the percentage of blastomeres undergoing apoptosis in response to heat shock (Chapter 3) Moreover, addition of CSF2 to culture medium Days 5 to 7 after insemination improve d the competence of in vitro produced embryo s to establish pregnancy after transfer to a recipien t female while also r educ ing the probability of fetal loss after Day 30 35 of pregnancy (Chapter 2). The primary objective of this study was to evaluate possible mechanisms by which CSF2 acts during Day 5 to 7 of development to improve embryonic and fetal survival. One hypothesis was that CSF2 causes increased secretion of interferon tau ( IFNT ) by the trophoblast of elongated conceptuses. CSF2 has been shown to stimulate production of IFNT by sheep trophoblast (Imakawa et al. 1993; Imakawa et al. 1997; Rooke et al. 2005) and by a bovine trophoblast cell line (CT 1 cells) (Michael et al. 2006) CSF2 might also affect elongation of the trophoblast because of evidence that treatment of embryos from Day 5 6 with CSF2 caused differential regulation of a large number of genes involved in developmental processes (Chapter 3). Analysis of the genes involved in developmental processes regulated by CSF2 indicates a propensity for CSF2 to upregulate genes involv ed in mesoderm formation or differentiation while

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104 decreasing expression of genes involved in neurogenesis (Chapter 3). This finding raises the possibility that CSF2 causes changes in gastrulation that could be reflected in changes in gene expression in the embryonic disk or trophoblast. A large proportion (~25%) of in vitro produced embryos that survive to Day 14 15 of gestation lose the embryonic disk (Fischer Brown et al. 2004; Fischer Brown et al. 2004; Block et al. 2007) and CSF2 might act to improve survival of the embryonic disc, either because of its antiapoptotic actions (apoptosis being more frequent in inner cell m ass) (Knij n et al. 2003; Fouladi Nashta et al. 2005; Pomar et al. 2005) or because of regulation of genes involve d in developmental processes. As part of this study, methods were developed to divide the elongated conceptus into 1) the embryonic disk and a small amount of adjacent trophoblast and 2) tissue containing trophoblast only. As a result, over 500 genes were identified that were preferentially expressed in the embryonic disk. These genes, or the proteins they encode, represent candidates markers for embryonic disk that should prove useful for studying the differentiation of the bovine conceptus through the pe riattachment period. Materials and Methods In vitro Production of E mbryos Embryo production was performed as described in Chapter 3 with modifications described below. Cumulus oocyte complexes (COCs) from ovaries from a mixture of beef and dairy cattl e were collected in Tissue Culture Medium 199 (TCM 199) with bovine steer serum containing 2 U/ml heparin (Pel Freez, Rogers, AR) 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocytes were allowed to mature for 20 22 h in groups of 10 in 50 l microdrops of TCM 199 (Invitrogen,

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105 2 g/ml estradiol 17 20 g/ml bovine follic le stimulating hormone (Folltropin V; Belleville, ON, Canada), 22 g/ml sodium pyruvate, 50 g/ml gentamicin sulfate, and 1 mM glutamine. Matured oocytes were then washed in HEPES TALP (Parrish et al. 1986) ( Caisson, Sugar City ID, USA) and transferred in groups of 50 to four well plates containing 600 L of IVF TALP supplemented with 25 L PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 M epinephrine in 0.9% [w/v] NaCl), and fertilized w ith 30 L Percoll purified spermatozoa (~ 1x10 6 sperm cells). Sperm were prepared from a pool of frozen thawed semen from three different bulls; a different set of bulls was generally used for each replicate). Fertilization proceeded for 18 20 h at 38.5 C in a humidified atmosphere of 5% (v/v) CO 2 in humidified air. Putative zygotes were removed from fertilization plates, denuded of cumulus cells by vortexing in HEPES TALP, and placed in groups of 30 in 45 l microdrops of KSOM BE2 (Soto et al. 2003) Embryos were cultured at 38.5 C in a humidified atmosphere of 5% CO 2 or 5% CO 2 5% O 2 and 90% N 2 (v/v). C leavage rate was assessed at Day 3 after insemination. At Day 5 after insemination, 5 l of KSOM BE2 or 5 l of KSOM BE2 containing 100 ng/ml recombinant bovine CSF2 (a gift from Novartis, Basle Switzerland) were added to each drop to achieve a final CSF2 concentration of 0 or 10 ng/ml. Transfer I nto R ecipients Morula, blastocyst and expanded blastocyst stage embryos (6, 9 and 5 in the control group and 5, 5 and 5 in the CSF2 group, respectively) classified as Grade 1 (Robertson and Nelson, 1998) were harvested on Day 7 after insemination and loaded into 0.25 ml straws in 250 l HEPES TALP supp lemented with 10% (v/v) fetal calf

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106 serum and 50 M d ithiothreitol (S igma Aldrich, St. Louis MO, USA) Straws containing selected embryos were then placed horizontally into a portable incubator (Cryologic, Mulgrave, Vic, Australia) at 38.5C and transported to the farm. Thirty five multiparous lactating Holstein cows were used as recipients. Cows were housed in a free stall barn equipped with fans and a sprinkler system at a commercial dairy in Bell, F lorida ( 29.75578N, 82.86188W). Overall, 4 replicates w ere completed with 5 11 recipients per replicate from June to October of 2009 For each replicate, eligible cows were synchronized for embryo transfer using the Ovsynch 56 procedure ( Chapter 2 ). Day 0 was considered the Day of expected ovulation. Hormone treatments consisted of 100 g gonadotrophin releasing hormone (GnRH ; Merial; D uluth, GA, USA) i.m. on Day 10; 25 mg PGF (Pfizer; New York, NY, USA) i.m. on Day 3; and 100 g of GnRH i.m. 56 hours after PGF Cows were diagnosed for the presence of a corpus luteum (CL) at Day 7 after anticipated ovulation ( Day of ovulation was considered Day 0) using an Aloka 500 ultrasound equipped with a 5 MHz linear array transducer. Cows diagnosed with a corpus luteum were given epidural anesthesia [5 ml of 2% (w/v) lidocaine] and a single embryo transferred to the uterine horn ipsilateral to the ovary via t he transcervical route. Embryo Recovery and E valuation At Day 15 after expected ovulation, the ovary ipsilateral to the uterine horn that received the embryo was examined using ultrasonography to confirm the pres ence of the corpus luteum. C ows that did no t have a visible CL were not flushed E mbryos were recovered transcervically with a 20 French Foley catheter inserted with a s tainless steel stylet and held in position by inflating the cuff at the end of the catheter The uterine horn ipsilateral to the C L was flushed with DPBS (Sigma Aldrich St. Louis MO, USA)

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107 with 1% (v/v) p o lyvinyl alcohol (PVA) Flushing involved multiple injections and recovery of 60 ml DPBS using a 60 cc syringe attached to the Foley catheter. The procedure was continued until either an embryo was identified in the flush or 180 to 240 ml of DPBS PVA had been flushed The flushings recovered from the first 60 ml flush were centrifuged at 500 rpm for 10 min and stored at 20 o C for analysis of antiviral activity Following recovery, ea ch embryo was assessed for length, stage, and the presence or absence of an embryonic disc ( ED ) by light microscopy using a stereomicroscope. S tage of development was classified based on shape as ovoid, tubular, or filamentous. After all measurements were recorded, embryos were washed on ce in DPBS PVA and the embryo was dissected to produce a piece of tissue containing the ED and some nearby trophoblast (termed ED) and two p ieces of trophoblast (Tr) that were on either end of the ED ( Figure 4 1 ) T issues were immediately snap frozen separately in liquid nitrogen. An tiviral A ssay The quantity of biologically active IFNT in uterine flush ings was determined indirectly using an antiviral assay based on the inhibition of vesicular stomatitis virus (VSV) induced lysis of Madin Darby bovine kidney (MDBK) cells The assay was performed as described by Micheal et al. ( 2006) Briefly, 1:3 serial dilutions of the uterine flushings was performed using DMEM containing 10% (v/v) FBS in 100 l volume in 96 well plates. MD BK cells were resuspended in DMEM FBS and 50 l of cell preparation ( ~ 500.000/ml) was added to each well. The plate was incubated at 37C and 5% CO 2 in air for 24 h. Medium was then aspirated from wells and VSV diluted in DMEM (without FBS) was added to ea ch well. The plate was incubated for 1 h at 37C.

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108 The VSV solution was aspirated, replaced by 100 l DMEM FBS and the plate was incubated for 24 h at 37C. After aspiration of medium, cells were fixed with 70% (v/v) ethanol and stained with 0.5% (w/v) gent ian violet stain in 70% (w/w) ethanol (Fisher Scientific, Pittsburgh, PA, USA). Plates were washed two times in water and allowed to dry. Antiviral activity of each sample was assessed visually based on inhibition of lysis of MDBK cells. The a ntiviral acti vity ( units/ml ) was defined as the dilution of flushing that reduce d by 50% the destruction of the monolayer by virus. Antiviral units were converted to IFN ( IU/ml ) using a standard curve with known amounts of human IFN standard included in th e assay (EM D Biosciences, San Diego, CA). All samples (from cows with and without an embryo) were analyzed in duplicate. Analysis of the Transcriptome of T rophectoderm and Embryonic D isc Microarray analysis was performed using a subset of 8 ED and 8 T r samples that were randomly selected from among the filamentous embryos. T issues were disrupted by vortexing for 30 seconds and passed through a 20 gauge needle. Samples were homogenized using a QIAshredder (Qiagen Inc, Valencia, CA USA ). Total cellular RNA was extracted with the RNeasy Plus Micro kit (Qiagen instructions. Concentration of the input RNA was determined by Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA) and RNA integrity was determined by use o f the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa clara, CA USA ). Only samples that showed high RNA integrity (RIN > 7) were used for the microarray hybridization and quantitative PCR analysis. Extracted RNA was stored at 80 o C until microarray analysis.

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109 Microarray Hybridization Procedures were performed by the University of Florida Gene Expression Division of the Interdisciplinary Center for Biotechnology Research. Labeled aRNA was generated using Amino Allyl MessageAmp II Amplification kit (A mbion Inc Austin, TX, USA ). First strand cDNA was synthesized from 425 ng of total RNA. Half of the first protocol and the remainder was reserved for quantitative PCR (qPCR) ver ification. The microarray analysis was performed using the Bos taurus Two Color Microarray Chip from Agilent v 2 (Agilent Technologies, Santa Clara CA, USA). The 16 samples were distributed between the 2 microarray chips and the two dyes to avoid location biases A total of 825 ng of labeled aRNA per sample was used for the hybridization. Hybridization and washing were performed according to the manufacturer's protocol using the Gene Expression Hybridization Kit and Gene Expression Wash Buffers (Agilent). A rrays were scanned using a dual laser DNA microarray scanner (Model G2505C Agilent). Analysis of Microarray Data The microarray images were first analyzed with Agilent Feature Extraction Software v10.1 (Agilent Technologies, Inc). Spot signal intensitie s were adjusted by subtracting local background and normalizing using within array lowess approach for dye bias correction. The quantile approach was then used for between array normalization. Statistical tests were performed using BioConductor statistical software ( http://www.bioconductor.org/ ), which is an open source and open development software project for analysis o f microarray and other high throughput data based primarily on the R programming language (Gentleman et al. 2004)

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110 Differentially expressed genes were identified using Limma, a software package that implements linear models for microarray data (Smyth et al. 2005) .In Limma, p values are obtained from moderate d t statistics or F statistics using empirical Bayesian methods. The primary analysis comparing differential expression was a 2 2 factorial design with tissue (ED, Tr) and treatment (control, CSF2) as main effects and the tissue x treatment interaction as another effect. Pairwise comparisons of interest were also performed to test for significant differential expression. The Benjamini Hochberg procedure (Benjamini and Hochberg 1995) was used to control false discovery rate (FDR) at the 0.01 level. Genes meeting this statistical threshold and showing a fold change equal or greater than 2 were considered as differentially expressed. Ingenuity pathway analysis software ( http://www.ingenuity.com ) was used to identify can onical pathways associat ed with exact test was used to determine the probability that the association between the genes and the pathway was explained by chance alone. Quantitative Real Time PCR Quantitative Real T ime PCR analysis (qPCR) of 11 differentially expressed genes and one housekeeping gene ( GAPDH ) Specific primers ( Table 4 1 ) were designed using Integrated DNA technologies software ( http://idtdna.com ). The SsoFast EvaGreen Supermix (Bio Rad Laboratories, Hercules, CA, USA) reaction chemistry and a CFX 96 Real Time PCR Detection System (Bio Rad) were used t o quantify mRNA concentrations. After an initial activation step (95C for 30 sec), 45 cycles of a two step amplification protocol (95C for 5 sec; 60C for 5 sec) were completed.

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111 The amplification of a single product was verified by performing dissociation curve analysis (65 95C) in the thermocycler. In additional, agarose gel electrophoresis of the PCR product was performed. Identity of the amplicon was confirmed by sequencing the PCR product of the band product. The DNA was extracted from the gel fragment using the PureLink Quick ge l Extraction and PCR Purification Combo Kit (Invitrogen Carlsbad, CA, USA). First, the gel with the DNA fragment was dissolved using Solubilization Buffer for 15 min at 50 C. The gel mixture was centrifuged for 1 min at 10.000 x g in a PureLink clean up sp in column. After a wash, the DNA was extracted from the tube membrane by centrifuging the column with Elution Buffer for 1 min at 10.000 x g. The purified DNA was sequenced by the University of Florida Gennomics Division of the Interdisciplinary Center for Biotechnology Research. Each sample was analyzed in duplicate reactions All C T values f or genes of interest were normalized to the housekeeping gene GAPDH T method. T T of th e gene of interest for control embryos from the value for each individual embryo. Fold change was determined by solving for 2 Statistical Analysis Categorical data w ere analyzed by logistic regression using the LOGISTIC procedure of the Statistical Analysis System ( SAS 9.1.3 SAS Inst., Cary, NC USA ) with backward selection (P=0.2). The statistical model included replicate, stage of the embryo at the time of transfer, and treatment. Effects on continuous variables were analyzed by least squares analysis of variance using the General Linear Models procedure of SAS Various mathematical models were utilized. All included treatment and, in addition, included replicate, stage of the embryo at the time of transfer, stage of the embryo at

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112 Day 15 and, for embryo length, whether the embryo was intact. Data for embryo length and antiviral activity were subjected to log transformation before analysis to account for heterogeneity of variance. In addition and median nonparametric tests with the NPARWAY1 procedure of SAS. Results Embryo Survival After T ransfer Results are presented in Table 4 2 A total of 80% of cows receiving a control embryo and 93% of cow s receiving a CS2F treated embryo had a CL detected by ultrasound. For the other cows, the CL was either absent or small and regressing. Cows without a CL represent either cows that were not successfully synchronized, so that the presumed Day 15 was actual ly later in the estrous cycle, or cows in which the CL had undergone luteolysis by Day 15. When all cows were considered, including those without a large corpus luteum, there was a tendency (P=0.07) for the proportion of cows with a recovered embryo to be higher for those receiving a CSF2 treated embryo (35 % for control vs. 66 % for CSF2 ) The same trend was apparent when only cows with a CL were considered (44% for control vs 71% for CSF2) but the difference did not approach significance. One cow in the c ontrol group did not have a detectable embryo but there was abundant antiviral activity in uterine flushings (59.049 IU IFN /ml). This cow either lost its pregnancy after the embryo initiated large scale IFNT secretion or the embryo was not recovered in the flushing. When this cow was considered pregnant, differences in survival between control and CSF2 groups remained but did not approach significance ( Table 4 2 ).

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113 Embryonic G rowth and D evelopment Five of 7 c ontrol embryos (71%) and 8 of 10 (80%) CSF2 treated embryos were filamentous. The embryonic disc was visible in 6 of 7 ( 83% ) of c ontrol embryos and 6 of 10 ( 60% ) of CSF2 treated embryos. Differences between treatments in these two variables were not signif icant As illustrated in Figure 4 2 there was great variability in embryo length, even if the dataset was restricted to embryos that were filamentous. While there was no significant effect of treatment, CSF2 treated embryos tended to be longer than contr ol embryos regardless of whether all embryos were considered, only those recovered intact were considered (4 embryos were recovered in pieces and were likely to be larger than measured), or only filamentous embryos were recovered. Overall, the average leng th was 39 mm for control embryos and 62 mm for CSF2 treated embryos. Antiviral Activity in Uterine F lushings Antiviral activity was also highly variable, ranging from non detectable amounts (one cow in each group) to almost 9,000,000 IU IFN/ml ( Figure 4 2 ). For all cows, there was a nonsignificant trend for antiviral activity to be higher for cows receiving CSF2 treated embryos than for cows receiving control embryos. This difference approached significance (P=0.07) if only cows with detectable antivira l activity were considered. Changes in the T ranscriptome of Embryonic Disc and T rophectoderm To test the hypothesis that CSF2 alters the transcriptome of the filamentous embryo, gene expression was evaluated in two tissues embryonic disc (which also con tains adjacent trophoblast) and trophoblast using microarray technology. All 43.803 probes on the array produced signal above background. These probes represent 19 500 distinct genes or 88% of the bovine genome (btau 4. 0). Hierarchical ana lysis

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114 ( Figure 4 3 ) i ndicated that the 8 ED samples formed a distinct cluster separated from the T r T here was however, no such distinction between samples from control and CSF2 treated embryos. Using a and a 2 fold difference as criteria, there were no genes aff ected by treatment or the treatment x tissue type interaction. There was, however, a total of 627 genes that were differentially expressed between ED and Tr and 576 of these could be annotated O f th e annotated genes 538 genes were upregulated in ED and 38 genes were upregulated in Tr. One would expect a preponderance of differentially regulated genes that were upregulated in ED, and not in Tr, because the former tissue contains some Tr while the Tr sample was free of ED tissue. Characteristics of G enes D ifferentially E xpressed B etween E mbryonic D isc and T rophoblast Ingenuity software was used to identify p athway s that were significantly overrepresented in the set of genes that were differentially expressed between ED and Tr. Determination of signif icant pathways is based on the ratio of the number of genes in the differentially expressed data set divided by the number of genes in the pathway and the P value. A total of 31 canonical pathways were identified as above the threshhold (P<0. 05; Table 4 3 ). The pathway with the highest score ( smallest P value) was Embryonic Stem Cell Pluripotency. Of the 148 genes in this pathway, 16 ( RAC2, NODAL, FGF2, FGFR1, SMAD6, FZD1, LEFTY2, TCF7, SOX2, NANOG, SMO, PDGFRA, BMP6, WNT11, POU5F1, FZD7 ) were represented in the list of differentially expressed genes Other pathways related to pluripotency were also significant, including Role of Oct4 in Mammalian Embryonic Stem Cell Pluripotency, with 7 of 45

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115 genes in the pathway being differentially expressed, and Role of N ANOG in Mammalian Embryonic Stem Cell Pluripotency, with 9 of 114 genes being differentially expressed. Several pathways that promote cellular differentiation were identified including pathways for Factors Promoting Cardiogenesis in Vertebrates, responsi ble for differentiation of the mesoderm into myocardium and endocardium, with 11 of 89 genes in the pathway being differentially expressed, Wnt/ catenin Signaling ( responsible for many differentiation events including cell polarity, neural tube patterning and cardiogenesis ) with 15 of 168 genes being differentially expressed and Axonal Guidance Signaling responsible for formation of neuronal connections, with 22 of 403 genes being differentially expressed. Another type of pathway that was well represented was for cell signaling. Among these were pathways for B asal Cell Carcinoma Signaling with 8 of 68 genes being differentially expressed, Ovarian Cancer Signaling with 10 of 135 genes, Leukocyte Extravasation Signa ling with 12 of 194 genes, Prolactin Signaling, with 6 of75 genes, CTLA4 Signaling in Cytotoxic T Lymphocytes with 7 of 94 genes, G Protein Coupled Receptor Signaling with 12 of 220 genes, HER 2 Signaling in Breast Cancer with 6 of 79 genes, TGFB Signa ling, with 6 of 83 genes, Acute Phase Response Signaling with 10 of 178 genes, and Colorectal Cancer Metastasis Signaling with 12 of 249 genes represented. Identification of Likely Candidate Genes for Use as Embryonic Disc Markers It is likely that man y of the genes that are differentially expressed between ED and Tr will prove useful in future studies for identifying ED and Tr. Two different criteria were used to identify some of the 576 differentially expressed genes that might be particularly

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116 useful as markers. The first criterion was to identify genes with the highest fold change between ED and Tr. The 15 genes with the highest fold difference for genes overexpressed in ED and the 15 genes with the highest fold difference for genes overexpressed in T r are shown in Table 4 4 The fold difference for the 15 highest genes overexpressed in ED ranged from 120 fold to 45 fold, with the most overexpressed gene being fatty acid binding protein 1 ( FABP1) Other genes with large fold increase for ED compared w ith Tr were transforming growth factor induced protein IG H3 ( TGFBI1) ( 95.2 fold increase ), sepallata3 ( SEP3) ( 89.9 fold increase ) and nanog homeobox ( NANOG) ( 75.7 fold increase ). In contrast, the fold difference for the 15 highest genes overexpressed in Tr ranged from 8.3 to 2.5, with the greatest fold change for r ibosomal protein L36a ( RPL36A ) ( 8.3 fold), r ibosomal protein L10a ( 5.2 fold) RAR related orphan receptor B ( RORB ) ( 4.7 fold). The second approach to identify candidate gene markers was to identify the differentially expressed genes that were the most abundant. The rationale was that abundant genes, or their protein products, would be more easy to identify using a variety of molecular and immunochemical procedures. A list of the 15 most abu ndant genes for ED and Tr, based on signal intensity on the microarray, is presented in Table 4 5 Validation of Microarray Results U sing qPCR A total of 11 genes that were differentially expressed between ED and Tr based on microarray analysis, were s ubjected to analysis by qPCR For all 11 of the genes, the fold change for ED relative to Tr was in the same direction for qPCR as the fold change as determined by microarray hybridization. Differences between ED and Tr

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117 were significant for 10 of the 11 genes, the exception being GJC1 For 7 genes ( BMPER, CLDN11, FGF2, GATA5, GDF3, IGFL1 and NODAL ), the fold change was of greater magnitude as determined by qPCR than as determined by microarray hybridization There was no effect of treatment (control vs CSF2) or treatment x tissue interaction on mRNA abundance as determined by qPCR. Discussion Exposure of bovine embryos to CSF2 from Day 5 to 7 of development can have a profound effect on the subsequent developmental fate o f the embryo. In particular, a greater percentage of embryos result in pregnancies at Day 30 35 of pregnancy and fewer of the pregnancies established at that point are lost thereafter (Chapter 2). The purpose of this experiment was to understand the mechan ism by which CSF2 increases embryonic survival as measured at Day 30 35 and reduces fetal mortality thereafter. Results suggest that higher pregnancy rates at Day 30 35 represent increased embryonic survival before Day 15 and a greater capacity of the embr yo to elongate and secrete IFNT at Day 15. Analysis of gene expression in filamentous embryos indicates little difference in transcription among this subset of embryos that survived to Day 15 and elongated successfully. Therefore, the reduction in embryoni c and fetal loss after Day 30 35 caused by CSF2 is probably not a direct reflection of altered gene expression at Day 15. Conclusions regarding embryonic survival, growth and IFNT secretion at Day 15 must be tentative because differences between control an d CSF2 were largely non significant. Nonetheless, it seems more likely that these traits were affected by CSF2. The proportion of transferred embryos that were recovered at Day 15 was twice as high for cows receiving CSF2 embryos (35% in control vs 66% in CSF2; P<0.07). Embryonic

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118 length and antiviral activity in the uterus (a measure of IFNT bioactivity) (Maneglier et al. 2008) were highly variable, as is typical at this stage of pregnancy (Bilby et al. 2004; Bilby et al. 2006) but the largest embryos were in the CSF2 group and antiviral activity among cows that had detectable activity (excluding cows wi th tubular embryos where IFNT secretion would be low (Short et al. 1991) tended (P<0.07) to be greater for cows receiving CSF2 embryos. Based on these findings, it can be postulated that CSF2 impro ves embryonic survival at two periods. The first is in the 8 Day period between transfer at Day 7 and flushing at Day 15. Embryos treated with CSF2 have large inner cell mass at Day 7 (Chapter 2) and are less prone to undergo apoptosis in response to heat shock (Chapter 3). The larger size of the inner cell mass and resistance to stress could promote early survival of the embryo. The second period is the period of maternal recognition of pregnancy, around Day 15 18, when the embryo acts to prevent luteoly sis through secretion of IFNT (Thatcher et al. 2001) The larger size and greater IFNT secretory activity of the CSF2 treated embryos (as determined by uterine antiviral activity) would make the CSF2 embryo better able to prevent luteolysis and allow continued development of the embryo. S ize of the embryo at this period does affect IFNT secretion (Bilby et al 2004; Bilby et al. 2006) Indeed, the trend for a lower incidence of cows without functional CL (based on ultrasound) in cows receiving CSF2 treated embryos may reflect the greater antiluteolytic capacity of the CSF2 treated embryo. The mechanism by which CSF2 treatment from Day 5 7 results in greater embryonic growth and IFNT secretion at Day 15 is not known. However, CSF2 can

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119 increase expression of IFNT in sheep trophoblast (Imakawa et al. 1993; Imakawa et al. 1997; Rooke et al. 2005) and bovine trophoblast cells (CT 1 cells) in vitro (Michael et al. 2006) In addition, embryos treated with CSF2 at Day 5 had altered expression of genes involved in developmental processes at Day 6 (Chapter 3) characterized by an increase in expression of genes in volved in mesoderm, mesenchyme and muscle formation and a decrease in genes involved in neurogenesis. Perhaps, effects of CSF2 on gastrulation lead to increased growth of trophoblast between Day 7 and 15 of development. A relatively large number of embry os at Day 14 15 do not have a detectable embryonic disc (Bertolini et al. 2002; Fischer Brown et al. 2002; Fischer Brown e t al. 2004) and these embryos all eventually die (Fischer Brown et al. 2004 ) Despite its effects on inner cell mass number, however, CSF2 did not affect the incidence of embryos without a disk and, numerically, the percentage of embryos without a disk was lower in the CSF2 group. It can be concluded from these observations that CSF2 is not improving embryonic survival by increasing the likelihood that the embryonic disc survives to Day 15. There was no effect of CSF2 on expression of genes in either the ED or Tr at Day 15. The embryos used for this analysis were all filamentous embryos. Thus, CSF2 has no discernable effect on embryonic gene expression at Day 15 for the subset of embryos t hat has developed normally up to that point. It is unlikely, therefore, that the reduction in embryonic or fetal loss caused by CSF2 is the result of global changes in transcription. Instead, perhaps, altered trajectory of development caused by CSF2, as c haracterized by changes in expression of developmentally important genes at Day 6 (Chapter 3) could lead to formation of a fetus that is less likely to experience a

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120 developmental defect leading to pregnancy loss after Day 35. It is also possible that CSF2 causes epigenetic changes in the developing embryo that result in altered gene expression at time points later than studied here These changes would affect late embryonic and fetal loss. One benefit of the transcriptomal analysis was that a large number of genes that are overexpressed in ED were identified. These genes represent good candidates for markers of the embryonic disc. The developmental processes involved in developmental events after embryonic hatching, including gastrulation and organ development, are not well understood in cattle and is the subject of investigation by several groups (Maddox Hyttel et al. 2003; Alexopoulos et al. 2005; Tveden Nyborg et al. 2005; Vejlsted et al. 2006) Identification of markers of the embryonic disc could simplify the study of post hatching development. The genes that were differentially regulated between ED and Tr included large numbers of pluripotency genes, genes controlling differentiation and development and cell signaling genes. This is to be expected given the nature of the developmental processes underway in the ED at Day 15. Three genes involved in pluripotency serve as examples of the functional importance of the genes overexpressed in ED. NANOG was overexpressed 75 fold in the ED. In the mouse and human blastocyst, NANOG expression is limited to inner cell mass and epiblast and is necessary for pluripotency maintenance (Marikawa and Alarc n 2009; Guo et al. 2010) NA NOG is downregulated in trophoblast by CDX2 (Strumpf et al. 2005) In the bovine, NANOG mRNA and protein are found in both inner cell mass and trophectoderm of the blastocyst, although NANOG expression is greater in the ICM and epiblast (Degrelle et al. 2005; Mu oz et al. 2008) Another gene that

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121 serves as a marker of ICM and epiblast in mouse human, and bovine, SOX2 (Degrelle et al. 2005; Guo et al. 2010) was upregulated 57 fold in the ED compared with Tr in the present study. SOX2 is the firs t transcription factor to appear in the late morula and is a key factor for maintenance of pluripotency and for reprogramming of differentiated cells into induced stem cells (Takahashi and Yaman aka 2006) Finally, POU5F1 also known as Oct4 was increased 29 fold in ED compared with Tr. Expression of POU5F1 is limited to the ICM in human and mouse blastocysts (Zernicka Goetz et al. 20 09) but is in both ICM and Tr of in vitro and in vivo produced bovine blastocysts through Day 10 of development (Eijk et al. 1999; Kirchhof et al. 2000; Degrelle et al. 2005) POU5F1 is a transcription factor necessary for the maintenance of the pluripotency in the ICM and it prevents ICM transformation to TE (Zernicka Goetz et al. 2009). Some markers of trophobl ast were also identified although technical limitations of the procedure used to separate ED and Tr meant that many fewer of these were found than for ED. In conclusion, results support the idea that the increased survival of embryos exposed to CSF2 from Day 5 7 of development is the result of increased embryonic survival before Day 15 and a greater capacity of the embryo to elongate and secrete IFNT at Day 15. The reduction in embryonic and fetal loss after Day 30 35 caused by CSF2 is probably not a dire ct reflection of altered gene expression at Day 15.

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122 Table 4 1 Primers used for qPCR. Accession # Name Sequence Product size Melting temp NM_001077997 BMPER F 5' AGA GGA CTC CTA GTC CAA CAC TCT 3' 128 bp 77C R 5' GGA AAT GAG AGC AAG CAT GTA GAC C 3' NM_001030318 CAST F 5' AGA GGT CTA TGT GTT CCG TGC AGT 3' 136 bp 86C R 5' ACA GGC TTT CCG TCT TCT GGA TCT 3' NM_001035055 CLDN11 F 5' CAT TCT GTG AGC TGT CTT GAA GTG 3' 81 bp 75C R 5' ATC AGT GTT TGC ACC CGT AAA GCC 3' NM_174056 FGF2 F 5' TCT CTC GGG AAA CTG CTG ACT TGT 3' 110 bp 74C R 5' CCC ACT GTT TCA CTC ATA CAG AAT TT 3' NM_001034221 GATA5 F 5' TCA CAT TGT AAT CAT CGT GGA CCC G 3' 80 bp 77C R 5' CAG AAC AAG GAA GGC TCT TTA CTG CC 3' NM_001025344 GDF3 F 5' CTC CAT GCT CTA CCA GGA CAA TGA 3' 83 bp 78C R 5' ACC CAC ACC CAC ACT CAT CAA CTA 3' NM_001046076 GJC1 F 5' AGA GAA CGG GAA ACA CAG CGT TC 3' 80 bp 77C R 5' CTG GAA GAC ACA AAT GTA AAG TTC TGC AAC 3' BE664075 IGFL1 F 5' AGG CAC TCT GTG AAT TCT GCA ACC 3' 91 bp 79C R 5' TCT TGC CAC CTT TGG AAG TGG AGA 3' XM_609225 NODAL F 5' GAA GAC CAA GCC CTT GAG TAT GCT A 3' 91 bp 78C R 5' GCA CCC ACA TTC CTC CAC AAT CAT 3' NM_176646 SERPINA5 F 5' GGG ATT GTA CTG TCC TGT GGG TTA 3' 80 bp 80C R 5' TAT TCG CGT CAG GCC TCC ATT CTT 3' XM_001788143 TMEFF1 F 5' AAT AGA GGA CGA CGA CAG AAG CA 3' 80 bp 77C R 5' AAG TCA CCA GTT CAA ACC ATT CTG G 3' NM_001034034.1 GAPDH F 5' ACC CAG AAG ACT GTG GAT GG 3' 177 bp 88C R5' CAA CAG ACA CGT TGG GAG TG 3'

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123 Table 4 2 Estimates of effect of CSF2 on embryonic survival at Day 15 after expected ovulation Control CSF2 P value Number of cows receiving embryos 20 15 Number of cows ( percentage ) with CL on Day 15 16/20 (80%) 14/15 (93%) N.S. Number ( percentage ) of cows with a recovered embryo, of all cows 7/20(35%) 10/15 (66%) 0.07 Number ( percentage ) of cows with a recovered embryo, of cows with a CL 7/16 (44%) 10/14 (71%) N.S. Number ( percentage ) pregnant a of all cows 8/20 (40%) 10/15 (67%) N.S. Number ( percentage ) pregnant a of cows with a CL 8/16 (50%) 10/14 (71%) N.S. a Cows were considered pregnant if there was a detectable embryo or antiviral activity in the flushing

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124 Table 4 3 Canonical pathways containing a significant number of genes differentially expressed between embryonic disc and trophoblast. Pathway b P value Genes a Human Embryonic Stem Cell Pluripotency 0.000001 RAC2, NODAL, FGF2, FGFR1, SMAD6, FZD1, LEFTY2, TCF7, SOX2, NANOG, SMO, PDGFRA, BMP6, WNT11, POU5F1, FZD7 Factors Promoting Cardiogenesis in Vertebrates 0.000032 NODAL, CER1, SMO, FZD1, DKK1, BMP6, TCF7, WNT11, PRKD1, FZD7, PRKCB Coagulation System 0.000105 F10, PROS1, SERPINA5, PROC, A2M, SERPINF2, SERPIND1 catenin Signaling 0.000141 RAC2, GJA1, SFRP2, FZD1, SOX2, CDH2, DKK3, SMO, DVL3 PPP2R2C, SOX8, SFRP1, DKK1 WNT11, FZD7 Role of Oct4 in Mammalian Embryonic Stem Cell Pluripotency 0.000380 SOX2, TDH, NANOG, SPP1, PHC3 FBXO15, POU5F1 Basal Cell Carcinoma Signaling 0.000741 SMO, DVL3 FZD1, BMP6, GLI1, TCF7, WNT11, FZD7 Role of Osteoblasts, Osteoclasts and Chondrocytes in Rheumatoid Arthritis 0.001738 RAC2, SPP1, SFRP2, SMAD6, FZD1, TCF7, DKK3, NGFR, SMO, SFRP1, DKK1 BMP6, WNT11, TNFRSF11B, FZD7 Hepatic Fibrosis / Hepatic Stellate Cell Activation 0.001778 COL1A2, IGFBP4, CD40 FGF2, NGFR, FGFR1, PDGFRA, EDNRA, MMP2, A2M, TNFRSF11B Axonal Guidance Signaling 0.001778 DPYSL2, PRKACB, FYN, RAC2, PLXNC1, NRP2, EPHB2, DPYSL5, FZD1, ROBO1, SDC2, CXCL12, NGFR, SMO, RASSF5, BMP6, GLI1, WNT11, PRKD1, FZD7, PRKCB, UNC5C Role of Macrop hages, Fibroblasts and Endothelial Cells in Rheumatoid Arthritis 0.004365 RAC2, SFRP2, FGF2, FZD1, TCF7, ROR2, DKK3, CXCL12, NGFR, SMO, ATF4 SFRP1, DKK1, WNT11, PRKD1, TNFRSF11B, FZD7, PRSS35, PRKCB

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125 Table 4 3 Continued Pathway b P value Genes a Pantothenate and CoA Biosynthesis Ovarian Cancer Signaling 0.004677 0.004786 DPYSL2, DPYS, CILP2, ENPP2 PRKACB, RAC2, GJA1, SMO, EDNRA, MMP2, FZD1, TCF7, WNT11, FZD7 Role of NANOG in Mammalian Embryonic Stem Cell Pluripotency 0.005888 SOX2, RAC2, NANOG, SMO, FZD1, BMP6, WNT11, FZD7, POU5F1 LPS/IL 1 Mediated Inhibition of RXR Function 0.006607 CYP2C9, APOC4, APOC2, FMO5, FABP2, ALDH1A1 UST, NGFR, FABP1, FABP7, ACSL1, MGST3, TNFRSF11B Leukocyte Extravasation Signaling 0.010965 CLDN10, RAC2, CLDN11, CLDN5, MMP25, CXCL12, MMP2, RASSF5, ITGAL, PRKD1, MMP19, PRKCB RAR Activation Glycerolipid Metabolism 0.012023 0.014454 PRKACB, RAC2, ALDH1A1, CRABP2, ADCY3, SMAD6, NCOR1 CRABP1, PRKD1, ADH4, PRKCB ADH6, PNLIPRP2, LIPA, ALDH1A1, LPL, DGAT2, APOC2, ADH4 Prolactin Signaling 0.016982 FYN, PRLR, PDK1, TCF7, PRKD1, PRKCB FXR/RXR Activation 0.017378 RAC2, APOB, PCK2, APOC2, HNF4A, FOXA3, MTTP LXR/RXR Activation 0.018197 APOC4, NGFR, LPL, NCOR1 APOC2, TNFRSF11B CTLA4 Signaling in Cytotoxic T Lymphocytes 0.018197 FYN, CD3G, RAC2, LCK, AP1S2, PPP2R2C, CD3D PXR/RXR Activation 0.020417 PRKACB, RAC2, ALDH1A1, CYP2C9, PCK2, HNF4A G Protein Coupled Receptor Signaling 0.021380 GPR161, PRKACB, FYN, RAC2, RASGRP1, RGS10, ADCY3, ATF4, EDNRA, DRD2, PDE4D, PRKCB HER 2 Signaling in Breast Cancer 0.026303 RAC2, NRG1, MMP2, PARD6G, PRKD1, PRKCB

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126 Table 4 3 Continued Pathway b P value Genes a Maturity Onset Diabetes of Young (MODY) Signaling 0.027542 FABP2, FABP1, HNF4A TGF 0.029512 ZNF423, NODAL, INHA, GSC, SMAD6, HNF4A Acute Phase Response Signaling 0.032359 RAC2, TF, NGFR, APOA2, CRABP2, A2M, SERPINF2, CRABP1, TNFRSF11B, SERPIND1 Calcium induced T Lymphocyte Apoptosis 0.033113 CD3G, LCK, CD3D, PRKD1, PRKCB Virus Entry via Endocytic Pathways 0.046774 FYN, RAC2, ITGAL, PRKD1, FOLR1, PRKCB Molecular Mechanisms of Cancer 0.047863 PRKACB, FYN, RAC2, DIRAS3, ADCY3, SMAD6, FZD1, RASGRP1, SMO, IHH, BMP 6, GLI1, PRKD1, BCL2L11, PRKCB, FZD7 Colorectal Cancer Metastasis Signaling 0.047863 PRKACB, RAC2, MMP25, DIRAS3, ADCY3, SMO, MMP2, FZD1, TCF7, WNT11, FZD7, MMP19 a Genes symbols in black are overexpressed in ED, genes symbols in red are overexpressed in Tr. b Pathways are from Ingenuity (www.ingenuity.com)

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127 Table 4 4 Genes with the grea test fold change for embryonic d isc (ED) compared with trophoblast (Tr). Intensity Means Accession # Name Fold Change T r ED Overexpressed in ED NM_175817 Fatty acid binding protein 1 ( FABP1 ) 119.93 4.90 610.36 XR_028016 Transforming growth factor Beta IG H3 ( TGFBI ) 95.21 24.99 2379.0 NM_001076949 Septin 3 ( SEP3 ) 89.88 7.44 669.04 NM_001025344 Nanog homeobox ( NANOG ) 75.69 11.51 1390.0 NM_174188 Secreted phosphoprotein 2 ( SPP2 ) 68.26 7.32 499.21 NM_001077921 Amyloid beta precursor protein A 2 ( APBA2 ) 64.85 7.25 470.18 NM_001105463 SRY (sex determining region Y) box 2 ( SOX2 ) 57.68 7.59 873.88 NM_001105411 GDNF family receptor alpha 1 ( GFRA1 ) 56.96 4.59 261.73 NM_177484 Transferrin ( TF ) 54.89 31.07 6954.9 NM_001040502 Alpha 1 acid glycoprotein ( AGP ) 52.29 16.37 1006.4 NM_001083369 Transmembrane protein 130 ( TMEM130 ) 50.70 4.74 240.07 NM_001078019 Solute carrier family 7, member 3 ( SLC7A3 ) 49.32 13.38 660.05 XM_001789128 DAZ interacting protein 1 ( DZIP1 ) 48.07 4.38 210.50 NM_001076945 Stearoyl CoA desaturase 5 ( SCD5 ) 46.19 7.65 460.06 XM_612940 Carboxypeptidase A6 (LOC540749) 45.16 4.53 238.31

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128 Table 4 4 Continued Intensity Means Accession # Name Fold Change T r ED Overexpressed in Tr XM_614458 CAP GLY domain containing linker protein 1 ( CLIP1 ) 2.53 333.15 131.55 XM_866711 Nuclear receptor co repressor 1 ( NCOR1 ) 2.57 184.07 71.62 XM_614825 Centrosomal protein ( CEP350 ) 2.59 104.34 40.32 NM_001034342 ATF4 activating transcription factor 4 ( ATF4 ) 2.60 342.05 131.44 XM_612376 Centromere protein F ( CENPF ) 2.64 345.30 130.74 XM_585315 Biorientation of chromosomes in cell division 1 ( BOD1L ) 2.71 296.27 109.40 NM_001102181 Cromodomain helicase DNA binding protein 2 ( CHD2 ) 2.81 287.45 102.12 NM_174436 Psrotein kinase, cGMP dependent, I ( PRKG1 ) 2.88 73.98 25.68 XM_580572 Dickkopf homolog 1 ( DKK1 ) 2.92 2519.12 887.33 NM_174239 Aldehyde dehydrogenase 1, member A1 ( ALDH1A1 ) 3.02 457.08 151.46 XM_581155 Filamin A interacting protein 1 ( FILIP1L ) 3.04 440.05 144.76 NM_001045901 Growth arrest and DNA damage ( GADD45G ) 3.56 2178.12 611.84 XM_606804 RAR related orphan receptor B ( RORB ) 4.74 36.98 7.79 XM_001256996 Ribosomal protein L10a (LOC790558) 5.23 81.89 15.65 XM_864189 Ribosomal protein L36a ( RPL36A ) 8.26 96.67 11.70

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129 Table 4 5 The 15 most abundant genes overexpressed in embryonic disc or trophoblast Accession # Name Intensity Means Embryonic Disc ED Tr Fold Change NM_001034790 Stathmin 1 (STMN1) 39876.8 8984.0 4.4 NM_001166505 Tubulin, alpha 1a (TUBA1A) 39837.2 4416.9 9.0 NM_175801 Follistatin (FST) 29163.8 13194.4 2.4 XM_001251618 GL12416 like (TUBA3D) 26547.3 2820.9 9.4 NM_176646 Serpin peptidase inhibitor, clade A, member 5 (SERPINA5) 18250.3 1213.3 15.0 NM_174580 POU class 5 homeobox 1 (POU5F1) 17887.4 489.3 29.2 NM_001098378 Transmembrane protein 88 (TMEM88) 17535.0 7907.4 2.2 NM_205802 Lysosomal protein transmembrane 4 beta (LAPTM4B) 13950.3 2251.5 6.1 NM_001075171 Coiled coil domain containing 109B (CCDC109B) 12955.7 3284.5 3.9 NM_001034231 Inhibitor of DNA binding 2 (ID2) 12754.3 4141.6 3.0 NM_001097568 Inhibitor of DNA binding 1 (ID1) 11206.3 2432.3 4.6 NM_174438 Protein S alpha (PROS1) 9820.1 2079.4 4.7 NM_001114857 Metallothionein 1E (MT1E) 9815.9 393.8 18.9 NM_001101149 Procollagen lysine, 2 oxoglutarate 5 dioxygenase 2 (PLOD2) 9500.8 4296.1 2.2 NM_001035046 Microsomal glutathione S transferase 3 (MGST3) 8855.9 1347.2 6.6 Trophectoderm ED Tr Fold Change XM_580572 Dickkopf homolog 1 (DKK1) 887.33 2519.12 2.92 NM_001045901 Growth arrest and DNA damage inducible, gamma (GADD45G) 611.84 2178.12 3.56 XM_581232 Rho GTPase activating protein 21 (ARHGAP21) 392.39 841.24 2.19 NW_877106.1 Retrovirus related Pol polyprotein from transposon 297 (LOC608201) 339.91 783.89 2.31 NM_174239 Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) 151.46 457.08 3.02

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130 Table 4 5 Continued Accession # Name Intensity Means Trophectoderm ED Tr Fold Change XM_581155 Filamin A interacting protein 1 like (FILIP1L) 144.76 440.05 3.04 NM_174824 Protein phosphatase 1, regulatory (inhibitor) subunit 16B (PPP1R16B) 182.95 386.36 2.11 XM_612376 Centromere protein F, 350/400ka (CENPF) 130.74 345.30 2.64 NM_001675.2 ATF4 activating transcription factor 4 (ATF4) 131.44 342.05 2.60 XM_614458 CAP GLY domain containing linker protein 1 (CLIP1) 131.55 333.15 2.53 XM_585315 Biorientation of chromosomes in cell division 1 like (BOD1L) 109.40 296.27 2.71 NM_001102181 Chromodomain helicase DNA binding protein 2 (CHD2) 102.12 287.45 2.81 NM_001083753 SH3 domain containing ring finger 2 (SH3RF2) 115.48 236.26 2.05 XM_589712 Tetratricopeptide repeat domain 17 (TTC17) 94.55 219.57 2.39 NM_001103246 N acetyltransferase 9 (NAT9) 82.36 202.23 2.46

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131 Table 4 6 Differences in expression of selected genes between embryonic disk and trophoblast as determined by microarray analysis and qPCR. Fold Change a Name Symbol Microarray qPCR P value for qPCR BMP binding endothelial regulation BMPER 26.0 1389 <0.0001 Calpastatin CAST 2.1 2.4 <0.006 Claudin 11 CLDN11 36.0 205.0 <0.0001 Fibroblast growth factor 2 FGF2 8.9 118.6 <0.0001 Gata binding protein 5 GATA5 15.4 61.4 <0.0001 Growth differentiation factor 3 GDF3 36.7 167.7 < 0.0001 Gap junction protein, gamma 1 GJC1 11.4 3.2 N.S. IGF like family member 1 IGFL1 20.0 39.4 <0.0005 Nodal NODAL 16.8 608.9 <0.0001 Serpin peptidase inhibitor, clade A SERPINA5 15.0 12.8 <0.05 Transmembrane protein with EGF like and two follistatin like domain TMEFF1 11.1 11.3 <0.0001 a ED/Tr

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132 Figure 4 1 Separation of a Day 15 conceptus into embryonic disc and trophoblast. The left panel shows an entire Day 15 elongated embryo and the right panel shows an enlargement of the same embryo to visualize the embryonic disc. The embryo was bisected along the red lines.

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133 Figure 4 2 Individual values of antiviral acti vity in uterine flushings (top) and length of recovered embryos (bottom). Triangles represent tubular embryos and circles represent filamentous embryos. In the bottom panel, embryos that were not recovered intact are represented by open circles. The hori zontal bars represent the mean value for each treatment. Embryo length was not significantly affected by treatment. Antiviral activity was also not affected by treatment. However, when considering only those cows in which detectable antiviral activity was present, there was a tendency for antiviral activity to be greater for cows receiving CSF2 treated embryos (P=0.07).

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134 Figure 4 3 Hierarchical Cluster of the transcriptomes of samples of embryonic disc (ED) and trophoblast (Tr) for control and CSF2 treated embryos. ED samples form a separate cluster from Tr but there was no distinct clusters based on treatment.

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135 CHAPTER 5 GENERAL DISCUSSION Technological advances in manipulation of mammalian embryos outside the maternal environment have provi ded opportunities to study preimplantation embryo development and to optimize genetic selection and fertility. In vitro embryo production (IVP) has great potential to improve fertility and enhance breeding schemes in beef and dairy production systems. None theless, transfer of IVP embryos has not met its potential because these embryos are altered in terms of ultrastructure (Rizos et al. 2002) metabolism (Khurana and Niemann 2000) and gene expression (Lonergan et al. 2006) These differences are associated with reduced embryo survival rates following transfer (Farin and Farin 1995; Drost et al. 1999; Hasler 2000) increased pregnancy loss (Hasler 2000; Block et al. 2003) and increased proportion of calves with congenital malformations and neonatal abnormalities (Farin et al. 200 6) One approach to improve post transfer survival of IVP embryos is to modify culture medi um with growth factors to more closely mimic the microenvironment found in vivo Modification of embryo culture conditions can significantly impact the competence f or development of the resulting blastocyst. Many attempts have been made to mimic aspects of the uterine environment in culture and decrease alterations in embryo morphology and physiology. Several growth factors and cytokines have been tested for their a ctions on bovine embryonic development in vitro including epidermal growth factor (Sagirkaya et al. 2007) interleukin 1 (Paula Lopes et al 1998) IGF 1 (Block et al. 2003; Jousan and Hansen 2004; Lima et al. 2006) and CSF2 (de Moraes and Hansen 1997 a ) Only IGF 1 has been tested for improvement of post transfer survival of bovine IVP embryos. In this case, IGF 1 treated embryos had increased survival rates after

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136 transfer when transfers were performed in heat stressed cows but not when performed in cows not subject to heat stress (Block et al. 2003; Block and Hansen 2007) In this dissertation, we have identified CSF2 as another maternally derived cytokine that can alter embryonic development in a way that increases the likelihood that a transferred embryo will continue to term. The actions of CSF2 on embryonic development that promote survival include changes in gene expression at Day 6 of development that block apoptosis and could conceivably result in alterations in gastrulation. In addition, CSF2 tended to increase embryonic survival between transfer and Day 15 and to i ncrease the competence of the Day 15 embryo to exerts its antiluteolytic actions on the endometrium ( Figure 5 1 ). In Chapter 2, the ability of CSF2 to increase blastocyst development and post transfer survival of bovine IVP embryos, as well as cell number cell allocation and apoptosis were analyzed. The results indicate that addition of CSF2 to the culture medium at Day 5 after insemination increases blastocyst development, pregnancy rate at Day 35 and decreases pregnancy loss thereafter On the other han d, addition of CSF2 at Day 1 increased blastocyst development at a lesser extent and did not have an effect on pregnancy rate. The differential effect of CSF2 may be dependent on stage of development of the embryo. Early addition of CSF2 could cause intern alization and degradation of the ligand receptor complex before the embryo has overcome the EGA process and is not ready to respond to the beneficial effects of CSF2. Another effect of CSF2 that may be related to increased embryonic survival is a prefere ntial increase in the number of cells in the ICM The number of cells in the ICM correlates strongly with viability after transfer in mouse embryos (Lane and Gardner

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137 1997) The ICM/TE ratio between bovine embryos produced in vitro differs considerably from embryos generated in vivo (Iwasaki et al. 1990; Crosier et al. 2001) Our results show that CSF2 can minimize the detrimental effects of in vitro culture by increasing the ICM/TE ratio. Another particularity of this study is th at embryos were produced using X sorted semen. It is known that the female embryo undergoing the transition from the morula to b lastocyst stage does not tolerate glucose as well as the male embryos (Gutirrez Adn et al. 2000) In addition, male embryos develop faster in culture than female embryos (Gutirrez Adn et al. 2000; Kimura et al. 2005) The gender difference in growth rate and sensitivity to glucose is probably due to differential expression of the X chromosomes. In the female embryo, both X chromosomes are active at the cleavage stage (Mak et al. 2004) and the gene for glucose 6 phosphate dehydrogenase, an enzyme that controls the entry of glucose into the pentose phospate pathway, is located on the X chromosome Another gene, h ypoxanthine phosphoribosyl transferase 1, wich encodes an enzyme involved in controlling the amount of oxygen radicals, is also on the X chromosome (Goldammer et al. 2003) The excess in free radical and the poor tolerance to glucose could retard the dev elopment of female embryos. Since the primary action of CSF2 is to promote cellular survival there is a possibility that the effects of CSF2 in this study were amplified by the fact that at least 85% of the embryos were female. To further examine the chan ges in the embryo caused by CSF2 and the consequences of these changes into adulthood a follow up study to investigate the reproduction and health status of the heifers born from the CSF2 treated embryos would be applicable.

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138 Addition of CSF2 to mouse cul ture medium partially alleviates the long term adverse consequences for postnatal growth caused by IVP. Adult mice that originated from embryos cultured in medi um without any cytokine have increased body weight, increased central fat and increased fat rela tive to total body mass compared with mice that originated from embryos cultured in the presence of CSF2 or embryos developed in vivo (Sjblom et al. 2005) When pregnant, female mice generated from in vitro culture have a larger placenta, which decreases the placenta/fetus weight ratio, compared with mothers that were developed from embryos produced in vivo or cultured in vitro in the presence of CSF2 (Sjblom et al. 2005) Thus far, two molecules, IGF1 and CSF2 have been reported to improve the post transfer survival of bovine embryos, with IGF1 only being effective during the hot months of the year. This peculiarity of IGF1 was not specific ally tested for CSF2; however the increased pregnancy rates found with addition of CSF2 at Day 5 after insemination were from transfers done in both the fall and winter seasons. While the transfer of embryos that were treated with CSF2 at Day 1 after insem ination (when pregnancy rates were not increased) were done only during the summer season. The combination of IGF1 and CSF2 plus other growth factors and cytokines (IGF2, FGF2, TGFB and LIF) have been tested for embryo development and cell number. The addi tion of a single cytokine as well as the combination of all cytokines improved development and increased blastocyst cell number compared with medium alone (Neira et al. 2010) However this study wa s not able to clarify whether the combination of multiple cytokines is better than the addition of a single cytokine since all the possible combinations were not compared at simultaneously. It is possible that the production of

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139 bovine embryos in the presen ce of both IGF1 and CSF2 could compensate for the in ability of IGF1 to improve embryo survival during the winter and therefore result in greater post transfer survival irrespective of season. IGF1 acts as a mitogenic and cellular survival factor, but it fai ls to surmount the negative effects of cryopreservation on embryos (Velazquez et al. 2009) While CSF2 can also act as a mitogenic factor, its central role is cell survival (Trapnell et al. 2009) including being able to protect one cell mouse embryos from freezing damage and decrease the apoptotic index to zero (Desai et a l. 2007) It is unlikely that the absence of CSF2 would cause absolute infertility in the cow. it is likely that in the absence of CSF2, the receptors would be activated by o ne or both of these two cytokines (Hansen et al. 2008) Moreover, CSF2 deficient mice (CSF2 / ) can produce viable puppies (Robertson et al. 1 999) On the other hand, in human and mice, excess ive production of CSF2 has been reported to cause multiple pathologies, mostly related to autoimmune diseases (Hansen et al. 2008) An increased immune response, besides increasing the chances of diffuse inflammation, could also result in less tolerance for the fetus. In Chapter 3, the transcriptome of the CSF2 embryo was analyzed. Embryos produced in vitro have altered gene expression patterns when compared with embryos produced in vivo In vitro culture of bovine embryos results in increased abundance of the transcripts for heat shock protein 70.1, copper/zinc superoxide dismutase, glucose transporters 3 and 4 ) (Lazzari et al. 2002) altered levels of expression of X linked genes (King 2008) and differential expression of IGF family genes (Bertolini et al. 2002;

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140 Sagirkaya et al. 2006; Moore et al. 2007) Furthermore, blastocysts produced in vitro have higher expression of BCL2 associated X protein, a pro apoptotic protein, and arcosine oxidase, an oxidative enzyme and decreased expression of mitochondrial manganese superoxide dismutase, an important antioxidant defense in cells exposed to oxygen (Rizos et al. 2002) The effects from embryo culture in vitro persist into fetal life being presented as fetal abnormalities and large offspring syndrome. These deformitie s could be consequence of alterations in the transcripts at the preimplantation stage or an epigenetic effect as IVP embryos have decreased expression of transcripts for enzymes involved in the de novo methylation process (Smith et al. 2009) The results of Chapter 3 indicate that addition of CSF2 to embryo culture altered the expression of 3 major groups of genes (development and differentiation process, cell communication and a poptosis). The genes involved in the developmental process and differentiation were functioning to inhibit neurogenesis and stimulate mesoderm or muscle formation, and pluripotency. In cell communication process the signaling system that was most represent ative was the WNT system, analysis of the data indicates inhibition of catenin dependent WNT signaling. The apoptosis signaling pathway was also inhibited by CSF2. The biological effect of CSF2 on apoptosis genes was appropriately proved by submitting th e embryos to heat shock and analyzing the percentage age of apoptotic blastomers by a well known assay However, the differential expression of genes promoting mesoderm and delaying neural tissue development should be further studied. The pre gastrulation period is when the earliest structural decisions are being made; alterations of the genome during gastrulation may cause damage that will only

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141 manifest ed in subsequent cell generations (Rutledge 1997) Certain changes in early development can also induce mid to late gestation anatomic malformations and fetal lethality (Rutledge 1997) To further investigate the effects of CSF2 in the gastrula tion process a imunohistochemistry study of embryos recovered at different stages of development, such as Day 15 (when both mesoderm and neural tissue are beginning to form) and Day 21 (when gastrulation and neurulation are completed and somites are start ing to form), could allow for properly visualization of the structures controlled by the referred actin isomers from actin from non smooth muscle, cytok eratin for epithelial tissue and anti neurofilament for neural tissue could reveal the preferential development of one tissue over the other. If the changes in these early stages are confirmed, a histomorpholog ical study could be performed after the organs are formed ( Day 45) to define if the damage persist ed and if that would be one of the reasons for the pregnancy losses that happen after Day 35. The development and morphology of the placenta would also be of interest in this study. The effects of CSF2 to inhibit WNT signaling pathway could be examined in a comparative study with control embryos, CSF2 treated embryos and embryos submitted to WNT inhibitors. In C hapter 4, CSF2 treated embryos were tested for characteristics that could make them more like ly to establish and maintain pregnancy. CSF2 treated embryos were greater in size, secrete more IFNT and have a higher recovery rate. However, there was no significant effect of treatment when expression of the embryonic disc and

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142 trophectoderm dissected from elongated control and CSF2 embryos were. The lack of difference may be due to the fact that only filamentous embryos were selected in both groups. Perhaps if we had analyzed all the embryos recovered this bias could have been avoided. Ano ther possibility is that the development of the placenta is facilitated by CSF2 as seen by a longer trophectoderm, and such an effect could improve implantation and possibly decrease embryonic loss. The last study also resulted in the identification of over 500 genes preferentially expressed in the embryonic disc. Many of these have been described earlier as be ing ICM or trophectoderm markers in mouse and human embryos. Identification of new markers of ICM and TE could be used to facilitate developmen t of the bovine e mbryonic stem cell and advance the understanding of the embryonic developmental process. Lastly, t he study of different DNA methylation patterns may provide more insight into the actions of CSF2 to promote embryo development and survival. The knowledge at tained by these studies could be pertinent for the development of more efficient systems in the production of embryos in vitro which in turn will result in enhanced pregnancy success following embryo transfer in cattle. Understanding the mechanisms that r egulate early bovine embryonic development may prove useful to study infertility in other species, including human.

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143 Figure 5 1 Summary of effects CSF2 on embryo development and po st transfer survival. Treatment of embryos with CSF2 from Days 5 7 after insemination increases development of oocytes to blastocyst increases the number of cells in the inner cell mass, decreases apoptosis, increases expression of genes regulating mesoderm formation and epithelial mesenchymal transition an d decrease expression of genes regulating neural cell development. CSF2 treatment increases recovery of embryos on Day 15, length, IFNT secretion and expression of IFNT and KRT18 mRNA. Recipients that receive an embryo treated with CSF2 have increased preg nancy rates at Days 30 35, decreased pregnancy losses after Days 30 35 and increased calving rate.

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172 BIOGRAPHICAL SKETCH B rbara Loureiro was b orn in 1980 in Linhares, Esp rito Santo, Brazil, to Maria Jos and Carlos Alberto Barbosa Loureiro. The elder of two children, Brbara lived her whole life in S o Mate us, until she graduated from high school in 1997. Following graduation from high school, she entered the School of Veterinary Medicine at Universidade Federal Rural de Pernambuco, Brazil There she was a warded the researc h scholarship Research Program (PIBIC/CNPq) During the three years of the scholarship she developed research in the Bovine Clinic and acquired good experience in scientific work. During her time in c ollege she was also interested in the educational process and she served as president of the Veterinary Students Association. She helped develop the academic program for the School of Veterinary Medicine and represented the students as a board member at t he Educational Council of the S c hool of Veterinary Medicine and at the Administrati ve Council of the Veterinary Department She received her degree in Veterinary Medicine in July of 2003. C oncomitant with Veterinary S chool she received education in the Special Education Program focused on te a ching Agricultural Science begning in 2002 There s he had the opportunity to teach at the Federal Agricultural Technical School of Pernambuco in the Animal Science program and develop extension projects in Enviromen tal Health. She received her Teaching Specialist degree in May of 2004. Immedialty afte r graduation from Veterinary S chool she entered a Master of Veterina ry Science program at Universidade Federal Rural de Pernambuco O ne year into her m aster s he received the opportunity to do an intership with Dr P.J. Hansen at the University of Florida. Her research was focused on apoptosis i n the

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173 preimplanta t she was awarded a CAPES/F ulbright scholarship to persue a Doctor of Philosophy Degree in the Anim al Mollecular and Cell ular Biology Graduate Program and continue her work with Dr Hansen In 2010 she received the Sigma Xi Gra d uate Research Award After completin g the requirements for th e doctoral degree Brbara will continue her career as a research scientist back in Brazil.