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© 2013 Juanmahel Davila
THE ROLE OF RAS-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1 (RAC1)-
DEPENDENT PATHWAY IN THE REGULATION OF UTERINE FUNCTION DURING
EARLY PREGNANCY
BY
JUANMAHEL DAVILA
DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in VMS - Comparative Biosciences
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2013
Urbana, Illinois
Doctoral Committee:
Professor Indrani C. Bagchi, Chair and Research Director
Professor Emerita Janice M. Bahr
Professor Jodi A. Flaws
Professor Paul S. Cooke, University of Florida-Gainesville
Research Assistant Professor Quanxi Li
ii
ABSTRACT
During implantation, uterine stromal cells differentiate into specialized decidual cells, which
become secretory and support the growth of the embryo before placentation. At the same time,
new blood vessels begin to form in the maternal decidua from pre-existing vasculature. This
process termed angiogenesis is critical for the establishment of the placenta and the maintenance
of pregnancy. Although it is clear that decidualization and angiogenesis play a crucial role during
early pregnancy, the complex molecular pathways underlying these processes remain largely
unknown. Our recent studies revealed that RAC1 (Ras-related C3 botulinum toxin substrate 1), a
pleiotropic signaling factor belonging to the Rho family of GTPases, is induced in the uterine
stroma during decidualization and angiogenesis, suggesting a role for RAC1 in these critical
processes. To address the role of RAC1 during pregnancy, we created a conditional knockout of
the Rac1 gene in the uterus by crossing mice carrying floxed Rac1 with PgrCre mice, thereby
generating the conditional mutation termed as Rac1d/d. A six-month breeding study indicated a
severe defect in fertility of the Rac1d/d females. Further analysis revealed that while embryo
attachment appeared to be unaffected, differentiation and secretory functions of decidual cells were
impaired in Rac1d/d uteri. This abnormality of decidual cells led to multifactorial consequences,
including compromised uterine vascular remodeling and angiogenesis, maternal blood vessel
hemorrhage and placental abnormalities resulting in embryo loss around days 10 to 12 of
pregnancy. Fetuses that survived to day 15 of pregnancy showed indications of intrauterine growth
restriction. Based on the results obtained in this dissertation, we propose that RAC1 regulates
stromal differentiation and secretory functions, which produce and secrete critical local factors
that regulate vascular remodeling at the maternal-fetal interface and promote proper formation of
the placental disk. Therefore, RAC1 induced in the uterine stroma during decidualization plays a
critical role in maintaining proper function of the placental vascular bed and supporting embryo
growth and development during early and mid-stages of pregnancy.
iii
To Aurora, Mom, Dad, and Brother
iv
ACKNOWLEDGEMENTS
This dissertation would have never been possible without the generous help and support of many
people that I have encounter through my time in Illinois.
I will like to start by expressing my most sincere and deepest appreciation to my current and former
research advisors and mentors, Drs. Indrani Bagchi, Milan Bagchi and Paul Cooke. I owe great
debt of gratitude to these people because thanks to their patient guidance, continuous support,
constant challenge and encouragement, and mentorship, I have reached this far. All three have
been extremely instrumental in guiding me along the many challenges that were presented to me
during my academic journey, and have been key elements in my personal and scientific growth,
as well as achievements. I am also appreciative for the support of my former (Drs. William
Helferich and O. David Sherwood) and current (Drs. Janice M. Bahr, Jodi A. Flaws, and Quanxi
Li) members of my doctoral committee for their continuous support, motivation, and guidance
throughout these years. I have been very fortunate to have these three wonderful human beings as
pillars of my academic learning.
I am extremely thankful to all the people with whom I had interactions at the University of Illinois.
The former members of the Cooke laboratory (Drs. Liz Simon, Melissa Cimafranca,
Santhipriyadarsini Sridharan, Gaurav Tyagi, Heather Schlesser, Jaspreet Kaur, Mr. Daryl Meling,
and Ms. Gail Ekman), which were instrumental in my early scientific carrier providing me with
technical support and training, as well personal support and friendship. The former and current
members of the Bagchi laboratories, especially Hatice Kaya, Janelle Mapes, Sandeep Pawar, Dr.
Yuechao Zhao, Dr. Shanmugasundaram Nallasamy, and Alison Hantak, which have been also
supportive in my latest stages of scientific growth, as well as providing me with their friendship. I
will like to express a special appreciation to Drs. Mary J. Laws, Quanxi Li, and Athilakshmi
Kannan, my colleagues and dearest friends, who provided me with extensive support, mentorship,
assistance, and guidance during my transition and establishment to the Bagchi laboratory. Also, a
special thanks to all the wonderful past and current colleagues of the Department of Comparative
Biosciences, especially Dr. Denise Archambeault, Dr. Amrita Das, Thushara Chakkath, Snehita
Sri Varma, Joseph Cacioppo, Dr. Megan Mahoney, Dr. Leslie McNeil, Dr. CheMyong “Jay” Ko,
v
Dr. Jing Yang, Dr. Duncan Fergusson, Dr. David Bunick, Dr. Rex Hess, Dr. Marie-Claude
Hofmann, Dr. Melissa Clark, Dr. Zelieann Craig, Dr. Ju Lan Chun, Dr. Thomas Garcia, Patrick
Hannon, and Dr. Jackye Peretz, for a supportive, fun, and educational work environment. Finally,
I most express my gratitude to the administrative support of the former and current members of
the main office of the Department of Comparative Biosciences, with special attention to Carrol
Bunick, Carla Manuel, Todd Blazaitis, Cindy Dillman, and Mona Nordbrock.
I will like to extend my appreciation to the principal investigator and members of the laboratories
of Drs. Milan Bagchi, Duncan Ferguson, David Bunick, Marie-Claude Hofmann and Rex Hess
(former CB investigators), Jodi Flaws, and Jing Yang, for technical assistant and for allowing me
to use critical instrument that made part of my work possible; specifically acknowledging Dr.
Zelieann Craig for assistance with ELISA (Flaws’ Lab); Hatice Kaya for assistance with western
blots and microarray analysis (M. Bagchi’s Lab); Drs. Zhigang Jin, Jin Wei "Kenneth" Chung, R.
Tyler Schwend for assistance with microscope and western blots (Yang’s Lab); Dr. Thomas Garcia
for assistance in microscopy and fluorescent imaging (Hofmann’s Lab); and David Bunick for
assistance with quantitative real-time PCR. Also, I will like to thank the histology laboratory,
especially Karen Doty, for technical assistance with immunostaining.
Last but not least, I will like to thank my family and closest friends (Jeff Brown, Sylvia Choi, and
Ranjani Murali)! Which without their continuous support, encouragement, and confidence in me,
I would not be able to accomplish my career goals and achievements. I am extremely gratefully to
my brother Isamac Dávila, my mother Zelithez Lorenzo, my father Juan Dávila, my best friend
Emanuel Rivera, as well as my closest relatives, who inspired and pushed me to pursue my dreams
and to never give up without trying. I am in greatest debt to my loving fiancé Aurora M. Cruz,
who motivated, inspired, and supported me throughout all these years, as well as been one of my
strongest pillars in the pursuit of my doctoral degree. I am also, thankful to my new family
members, Wanda Torres, Andres Cruz (deceased), Andy Cruz, and Zoriann Cruz, for their
continuous encouragement and motivation.
vi
TABLE OF CONTENT
LIST OF FIGURES AND TABLES ........................................................................................... ix
LIST OF ABBREVIATIONS .................................................................................................... xii
CHAPTER 1: INTRODUCTION ................................................................................................ 1
CHAPTER 2: REVIEW OF RELEVANT LITERATURE ...................................................... 4
2.1 DEFINING THE PROBLEM: CLINICAL EVIDENCE FOR RECURRENT MISCARRIAGE AND PREGNANCY
LOSS ................................................................................................................................................. 5
2.1.1 FACTORS THAT MAY CONTRIBUTE TO MISCARRIAGE .............................................................. 5
2.1.1.1 CONGENITAL ABNORMALITIES ....................................................................................... 6
2.1.1.2 VASCULAR PROBLEMS AND BLEEDING DISORDERS ........................................................ 6
2.1.2 INTRAUTERINE GROWTH RESTRICTION ................................................................................... 7
2.1.3 ASSISTED REPRODUCTIVE TECHNOLOGIES .............................................................................. 9
2.2 UTERINE RECEPTIVITY, THE WINDOW OF IMPLANTATION, AND EARLY PREGNANCY IN THE MOUSE
......................................................................................................................................................... 9
2.2.1 CRITICAL EVENTS DURING PREGNANCY ................................................................................ 10
2.2.1.1 IMPLANTATION ............................................................................................................. 10
2.2.1.2 DECIDUALIZATION: A KEY EVENT FOR SUCCESSFUL ESTABLISHMENT OF PREGNANCY ..
...................................................................................................................................... 11
2.2.1.3 ANGIOGENESIS AND VASCULAR REMODELING ARE ESSENTIAL FOR MAINTENANCE OF
PREGNANCY ................................................................................................................................... 13
2.2.1.4 PLACENTATION ............................................................................................................. 17
2.2.1.4.1 PLACENTATION IN HUMANS AND MICE .................................................................... 17
2.2.1.4.2 REGULATION OF TROPHOBLAST INVASION AND PLACENTAL PATHOLOGIES ........... 18
2.2.1.4.3 THE MID-STAGES OF PLACENTATION IN THE MOUSE ................................................ 19
2.3 RHO GTPASES ................................................................................................................................ 19
2.3.1 THE RHO GTPASES ............................................................................................................... 20
2.3.2 ROLE OF RHO GTPASES IN ACTIN DYNAMICS ....................................................................... 20
2.3.2.1 FUNCTIONS BEYOND ACTIN DYNAMICS ........................................................................ 21
2.3.3 REGULATORS OF THE RHO GTPASES .................................................................................... 21
2.3.4 RHO GTPASES AND DISEASE ................................................................................................. 23
2.3.5 RAS-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1 (RAC1) ............................................... 23
2.3.6 AVAILABLE MOUSE MODELS TO STUDY RAC1 FUNCTION .................................................... 26
vii
2.4 THE AIMS OF THE DISSERTATION ................................................................................................... 27
2.5 CHAPTER FIGURES ......................................................................................................................... 28
CHAPTER 3: GENERATION AND PHENOTYPIC ANALYSIS OF RAC1D/D MUTANT
FEMALE MICE.......................................................................................................................... 38
ABSTRACT............................................................................................................................................... 39
INTRODUCTION ....................................................................................................................................... 40
MATERIALS AND METHODS .................................................................................................................... 42
RESULTS AND DISCUSSION ..................................................................................................................... 48
RAC1 IS EXPRESSED IN UTERINE STROMA DURING DECIDUALIZATION, BUT ITS EXPRESSION IS
INDEPENDENT OF STEROID HORMONES .............................................................................................. 48
THE RAC1D/D MOUSE MODEL ............................................................................................................... 49
CONDITIONAL DELETION OF UTERINE RAC1 LEADS TO SEVERE SUBFERTILITY ................................. 49
CONDITIONAL DELETION OF RAC1 HAS NO EFFECT ON OVARIAN FUNCTION ..................................... 50
THE SEVERE SUBFERTILITY IN RAC1D/D IS DUE TO MATERNAL BUT NOT EMBRYONIC DEFECT ........... 52
CHAPTER FIGURES AND TABLES ............................................................................................................ 54
CHAPTER 4: RAC1 IS A CRITICAL REGULATOR OF UTERINE ANGIOGENESIS
DURING EARLY PREGNANCY IN THE MOUSE .............................................................. 78
ABSTRACT............................................................................................................................................... 79
INTRODUCTION ....................................................................................................................................... 80
MATERIALS AND METHODS .................................................................................................................... 82
RESULTS AND DISCUSSION ..................................................................................................................... 87
MICROARRAY ANALYSIS REVEALS IMPAIRED ANGIOGENIC NETWORK IN RAC1-ABLATED UTERI ..... 87
ABLATION OF RAC1 IN THE UTERUS LEADS TO A DECREASED SECRETION OF PRO-ANGIOGENIC
FACTORS ............................................................................................................................................. 89
CONDITIONAL DELETION OF RAC1 IN THE UTERI LEADS TO SEVERE HEMORRHAGE .......................... 90
CHAPTER FIGURES .................................................................................................................................. 92
CHAPTER 5: CONCLUSIONS AND WORKING MODEL ............................................... 101
CHAPTER FIGURE ..................................................................................................................... 105
CHAPTER 6: FUTURE DIRECTIONS ................................................................................. 106
REFERENCES .......................................................................................................................... 109
viii
APPENDIXES ........................................................................................................................... 134
APPENDIX A: BREEDING STRATEGIES FOR THE FOUNDER AND EXPERIMENTAL COLONY
..................................................................................................................................................... 135
APPENDIX B: GENE EXPRESSION VALIDATION OF OTHERS MOLECULES INVOLVED IN
STROMAL CELL DECIDUALIZATION AND ANGIOGENESIS ........................................... 141
APPENDIX C: PRELIMINARY DATA FOR FUTURE DIRECTIONS ............................................ 143
C1. BACKGROUND INFORMATION AND THE RATIONALE................................................................. 143
C2. MATERIALS AND METHODS ...................................................................................................... 145
C3. PRELIMINARY DATA .................................................................................................................. 148
APPENDIX D: GENOTYPING PRIMERS AND PROTOCOL .......................................................... 156
APPENDIX E: ANTIBODY DILUTIONS .......................................................................................... 158
APPENDIX F: PRIMER SEQUENCES FOR GENE EXPRESSION ASSAYS .................................. 160
ix
LIST OF FIGURES AND TABLES
FIGURE 2.1: HORMONE PROFILE DURING PREGNANCY IN MICE. ...................................................... 28
FIGURE 2.2: COMPARISON OF THE EARLY EVENTS OF PREGNANCY IN HUMANS AND MICE.............. 29
FIGURE 2.3: MORPHOLOGICAL CHANGES DURING STROMAL CELL DIFFERENTIATION..................... 30
FIGURE 2.4: MAJOR EVENTS DURING IMPLANTATION AND DECIDUALIZATION. .............................. 32
FIGURE 2.5: DEVELOPMENTAL STAGES OF THE MOUSE PLACENTA. ................................................ 33
FIGURE 2.6: STRUCTURAL COMPARISON BETWEEN MEMBERS OF THE HUMAN RHO FAMILY OF
GTPASES. ............................................................................................................................... 34
FIGURE 2.7: REGULATORY MECHANISMS OF THE RHO FAMILY OF GTPASES. ................................ 35
FIGURE 2.8: PROTEIN SEQUENCE COMPARISON BETWEEN HUMAN AND MOUSE RAC1. .................. 36
FIGURE 3.1: BREEDING SCHEME TO GENERATE THE CONDITIONAL MUTANT FOR RAC1 USING CRE-
LOXP STRATEGY. .................................................................................................................... 54
FIGURE 3.2: EXPRESSION PROFILE FOR RAC1 GENE AND PROTEIN IN MICE. .................................... 56
FIGURE 3.3: RAC1 IS EFFICIENTLY DELETED FROM THE UTERUS USING PGRCRE. ............................ 58
TABLE 3.1: IMPACT OF RAC1 DELETION ON FEMALE FERTILITY ...................................................... 60
FIGURE 3.4: CONDITIONAL DELETION OF RAC1 DOES NOT AFFECT OVARIAN FUNCTION. ................ 61
FIGURE 3.5: CONDITIONAL DELETION OF RAC1 HAS DOES NOT PREVENT EMBRYO ATTACHMENT AND
INVASION INTO THE ENDOMETRIUM. ....................................................................................... 63
FIGURE 3.6: CONDITIONAL DELETION OF UTERINE RAC1 DOES NOT AFFECT THE FORMATION OF THE
DECIDUOMA, BUT DISPLAYS DYSREGULATION IN ALPL ACTIVITY. ........................................ 65
FIGURE 3.7: CONDITIONAL DELETION OF RAC1 LEADS TO A DYSREGULATED STROMAL CELL
DIFFERENTIATION. .................................................................................................................. 66
FIGURE 3.8: ACTIVATION OF THE DECIDUAL CELL RAC1 PATHWAY IS NECESSARY FOR STROMAL
CELL MORPHOLOGY AND NORMAL DIFFERENTIATION. ............................................................ 70
FIGURE 3.9: REPRESENTATIVE UTERI SAMPLES AT DIFFERENT STAGES OF PREGNANCY. ................ 71
FIGURE 3.10: SEVERE HEMORRHAGE IS OBSERVED IN IMPLANTATION NODULES FROM RAC1D/D
UTERI. ..................................................................................................................................... 72
TABLE 3.2: SINGLE OR EPITHELIAL-SPECIFIC RAC1 ALLELE DELETION DOES NOT IMPACT FERTILITY
............................................................................................................................................... 75
FIGURE 3.11: PREGNANCY DEFECT OBSERVED IN RAC1 CONDITIONAL-NULL UTERI IS NOT DUE TO
AN EMBRYONIC DEFECT. ......................................................................................................... 76
x
FIGURE 4.1: BIOLOGICAL PROCESS LIST OF GENES THAT WERE DOWN-REGULATED LESS THAN 1.5
FOLD ON MICROARRAY. .......................................................................................................... 92
FIGURE 4.2: CONDITIONAL DELETION OF RAC1 IN THE UTERUS LEADS TO DOWN-REGULATION OF
SEVERAL GENES THAT ARE CRITICAL FOR UTERINE ANGIOGENESIS. ........................................ 93
FIGURE 4.3: EXPRESSION OF TWO WELL-KNOWN GENE TARGETS OF RAC1 ARE UNAFFECTED. ...... 94
FIGURE 4.4: SUPPRESSION OF THE RAC1 PATHWAY COMPROMISES THE FORMATION OF THE
DECIDUAL VASCULAR PLEXUS. ............................................................................................... 95
FIGURE 4.5: CONDITIONAL DELETION OF UTERINE RAC1 IMPAIRS ANGIOGENESIS. ......................... 97
FIGURE 4.6: CONDITIONAL DELETION OF RAC1 IMPAIRS VEGFA TRANSPORT AND SECRETION OF
UTERINE ENDOMETRIAL CELLS. .............................................................................................. 98
FIGURE 4.7: RAC1D/D UTERI DISPLAY EXTRAVASATION OF RED BLOOD CELLS. ................................ 99
FIGURE 4.8: DECIDUA FROM RAC1D/D MICE SHOW SEVERE BLOOD HEMORRHAGE IN THE LATERAL
BLOOD SINUSES. ................................................................................................................... 100
FIGURE 5.1: WORKING MODEL FOR THE ROLE OF RAC1 IN THE UTERUS DURING EARLY EVENTS OF
PREGNANCY. ......................................................................................................................... 105
FIGURE A.1: BREEDING STRATEGY FOR GENERATING THE RAC BREEDING COLONY. ................... 135
FIGURE A.2: EXPECTANCY FREQUENCIES OF ANIMALS/GENOTYPE BASED ON THE BREEDING
STRATEGY FOR GENERATING THE FOUNDING ANIMAL COLONY. ............................................ 136
FIGURE A.3: BREEDING STRATEGY FOR GENERATING THE EXPERIMENTAL ANIMALS ................... 137
FIGURE A.4: EXPECTANCY FREQUENCIES OF ANIMALS/GENOTYPE BASED ON THE BREEDING
STRATEGY FOR GENERATING THE BREEDING COLONY. .......................................................... 138
FIGURE A.5: BREEDING STRATEGY FOR THE SIX-MONTH BREEDING STUDY. ................................ 139
FIGURE A.6: EXPECTANCY FREQUENCIES OF ANIMALS/GENOTYPE BASED ON THE SIX-MONTH
BREEDING STUDY. ................................................................................................................. 140
FIGURE B.1: GENE EXPRESSION OF OTHER CRITICAL MOLECULES, IMPLICATED IN STROMAL CELL
DIFFERENTIATION, WERE NOT AFFECTED BY THE CONDITIONAL RAC1 MUTATION. ............... 141
FIGURE B.2: GENE EXPRESSION OF OTHER MOLECULES IMPLICATED IN UTERINE ANGIOGENESIS. 142
FIGURE C3.1: CONDITIONAL DELETION OF RAC1 IN UTERINE DECIDUAL CELLS, DOES NOT PREVENT
EMBRYO IMPLANTATION, BUT LEADS TO TROPHOBLAST OVERGROWTH IN VIVO. ................... 148
FIGURE C3.2: GENE EXPRESSION PROFILE OF REGULATORS OF TROPHOBLAST EXPANSION. ......... 150
xi
FIGURE C3.3: CONDITIONAL DELETION OF RAC1 IN UTERINE TISSUES LEADS TO PLACENTAL
ABNORMALITIES AND INDICATIONS OF INTRAUTERINE GROWTH RESTRICTION. .................... 151
FIGURE C3.4: CONDITIONAL DELETION OF RAC1 IN UTERINE TISSUES LEADS TO PLACENTAL
ABNORMALITIES THAT DISPLAY IMPAIRED VASCULAR REMODELING. ................................... 153
xii
LIST OF ABBREVIATIONS
ADM Adrenomedullin
ALPL Liver/kidney/bone alkaline phosphatase
AM Anti-mesometrial decidua
ANGPT1 Angiopoietin 1
ANGPT2 Angiopoietin 2
ANGPT4 Angiopoietin 4
ANGPTs Angiopoietins
ANOVA Analysis of variance
ART Assisted reproductive technologies
ATP Adenosine triphosphate
BLAST Basic local alignment search tool
BMP2 Bone morphogenetic protein 2
BZ Basal zone
CDC42 Cell division control protein 42 homolog
CLDN3 Claudin 3
CS Circular smooth muscle
CZ Compact zone
DAVID Database for annotation visualization and integrated discovery
DC Decidual zone
E Embryo
E2 Estrogen (17β-estradiol )
ECM Extracellular matrix
ELISA Enzyme-linked immunosorbent assay
EPAS1/HIF2α hypoxia inducible factor 2α
ESR1/ERα Estrogen receptor α
F13A1 Coagulation factor XIII subtype A1
GAP GTPase-activating proteins
GDI Guanine nucleotide-dissociation inhibitors
GDP Guanosine diphosphate
GEF Guanine nucleotide exchange factors
GJP1/CX43 Connexin 43
GTP Guanosine triphosphate
GTPases Guanosine triphosphatases
HBSS Hank's balanced salt solution
xiii
HIF1α hypoxia inducible factor 1α
HIFs hypoxia inducible factors
HRP Horseradish peroxidase
IGF1 Insulin-like growth factor 1
IGF2 Insulin-like growth factor 2
IGFBP1 IGF binding protein 1
IGFBP4 IGF binding protein 4
IGFBPs IGF binding proteins
IGFs Insulin-like growth factors
IgG Immunoglobulin G
IUGR Intrauterine growth restriction
MKI67 Proliferation-related MKi-67 antigen
L Lumen
LDZ Lateral decidual zone
LS Lateral blood sinusoids (also known as vascular web)
LSMeans Least square means
M Mesometrial decidua
MG Metrial gland
MMP9 Matrix metalloproteinase 9
MMPs Matrix metalloproteinases
MT Mesometrial triangle
MY Myometrium
NBF Neutral-buffered formalin
NCBI National center for biotechnology information
NRP1 Neuropilin 1
P4 Progesterone
PBR Polybasic region
PBS Phosphate buffered saline
PECAM1/CD31 Platelet endothelial cell adhesion molecule 1
PGR Progesterone receptor
PI3K Phosphoinositide 3-kinase
PIP5K Phosphatidyl-dependentinositol-4-phosphate5-kinase
PREX1 PI3K-dependent Rac exchange factor
PRL3C1/PLPJ Prolactin family 3, subfamily c, member 1; PLP-J
PRL8A2/dPRP Prolactin family 8, subfamily a, member 2; decidual prolactin-related
protein
xiv
qPCR Quantitative real-time PCR
RAC1 Ras-related C3 botulinum toxin substrate 1
RAC1d/d Mice containing a conditional deletion for Rac1 gene at specific sites
where PGR is expressed
RAC1f/f Mice containing Rac1 gene flanked by LoxP sites
RAC2 Ras-related C3 botulinum toxin substrate 2
RAC3 Ras-related C3 botulinum toxin substrate 3
rER Rough endoplasmic reticulum
RHO Ras homologs
RHOA Ras homolog gene family, member A
RUNX1 Runt-related transcription factor 1
S.E.M Standard error of the mean
siRNA Small interfering RNA
SLC2A4/GLUT4 Glucose transporter type 4
SPHK1 Sphingosine kinase 1
TEK/TIE2 ANGPT 1/2 receptor
TIMPs Tissue inhibitors of MMPs
VEGF(A) Vascular-endothelial growth factor (A), also referred to as VEGF
VEGFR1/FLT1 VEGF receptor 1
VEGFR2/FLK1 VEGF receptor 2
WNT4 Wingless-related murine mammary tumor virus integration site 4
WNT5A Wingless-related murine mammary tumor virus integration site 5a
WNT7A Wingless-related murine mammary tumor virus integration site 7a
1
CHAPTER 1: INTRODUCTION
2
Implantation is a fundamental biological process during which the embryo attaches to the uterine
epithelium, invades the underlying stroma and promotes differentiation of the stromal cells to
decidual cells, which support embryonic growth and survival. It is a critical step for successful
establishment of pregnancy [1-11]. In humans, infertility is one of the most common disturbances
of reproductive health, with 10-15% of couples finding it difficult or impossible to conceive. [4,
5, 8, 12-16]. Despite significant advances in assisted reproductive technologies (ART), many
couples experience infertility as a result of failed implantation of the fertilized embryos into the
uterus and subsequent loss of these embryos. The implantation rates in ART remain low, even with
high-quality embryos, pointing to the importance of this process as a major cause of pregnancy
failure and infertility [1-3, 15, 17-21]. Understanding the signaling mechanisms central to embryo
implantation has the potential to alleviate many problems associated with infertility and improve
the clinical success of increasingly used ART.
This dissertation focuses on the newly identified role of RAC1 (Ras-related C3 botulinum toxin
substrate 1), a pleiotropic signaling molecule, in endometrial function during embryo implantation.
Elucidating the role of RAC1 signaling in the uterus may identify molecular targets for treating
infertility and improving the outcome of ART.
Implantation is a fundamental biological process during which the embryo comes into intimate
contact with the uterine epithelium, invades the stroma and establishes the placenta [1-5, 8-10, 22-
28]. This complex multistage process is pivotal for successful establishment of pregnancy. In
rodents and humans, as the embryo breaches the luminal epithelium following attachment, the
underlying stromal cells undergo a dramatic transformation to form a specialized tissue, known as
the decidua [5, 7-9, 22-25]. During this process, known as decidualization, fibroblastic stromal
cells proliferate and then differentiate into the secretory decidual cells that support embryo growth
and survival until placentation ensues. The decidual tissue also undergoes extensive remodeling to
control embryo invasion and accommodate the growing embryo. As the decidua forms around the
implanted embryo, the uterine endothelial cells proliferate to create an extensive vascular network,
which is embedded in the decidua and critical for embryo development [23, 29-33]. Even though
it is clear that decidualization is a key event in establishment of early pregnancy, the signaling
molecules that participate in the formation and function of this tissue remain poorly understood.
3
To gain an insight into the signaling pathways that are involved in the decidualization process, we
performed gene expression profiling of RNA collected from mouse uteri before and after decidual
stimulation. Our studies revealed that the gene encoding the Rac1, a pleiotropic signaling
molecule, is induced in uterine stroma at the time of decidualization. RAC1 is a member of the
Rho family of GTPases that regulate a wide range of cellular processes, including cell
proliferation, differentiation, cell-cell adhesion, endothelial cell function, and cell motility via actin
cytoskeleton remodeling. Because decidualization encompasses many of these physiological
events, it is possible that RAC1 regulates some of these processes during early pregnancy. In
support of this notion, a recent study has shown that invasion of the human embryo into
endometrial stroma in vitro involves motility of human endometrial stromal cells and that this
motility is dependent on the activity of the Rho GTPase RAC1 [7, 26].
To address the role of RAC1 in vivo during decidualization, we employed a loss-of-function
approach using genetically engineered mice. In this project, we generated a mutant mouse model
in which Rac1 gene is ablated from uterine stroma, characterized the mouse model and identified
the pathways that mediate RAC1 function in the uterus during decidualization. An in depth
analysis of this new mouse model is predicted to improve our understanding of the cause and
consequence of pregnancy complications involving impaired implantation, dysregulated
decidualization and placental complications in the human.
4
CHAPTER 2: REVIEW OF RELEVANT LITERATURE
5
2.1 Defining the problem: Clinical evidence for recurrent miscarriage and pregnancy loss
In recent years, worldwide, there has been a marked increase in human infertility, as well as the
use of assisted reproductive technologies (ART) [34-37]. The increased use of ART and the trend
towards low fertility has been attributed to the socioeconomic status, career demands in women,
improvement in contraception, or education, however biological factors that might contribute to
this decline need consideration [34]. In fact, clinically it is recognized that miscarriage and early
implantation failure are amongst the most common complications in pregnancy [6, 7, 26, 38, 39].
In many cases, this goes unnoticed because most miscarriages occur within the first 10 to 20 weeks
of gestation. Although pre-implantation pregnancy loss is considered to be normal in mammals as
an evolutionary advantage to select the best possible embryos, dysregulation of any of the events
before, during, or after implantation will lead to termination of pregnancy [1]. This concept has
created a challenge to researchers and clinicians who are trying to uncover ways to circumvent
human infertility, and at the same time, selecting for healthy offspring.
Recurrent miscarriage, which is defined clinically as pregnancy loss in more than three
reproductive cycles, is becoming more frequent in our society and in many cases (approximately
50%) the reasons leading to pregnancy loss remains unknown [40, 41]. The available data of a
large prospective register linkage study have shown that maternal age at conception is a strong risk
factor for miscarriage [42], and the risk of fetal loss increases steeply after the age of 35 years,
rising from 9% at 20–24 years to nearly 75% at 45 years and older [42-44]. Certainly there is an
urgent need to gain an understanding of the genetic- and molecular-basis of miscarriage, with
particular emphasis on the maternal environment that leads to pregnancy failure. A literature
review was undertaken to obtain current knowledge about certain maternal factors that may
contribute to miscarriage, as well as the influence of the uterine environment, and current
treatments for recurrent miscarriage.
2.1.1 Factors that may contribute to miscarriage
Recurrent miscarriage is clinically defined, by many clinicians, as the loss of three or more
consecutive pregnancies, and depending on how conservative the clinicians are when diagnosing
6
a patient with recurrent miscarriage, the incidence can affect anywhere from 1 to 5 % of couples
trying to conceive. It has been attributed to either genetic, structural, infections, endocrine,
immune, or unexplained causes [40, 44, 45], and in many cases, miscarriage is also associated with
psychological morbidity and stress for the patients. Very often women, more than men, attending
fertility clinics are clinically depressed, and about 20% of these women have anxiety levels that
are similar to those in psychiatric outpatient populations [17-20, 46, 47].
2.1.1.1 Congenital abnormalities
Although several known factors put some women at risk for miscarriage including malformation
of the uterus, diabetes, obesity, and alcohol use, the relationships between these conditions and
recurrent miscarriage have not been extensively studied and remain uncertain. Congenital uterine
abnormalities are commonly reported in women with recurrent miscarriage [48-51]. However,
some have argued that congenital uterine abnormalities might not represent a barrier anymore,
because there are women with structural abnormalities that have conceived children without
complications. The fact is that early miscarriages, especially the sporadic ones, are typically
associated with chromosome abnormalities of the embryo, which result in implantation failure or
growth arrest of the embryos leading to termination of pregnancy. Miscarriages without fetal
chromosomal abnormality or malformation are often thought to be a result of abnormal trophoblast
invasion and development [45, 52-54]. There is also evidence to suggest that impaired trophoblast
growth and invasion may be caused by an increased local or systemic immunological reaction to
the fetus or trophoblast [6]. In contrast to sporadic miscarriage, recurrent miscarriage tends to
occur even when the fetus has a normal chromosome complement, suggesting that the uterine
environment plays a role in determining pregnancy outcome. Therefore, recurrent miscarriage
undoubtedly is as complex disease, which may or may not have an underlying genetic component.
2.1.1.2 Vascular problems and bleeding disorders
It is well established that proper vascularization of decidua is critical for the success of pregnancy,
and it has been suggested that vascular disorders may play a role in pregnancies with complications
[55-58]. More recently, however, hypercoagulation disorders have been implicated in recurrent
7
pregnancy loss, which widens the scope of investigations and potential management to treat
recurrent miscarriage. However, data on the frequency of genetic thrombophilia are limited by the
small number of individual studies [44].
In some cases of early pregnancy failure, miscarriage has been attributed to the onset of a
precocious maternal blood flow leading to increased oxidative stress [52, 59]; and this is
independent of the karyotype of the conceptus [60]. Moreover, the early onset of maternal
circulation, which results in increased oxygen levels, can lead to widespread trophoblast damage
and placental degeneration [52, 59]. Furthermore, oxidative stress may modulate the architecture
of vasculature and the expression of angiogenic factors resulting in a dysregulated vascularization
[61]. Consistent with this observation, it has been reported that in women with miscarriages, there
is a reduced expression of vascular endothelial growth factor (VEGF) and its receptors [62].
Similarly, the decidual tissues of women with recurrent miscarriages show imbalance of the
expression ratio of angiopoietins (Angpt2/Angpt1), in favoring Angpt1, compared with the
predominance of Angpt2 in normal pregnancies [63]. While angiopoietin 2 is maximally expressed
during the early stages of pregnancy (i.e. first trimester) and declines afterwards, Angpt1
expression increases towards the third trimester, suggesting blood vessel stability [64]. Overall,
the data suggest that the role ANGPT2 during the first trimester is in branching angiogenesis,
leading to elongating and sprouting vessels and complex vascular network, whereas ANGPT1
predominates in the third trimester enhancing non-branching angiogenesis, maturation and
stabilization of the vascular network [58, 63, 64]. Therefore, a strong association between vessel
density and miscarriages is not farfetched.
2.1.2 Intrauterine growth restriction
Intrauterine growth restriction (IUGR) occurs with an incidence of 4 to 7% of all births, resulting
in low birth weight and preterm delivery. IUGR is associated with increased neonatal morbidity
and mortality because of placental problems leading to nutritional deficiencies [65, 66]. Several
risk factors have been associated with the pathogenesis of IUGR, which include chromosomal
abnormalities, infectious diseases, and defective vascularization of the uteroplacental unit.
Consistent with the latter, placental samples from women with IUGR showed a significantly lower
8
number of capillaries and a decreased capillary area in terminal villi of the placenta compared to
those with normal term pregnancies [67, 68]. Interestingly, previous studies have shown that in
pregnancies with IUGR, there is an apparent reduction in cytotrophoblast proliferation, poor
placental blood vessel development and an apparent increase in placental oxygen [52, 69, 70] [60].
Given that hypoxia promotes VEGF production, and thus angiogenesis, it has been suggested that
the relatively high oxygen levels in placentas with IUGR, will limit angiogenesis [69]. Consistent
with this notion, Khaliq et al. [71] reported a decreased expression of VEGF and an altered
expression of angiopoietins in IUGR placentas. The opposite is observed during the first trimester
of pregnancy where hypoxia upregulates VEGF secretion in the placenta. Given that trophoblasts
express the receptors for both VEGF and angiopoietins, which are known to influence behavior in
these cells, it is possible that a dysregulation of angiogenic factors can lead to perturbations in
proliferation and invasion of the trophoblast cells, as well as abnormal trophoblast migration and
differentiation (leading to placental abnormalities). Collectively, these result in an insufficient
exchange of oxygen and nutrients [72] in an otherwise normal embryo.
During the development of the placenta, trophoblast cells invade into the uterine tissues to gain
access to the maternal blood supply [73]. This process requires a balanced interplay between the
factors that promote and restrain trophoblast invasion. Consistent with this notion, early work has
suggested that the uterine microenvironment controls invasion of trophoblast, supporting the role
of the maternal decidual tissue as a restrain to trophoblast invasion [57, 74]. These studies also
imply that factors that promote trophoblast invasion are produced and secreted by the trophoblast
cells. Supportive of this model, it has been shown that the embryo secrete insulin-like growth
factor (IGF) 2, which drives trophoblast migration [1, 5, 9, 10, 29, 32, 75-77], as well as matrix
metalloproteinases (MMPs), which degrade the decidual extracellular matrix (ECM) [5, 78, 79].
To counteract these factors, the maternal decidua secretes tissue inhibitors of MMPs (TIMPs) [80,
81], as well as several IGF-binding proteins (IGFBP), including IGFBP1 [82-85] and IGFBP4 [65,
86-92]; molecules that are known to inhibit trophoblast invasion. Similarly, an interplay between
IGF and IGFBP exists, where IGF2 inhibits maternal decidual IGFBP1 and TIMP-3 expression,
supporting paracrine actions of trophoblast-derived IGF2 on decidual regulators of invasion [93].
Indeed, mounting evidence suggests that IGF and IGFBPs play critical roles during early
pregnancy, and it has been suggested that alterations of the IGF/IGFBP system can have serious
9
complications leading to IUGR [84, 85, 94]. In concordance with the importance of the IGF/IGFBP
system, overexpression of IGF1 [95] or IGFBP1 [94] in animal models leads to fetal loss or altered
placental development, respectively. In the clinics, women that are destined to deliver a growth-
restricted infant, display elevated levels of serum IGFBP4 compared to women with normal
pregnancies [65]. It is clear that the IGF family of proteins play an important role during pregnancy
overlapping the window of implantation and placentation [82, 85, 86, 90, 93, 96, 97]. Although
there are species differences, studies on transgenic mice have provided insights into the
IGF/IGFBP system during pregnancy [94, 95, 98-101].
2.1.3 Assisted reproductive technologies
The use of assisted reproductive technologies (ART) has increased dramatically in the last decade
[37, 39]. Many couples that have problems conceiving turn to ART to aide in this process. To
increase the likelihood of successful pregnancy, pre-implantation genetic screening of biopsied
blastomeres during in vitro fertilization has been recommended in order to prevent the transfer of
karyotypically abnormal embryos in couples with normal karyotypes. However, this practice may
not be effective in couples with recurrent idiopathic miscarriage due to limitations of genetic
testing [102]. Nonetheless, there is a need for research to investigate potential factors that could
predispose an individual to recurrent pregnancy loss.
2.2 Uterine receptivity, the window of implantation, and early pregnancy in the mouse
Steroid hormones, 17β-estradiol (E2) and progesterone (P4), regulate cell proliferation,
differentiation, and vascular remodeling of the endometrium by binding to their respective nuclear
receptors estrogen, ESR1/ERα (estrogen receptor α) and PGR (progesterone receptor). In each
reproductive cycle, under the influence of these hormones, the endometrium becomes competent
to receive an embryo during a short duration of time known as “window of receptivity” [11, 103-
106] (Figure 2.1). Intimate interactions between the uterus and fetus occur during this period that
promotes implantation leading to establishment of pregnancy [11, 107, 108]. About one third of
human pregnancies end in spontaneous abortion. Indeed, implantation failure still remains an
10
unsolved problem in reproductive medicine. It is a major cause of infertility [6, 37, 39, 45] and
represents a major challenge in ART.
2.2.1 Critical events during pregnancy
The concomitant actions of steroid hormones prepare the endometrium for the arrival of an embryo
and allow it to invade so that pregnancy is established. Whereas E2 initially regulates uterine
growth by stimulating epithelial cell proliferation and edema, P4 is mostly required for the
differentiation of stromal cells during pregnancy. Indeed genomic profiling of mouse and human
endometrium has uncovered a complex, yet highly conserved network of steroid-regulated genes
that supports stromal cell differentiation. In order to advance our understanding of the mechanisms
regulating critical aspects of pregnancy and better address the clinical challenges of infertility, it
is important to integrate the information gained from the human and mouse models [2, 109, 110].
Undeniably functional studies using transgenic mouse models over the past several years have
yielded valuable information regarding the molecular basis of pregnancy complications in humans
[25, 111]. Below we summarize some of the critical events required for the successful
establishment of pregnancy.
2.2.1.1 Implantation
Embryo implantation and placentation are critical events that support embryonic growth and
ultimately delivery of a live offspring in the mammalian system. [1, 25]. Failure of the
endometrium to provide a permissive growth environment can severely compromise the outcome
of pregnancy, leading to an impaired embryonic development or death. Despite some species
differences, in general, the principles of implantation and placentation are well conserved among
mammalian species [1, 5, 10, 14, 27, 28, 112, 113]. In species with invasive placentation,
implantation into the uterine endometrium is an absolute requirement and involves a series of
complex interactions between the implanting embryo and the endometrium [5, 41, 114-116].
In mice following fertilization, which typically occurs on the day the copulatory plug is found, is
termed as day 1 of pregnancy (Figure 2.2). The newly formed embryo then travels through the
11
oviduct and enters the uterine cavity on day 4 of pregnancy [41, 107, 117]. The embryo will then
begin an intimate crosstalk with the endometrium to initiate the implantation process (Figures 1.2
and 1.3) [1, 5, 116]. A transitory rise in E2, which in mice occurs on noon of day 4 pregnancy,
known as “nidatory estrogen”, is responsible for the initiation of implantation [1, 5, 8, 14, 118].
The necessity of the pre-implantation estrogen was established in the delayed implantation mouse
model, where ovariectomy before the nidatory estrogen surge prevents implantation. Replacement
of estrogen in these ovariectomized mice rescues implantation [103, 106]. Although is clear that
steroid hormones regulate embryo implantation in humans, it is still debatable whether a similar
E2 surge initiates embryo implantation in these species [14].
In a series of coordinated steps, i.e. apposition, adhesion, and invasion, the cells of the
trophectoderm penetrate into the stromal compartment of the endometrium and invasion into the
antimesometrial side of the uterus, i.e. the uterine side opposite to the mesometrial ligament starts
pregnancy [41, 117]. During this period, the uterus under the influence of P4 and the E2 becomes
receptive, a critical step for the initiation of this process (Figures 1.1 and 1.2). However, the uterus
will not remain permanently in a receptive phase and between 24 to 36 hours after the nidatory E2,
the uterus becomes refractory to implantation [11, 104, 105].
2.2.1.2 Decidualization: A key event for successful establishment of pregnancy
Maternal-fetal dialogue involving an intimate interaction between the specialized trophectodermal
cells of the embryo and the receptive uterine lining of the mother starts pregnancy [5, 10, 25]. The
attachment and breaching of the implanting embryo through epithelial barrier causes the
underlying fibroblastic stromal cells to undergo further proliferation and subsequent differentiation
into polyploid secretory decidual cells (Figure 2.3), in a process termed as decidualization [10,
108, 116, 119]. During decidualization, stromal cells transform into epitheloid cells that have
unique biosynthetic and secretory properties [23-25, 29, 33, 80, 84, 90, 91, 120-123]. These cells
are responsible for producing and secreting hormones, growth factors, and cytokines, which are
essential for tissue and vascular remodeling to support the growth and development of the embryo
until the establishment of the chorioallantoic (definitive) placenta. This typically happens on day
9 of pregnancy in rodents [1, 5, 9, 10, 23, 28, 29, 31, 78, 112, 124-133]. Another important feature
12
of the decidua is to resist the invasive capacity of trophoblasts by secreting products (such as
IGFBPs, TIMPs) that bind to trophoblast and modulate their migration and invasion [5, 80, 81, 83,
84, 86, 87, 122, 123, 134]. The invading trophoblasts secrete MMPs that degrade the decidual
ECM [5, 23, 78] and the decidual cells express high levels of TIMPs [135, 136], which control
trophoblast invasion. The maternal decidua allows trophoblast invasion far enough to access the
maternal blood supply, but not too far that the mother becomes endangered [23, 25, 29, 94, 130,
137, 138]. As embryo invades, it initiates a connection with the maternal vasculature and
ultimately, placentation ensues. Clearly, the formation of a functional placenta requires extensive,
but tightly regulated, invasion of the trophoblast through the endometrium [23, 25, 29, 94, 137,
138]. Insufficient trophoblast invasion can lead to number of placental complications such as
preeclampsia [139-143], whereas excessive invasion is associated with disorders such as placenta
accreta [125, 144-148].
The decidual cells are also thought to provide an immune barrier to the mother from the allogenic
embryo [23, 29, 149-151]. Mature decidual cells produce and secrete cytokines and chemokines
that maintain the trophoblast in its allogenic phenotype to avoid being targeted by the maternal
immune system. To this end, it has been thought that the modulation of the maternal immune
system is mediated by the recruitment of peripheral and resident leukocytes in the maternal
decidua, which provide local immunomodulation and support placental development [131, 143,
152, 153]. Indeed an immune environment is established in the endometrium, which is permissive
to the developing embryo [5, 130, 143, 152, 153].
The initiation of the stromal differentiation process can be monitored by the gene expression or
enzymatic activity of liver/kidney/bone alkaline phosphatase (ALPL, Figure 2.4) [154]. Around
day 4 midnight of pregnancy (or early day 5 morning), after the embryo has embedded itself into
endometrium, it is surrounded by the newly formed decidual cells of the primary decidual zone
[29, 155]. By day 6 and 7 of pregnancy, the stromal cells next to the primary decidual zone begin
to proliferate and then differentiate to form the secondary decidual zone (Figure 2.3B), forming
the decidua capsularis [1, 155]. As these cells differentiate, the cells of the antimesometrial
decidua, which differentiate first, will exit the cell cycle and begin expressing decidual prolactin-
related protein (PRL8A2/dPRP), which is typically used to assess the differentiation potential of
13
these cells [22, 156-158]. By day 8 of pregnancy, the differentiation program continues in the
mesometrial side of the uterus, i.e. the side that is closest to the principal maternal blood vessels,
and later forms the decidual basalis. These two decidual cell populations differ not only in their
morphology, but also by the genes they express and the putative roles they play in pregnancy [150].
The antimesometrial decidua is formed primarily by giant, polyploid, and closely packed decidual
cells, whereas the mesometrial decidua is formed by much smaller, loosely packed decidual cells.
It is relatively easy to separate these two sub-populations by the differences in their size and density
[150, 155]. Also, it is assumed that the mesometrial decidual cells regulate many important aspects
of vascular remodeling (Figure 2.3) [23, 29-32], energy storage and metabolism [159-161], as well
as placentation (Figure 2.5) [28, 31, 112]. By day 10 of pregnancy, however, the terminally
differentiated stromal cells undergo apoptosis to accommodate the growing embryo and
placentation ensues to maintain the conceptus until term [23, 150, 155]. Therefore, perturbations
in the decidual program will lead to pregnancy complications that may manifest in early pregnancy
loss. In humans, however, it should be noted that the stromal cell differentiation program is
initiated in the absence of embryo, but the extent of decidualization as well as vascular remodeling
increases significantly with the onset of implantation and establishment of pregnancy [33].
Experimentally, stromal cell differentiation into decidual cells can be studied independently of the
implanting embryo in an in vivo [154, 162, 163] as well as an in vitro model systems [164, 165].
In vivo, the experimentally-induced stromal differentiation is performed by ovariectomizing non-
pregnant mice, then replacing estrogen and progesterone, in a timely manner. Once the uterus is
primed, an external stimulus (most often oil injection) is given to one uterine horn, whereas the
other horn remains unstimulated, and serves as an internal control [163]. The in vivo
experimentally-induced differentiation model has been used extensively, by our laboratory, as well
as others, to establish the importance of steroid hormones in the regulation of stromal
differentiation during decidualization.
2.2.1.3 Angiogenesis and vascular remodeling are essential for maintenance of pregnancy
The establishment of an extensive angiogenic network during decidual transformation [23, 31, 57,
116, 166-168] is a key physiological event that supports early pregnancy and embryo development
14
(Figure 2.3). In mice, the initiation of vascular remodeling occurs soon after implantation and
manifests as an increase in microvascular permeability; only seen at the sites of embryo apposition
[116, 169-171]. Chicago blue dye injected via tail vein can be used to determine the sites of embryo
attachment on day 5 and 6 of pregnancy [169, 171]. This increase in vascular permeability is
thought to be a pre-requisite for embryo implantation [169, 172]. Under the influence of estrogen,
vascular-endothelial growth factor (VEGF) is produced and secreted as early as day 5 of pregnancy
[173-175]. This potent mitogen stimulates endothelial cells to proliferate and is thought to play a
major role in uterine angiogenesis during the decidualization process [176-178]. Because Vegf is
induced and maintained during the stromal cell differentiation process, it is considered as a marker
of decidualization [178, 179]. VEGF is a potent driver of angiogenesis, however, other cytokines
and growth factors are also recognized as angiogenic promoters. Therefore, one or more factor(s)
may be acting in concert with VEGF to mediate endometrial angiogenesis.
Vascular endothelial growth factor is a key regulator of angiogenesis [180, 181], and targeted
deletion of gene encoding Vegf results in embryonic death due to abnormal blood vessel formation
[181, 182]. VEGF has been shown to be one of the earliest genes activated during pre-implantation
embryo development [183]. Extensive analyses on the expression of VEGF, its steroid hormone
regulation, and involvement of specific receptors during pregnancy have suggested a critical role
for the VEGF system in pregnant uterus [175, 183]. VEGF is a heparin binding homodimeric
glycoprotein that mediates its action through tyrosine kinase receptors, namely VEGFR1/FLT1,
VEGFR2/FLK1, as well as the glycoprotein co-receptor neuropilin 1 (NRP1) [168, 178, 184, 185].
VEGFR2/FLK1 is the predominant VEGF receptor in the uterus [185], and binding of VEGF to
this receptor activates the VEGF signaling cascade. VEGFR1/FLT1 is also expressed in the uterus,
but is thought to have minimal to no effect during uterine angiogenesis [185]. Previous studies
have also shown that the human blastocysts are able to stimulate angiogenesis via the production
of significant amounts of VEGF [186]. Several studies have reported the expression of VEGF and
its receptors, in first-trimester human decidua basalis and parietalis, as well as in several cell types,
including endothelial cells, epithelial cells, macrophages and trophoblasts [187, 188]. VEGFA
(also indicated as VEGF), the predominant vascular endothelial growth factor that drives blood
vascular angiogenesis, has also been shown to stimulate trophoblast proliferation and invasion
15
[189]. Therefore, VEGFA may play an active role in trophoblast invasion and angiogenesis [187,
188].
Although it is clear that estrogen and progesterone play a major role during endometrial
angiogenesis [175, 178, 190], their specific roles in this process remain unsolved and controversial.
Whereas vascular permeability is under the regulation of estrogen [191], endothelial cells seem to
proliferate in response to progesterone [192, 193]. The importance of estrogen during angiogenesis
was recently studied in our laboratory [32, 194]. Laws et al. [32] clearly showed that ablation of
connexin 43 (Gjp1/Cx43), an estrogen-regulated gene from the decidual cells, caused a defect in
stromal cell differentiation and impaired vascular remodeling, primarily due to a marked reduction
in hypoxia inducible factors (Hif1α and Hif2α), Vegfa, angiopoietins (Angpt2 and Angpt4).
Similarly, Das et al. [194] reported that inhibition of local uterine estrogen can effectively impair
the production of pro-angiogenic factors, such as angiopoietins, which are well-known regulators
of angiogenesis [113].
During pregnancy, members of the angiopoietin family including Angpt1 and Angpt2, along with
Tek/Tie2, are expressed in the endometrium during stromal cell differentiation and modulate
angiogenesis [195, 196]. Angiopoietins are a class of angiogenic regulators that complement
VEGF signaling [180, 184, 197]. Angiopoietins mediate their actions by binding to the tyrosine
kinase receptor TEK/TIE2, and in the presence of VEGF, angiopoietins, especially ANGPT2,
promotes vascular remodeling in front of the invading vascular sprouts [184, 197, 198]. Although
it has been reported that steroids regulate the expression of these genes in the endometrium [192,
193], the exact mechanism and signaling pathways by which these hormones regulate angiogenesis
remain unclear.
Hypoxia is a detectable feature in the uterine environment during embryonic development [199-
201]. HIFs are major transcriptional regulators of Vegf. They play a critical role in maintaining the
physiological balance of oxygen tension and are closely associated with angiogenesis [1, 199, 202-
205]. Oxygen homeostasis is critical for cell survival and in response to hypoxic conditions, cells
activate the transcription of Hif1α to restore homeostasis [1, 206, 207]. Activation of HIF
stimulates Vegf expression, which promotes angiogenesis, thereby restoring oxygen homeostasis.
16
Both Hif1α and Hif2α transcripts are upregulated during the early stages of implantation and their
expressions are maintained during decidualization [1, 199, 202-204]. It has been reported that
EPAS1/HIF2α complements the action of HIF1α to regulate VEGF expression [205, 208].
Previous studies from our laboratory have demonstrated that both Angpts and Hif2α are regulated
by local endometrial E2 production during decidualization, thus contributing to VEGF regulation
and angiogenesis [194].
Whereas many of the early events leading to angiogenesis and vascular remodeling are regulated
by estrogen during pregnancy, progesterone also plays a major role in these events. Several in vivo
studies have suggested a pro-angiogenic role for progesterone [192, 193]. Human endometrial
endothelial cell cultures show an enhanced capacity to form tube-like structures and sprouts when
progesterone is added to the culture media [209]. It has been suggested that P4-induced
proliferation is most likely caused by a direct, but non-genomic effect of progesterone. Consistent
with this notion, Ma et al. [192] reported progesterone-mediated induction of endothelial cell
proliferation in ovariectomized mice and suggested the involvement of VEGF and its receptor
VEGFR2/FLK1 in conjunction with NRP1 in this process. Furthermore, progesterone-induced
endometrial endothelial cell proliferation is thought to be mediated by multiple angiogenic
mechanisms [32, 194, 210-212] and regulatory factors, acting in a paracrine manner between cells
within the endometrium [210-212]. These paracrine effects are absent in vitro, which could explain
the disparity between some in vitro and in vivo observations.
With the progression of decidualization and pregnancy, new blood vessels begin to form a vascular
plexus in the mesometrial side of the uterus (Figure 2.3) [23, 31, 57, 166, 167]. Moreover, the
specific pattern this plexus acquires is thought to be influenced by signals that originate from the
embryo [30, 130, 198]. In the experimentally-induced differentiation model the specific pattern of
vascular remodeling that is typically observed in pregnancy, is not acquired [30, 198]. It is believed
that the concerted actions of steroids, in addition to the signals originating from the embryo, are
thought to regulate the final outcome of uterine angiogenesis during pregnancy [23, 29, 30, 32, 57,
112, 130, 176, 194, 213].
17
2.2.1.4 Placentation
Whereas placentation is highly variable and species-dependent, the primary and major goals of the
placenta are to 1) anchor the conceptus in the maternal uterine wall, 2) establish a vascular supply
that enable optimal growth and development of the conceptus, 3) and protect the fetal allograft
from rejection [5, 9, 23, 28, 29, 31, 112, 124-128, 149-151, 214]. To achieve these goals, there are
complex molecular dialogues that take place between the maternal decidua and the developing
conceptus, as well as the placental structures [1, 5, 10, 23, 29, 78, 112, 127-133, 215].
During the early stages of gestation, it is thought that the developing embryo is supported by the
secretions of the luminal epithelium of the oviductal cells and those from the uterus [116, 216].
However, as the embryo grows in size and the trophectoderm begins to differentiate, the nutritional
requirements of the embryo change. Initially, the primary sources of energy for the early embryo
are pyruvate, lactate, and amino acids. But after compaction of the morula, glucose becomes the
primary source of energy for the embryo [28, 116, 217, 218]. At this point, the developing embryo
involving a dynamic crosstalk between trophoblasts and the maternal decidua begins attachment
and invasion through the luminal epithelium into the maternal endometrium [1, 5, 10, 29, 171,
219].
2.2.1.4.1 Placentation in humans and mice
Mice and humans exhibit hemochorial-type of placenta, and although there are some differences
in the architectural details of these placentas, their overall structures and the molecular
mechanisms underlying placental development are thought to be quite similar [28, 126-128, 132,
220]. As a consequence, the rodent system, especially the mouse, has been extensively used as a
model for studying the fundamentals of placental development. In mice, placental development
begins on day 4 of pregnancy when the blastocyst is formed (Figure 2.5) [5, 28, 126, 128]. Around
the time of invasion into the endometrium on day 5 of pregnancy, the mural trophectoderm cells,
i.e. those not in contact with the inner cell mass, proliferate and begin to give rise to two diploid
cell types: the extraembryonic ectoderm – presumably those which give rise to the chorionic
trophoblast forming the placental labyrinth and are analogous to cytotrophoblast of the human
18
placenta – and ectoplacental cone – presumably those which give rise to the spongiotrophoblast,
and are thought to support the developing labyrinth [28, 126, 128, 132]. The trophoblast cells that
are farthest from the inner cell mass stop dividing, but continue endoreduplication of the genome
without mitosis, and become the trophoblast giant cells (analogous to human extravillous
cytotrophoblast cells). The trophoblast giant cells form the interface with maternal decidua and are
capable of invasion into the endometrium [28, 31, 124, 132]. The cells from the ectoplacental cone
and extraembryonic ectoderm lineages thus establish the three layers of murine placenta: 1) The
labyrinth zone, which is the zone closest to the chorionic plate; 2) the juctional zone, which
supports and surrounds the labyrinth and is composed spongiotrophoblasts and trophoblast
glycogen cells; and the final layer 3) the trophoblast giant cells, which are at the maternal-fetal
interphase and a subgroup of these cells invade the maternal blood vessels [5, 10, 28, 31, 126, 127,
132, 214]. Although it is clear that there are interactions and crosstalk between the maternal and
embryonic cells, however, due to the complex nature of these interactions, the mechanism
underlying the placental formation is not clear.
2.2.1.4.2 Regulation of trophoblast invasion and placental pathologies
An important function of the decidua is to resist the invasive capacity of trophoblasts by secreting
factors that bind to trophoblasts and modulate their migration and invasion [23-25, 29, 33, 80, 84,
90, 91, 120-123, 127, 221]. Beyond the in vitro data, which strongly support the concept of a
“decidual barrier”, the results from classical embryo transfer experiments support the role of the
decidua in regulating trophoblast invasion. When mouse blastocysts are transferred to subepithelial
ectopic sites (such as kidney, testis, or anterior chamber of the eye), the embryo is able to penetrate
into the non-decidualized connective tissue much more deeply than into uterine decidua [222-225].
Similar examples of this phenomenon have been reported in human pregnancies established at
ectopic pregnancy sites. In oviductal pregnancies, for example, the trophoblast cells tend to be
more invasive [226]. It has been speculated that a potential reason for this excessive invasion is
because the pseudo-decidualized oviductal cells [129, 227] do not produce IGFBP1 [129].
Furthermore, it has been reported that excessive trophoblast invasion or ectopic pregnancy tends
to occur in tissues that exhibit impaired decidualization [129, 227]. Thus the unique feature of the
decidua to suppress uncontrolled trophoblast invasion, provides a possible explanation for why, in
19
pathological conditions where the embryo invades ectopic sites, trophoblast invasion tends to be
more invasive [127, 129, 226]. In more severe cases, where the decidual barrier is defective, little
or too much invasion can result in further complication to the mother. [39, 53, 54, 58-60]. One of
the hallmarks of preeclampsia is shallow trophoblast invasion, thought to cause an inadequate
spiral artery remodeling, leading to placental hypoxia. It is widely accepted that placental hypoxia
initiates the cascade of events that ultimately results in the maternal manifestations of high blood
pressure and proteinuria [61-64]. Similarly, in placenta accreta it has been proposed that either
primary deficiency of decidua or abnormal maternal vascular remodeling leads to excessive
trophoblastic invasion, resulting in massive postpartum hemorrhage and commonly leads to
emergency hysterectomy [65-70]. Nonetheless, the formation of the placenta is a critical process
during embryogenesis, and without a healthy placenta, the embryo will not survive and this
malformation can trigger a spontaneous abortion.
2.2.1.4.3 The mid-stages of placentation in the mouse
Day 10 of pregnancy is a critical stage in mouse placenta. At this point, the chorioallantoic
circulation becomes the primary support for the growing embryo. The placenta forms an essential
interface between the maternal and fetal layers that allow the exchange of oxygen, nutrients, and
waste products to provide a nourishing environment to the growing fetus [5, 9, 28, 31, 112, 176].
By day 12 of pregnancy, the fetal trophoblast cells start to invade into the maternal decidua to
reach spiral arteries and induce modifications with make high capacitance, low resistant blood
vessels [167, 228-232]. As a consequence of trophoblast invasion, the endometrial spiral arteries
undergo dilation, which results from the loss of smooth muscle cells surrounding the arterioles
[233]. Therefore, defective or improper invasion of fetal trophoblast cells into maternal decidua
can result in various clinical problems such as, spontaneous abortion, preeclampsia, and/or
intrauterine growth restriction [173, 174, 234-237].
2.3 Rho GTPases
The Ras Homology (Rho) family of guanosine triphosphatases (GTPases) are small signaling G
proteins (~21 kDa), and belong to the subfamily of the Ras superfamily of proteins (Figure 2.6).
20
Members of the Rho GTPase family regulate many aspects of actin dynamics and are found in
organisms raging from plants and Caenorhabditis elegans, to mice and humans. While their first
discovered role was regulation of actin dynamics in fibroblast cells [238], recent evidence has
suggested that the members of this family play important roles in signal transduction, cell
proliferation and differentiation, vesicle transport, cancer progression and metastasis, angiogenesis
and gene expression [238-248]. Clearly, these proteins play critical roles in many cellular
processes.
2.3.1 The Rho GTPases
Since the discovery of the first Rho member Rhoa in 1985, from a low stringency cDNA screening
[249], other members including Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rac2 in
1989 [250, 251], and cell division control protein 42 homolog (Cdc42) in 1990 were discovered
[252]. To date, more than 18 members in eight distinct subfamilies have been reported [244], and
of these three members of the family, RHOA, RAC1, and CDC42 [238, 249, 250, 253], have been
extensively studied. The Rho proteins are virtually found in all eukaryotic species examined so
far, and are highly conserved [241, 244, 247, 254-259]. In addition, given their close homology to
Ras GTPases, the Rho GTPases act as molecular switches to regulate signal transduction pathways
by alternating between an inactive GDP-bound state and an active GTP-bound conformational
states. The activities of these proteins are primarily controlled by growth factors or integrins, and
seven-transmembrane receptors [238, 239, 243, 244, 246, 254, 255, 258, 260-265].
2.3.2 Role of Rho GTPases in actin dynamics
It is clear that the actin cytoskeleton drives many dynamic processes in cells, including migration,
phagocytosis, endocytosis, vesicle trafficking to the plasma membrane, morphogenesis, and
cytokinesis [238, 241-244, 247, 248, 250, 260, 266-270]. Further, depending on their biological
function and location within the body, cells can acquire many shapes and forms. However, before
a cell can undergo cellular processes like mitosis or locomotion, a degree of polarity is required
[238, 244], which requires the cell to adopt a specific shape or direction rather than existing as an
amorphous cell. Each member of the Rho family, i.e. RHO, RAC, and CDC42, is responsible for
21
specific regulation of actin dynamics [238, 244]. One of the most well-known morphological
structure that is controlled by Rho proteins is the formation of lamellipodia and filopodia, which
look like “sheets” or “fingers”, respectively, and aid in the propulsion and sensing across surfaces
[238]. Rho GTPases are also responsible for the formation of stress fibers (originally thought to
arise from the effects of tension) [241, 270], important in cell motility, contractility, and adhesion
[243].
2.3.2.1 Functions beyond actin dynamics
Researchers working with Rho GTPases in the mid-1980s quickly realized their importance in
actin dynamics. However, more recent work has demonstrated that Rho GTPases do much more
than regulation of actin assembly, cell shape and migration. Over the last 20 years using a
combination of techniques, such as: 1) use of dominant-negative mutants of Rho, 2) use of RAC
and CDC42 toxins such as C3 (Rho inhibitor) or toxin B (RHO, RAC and CDC42 inhibitor), 3)
analysis of transgenic model organisms, and 4) use of siRNA have demonstrated how critical each
of these molecules are for the proper functioning of many cellular processes within a cell. Several
lines of evidence seem to suggest that members of the Rho family can regulate a wide range of
cellular functions including, cell-cell communication, gene expression, activation of the NADPH
oxidase complex, maintenance of cell polarity, cell proliferation/differentiation, angiogenesis, and
cancer progression and metastasis [113, 242, 246-248, 250, 254, 261, 262, 264, 269-282]. Signal
transduction pathways from the Rho proteins have been also implicated in the regulation of gene
transcription. For example, RHO is known to activate serum-response factor, whereas RAC1 and
CDC42 activate the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways
[244].
2.3.3 Regulators of the Rho GTPases
Under normal physiologic conditions, GTPases, including Ras and Ras-like proteins, are
predominantly regulated at the post-translational level. Promoter analysis of the human Ras
oncogene and Rac1 has revealed that the gene architecture resembles that of a housekeeping gene.
The promoters lack regulatory elements, like the TATA box and CAAT box, have multiple
22
initiation start sites, and a rich CG content, with CpG islands surrounding the transcription
initiation sites [283, 284]. However, use of in silico assays to study the promoter of the human
Rac1 gene indicated consensus binding sites for several transcription factors, including E2F-4, as
well as several zinc finger transcription factors, such as SP1, KLF15, MAZ, MZF1, PLAG1, and
WT1 [285]. The discrepancies in these results could be due to the use of different human Rac1
gene sequences, but clearly, further studies are needed.
The post-transitional modifications that control intracellular signaling pathways regulated by Rho
GTPases, act by affecting the state of the molecular switch. Alternating between GDP-bound and
GTP-bound forms, affect the conformation of the GTP-binding switch 1 and switch 2 regions
(Figure 2.7) [250, 254, 261, 264, 273, 274, 280, 286, 287]. The GDP-bound form is considered as
the inactive state, whereas the GTP-bound state activates downstream pathways by binding to
effectors. The cycling of Rho GTPases between the active/inactive states is regulated by three
families of proteins, guanine nucleotide-exchange factors (GEFs), GTPase-activating proteins
(GAPs) and guanine nucleotide-dissociation inhibitors (GDIs) [250, 254, 255, 261, 264, 273, 274,
286]. GEFs, however, activate Rho GTPases, GAPs and GDIs act as inhibitors of the protein
activity [244, 248, 255, 258, 260, 264, 287].
Because GDP binds tightly and the GTPase capacity of the molecule is slow, small GTPases
require GEFs to facilitate dissociation of GDP in exchange for a GTP molecule. Activated Rho
GTPases are known to interact with close to 80 effector molecules (i.e. kinase, phospholipases,
and scaffold proteins), which regulate downstream signaling events, including cell proliferation
(by contributing to spindle orientation), differentiation (by regulating gene expression, and perhaps
cell geometry and shape [288, 289]), cell migration (by affecting actin dynamics), and gene
expression (via downstream protein-protein activation). Soon after the GTPase becomes active,
GAPs associate with the protein and promote the GTPase activity leading to the hydrolysis of GTP
to GDP [287]. Hence, the transition (or the molecular switch) between one state and the other is
carefully balanced by the activation or suppression of GEFs and GAPs. Finally, the activity of Rho
proteins can be further attenuated by their interaction with GDIs, which shields the hydrophobic
tail that is incorporated in the C-terminus, and prevents the interaction with downstream effectors
and confines the GTPase to the cytosol (Figure 2.7) [287]. The hydrophobic tail allows the
23
anchoring and incorporation of the GTPase into the cell membrane when active (Figure 2.7) and
permits the interactions with effector proteins. Interestingly, not all known GTPases have the
capacity to hydrolyze GTP [255, 260]. Other mechanisms regulating Rho GTPases also exist, i.e.
phosphorylation, ubiquitination, and degradation, but these are minor, compared to those
mentioned above.
2.3.4 Rho GTPases and disease
The founding member of the Ras superfamily was intensely studied during the 1980s, since it was
found to be mutated in 30% of all human cancers. Further studies showed that a single amino acid
substitution was responsible for the protein’s insensitivity to GAPs and therefore remained
constitutively active [244]. Sequencing information derived from human cancers has uncovered
no similar mutations in Rho GTPases. However, genetic alterations in the Rho GTPase signaling
pathways have been reported in several human diseases. For instance, alterations in Rac2 gene
sequence have been associated with immunological disorders such as leukocyte adhesion
deficiency [245, 267, 290]. Similarly, other mutations in GAPs and GEFs have been reported [240,
244, 291]. To date, Rac1 has been shown to be abnormally expressed in several tumor samples
[203, 240, 265, 291]. Although there is evidence for polymorphism in the Rac1 gene sequence,
most are intronic, and thus may not contribute to any genetic dysregulation [285]. However, a
recent report has shown that a possible functional mutation due to a polymorphism in the Rac1
gene could result in drug resistance [285]. In addition, an alternatively spliced variant of Rac1,
Rac1b, containing an additional exon (exon 3b), and encoding a protein with 19 additional amino
acid residues, has been described. Rac1b gene, primarily found in cancers [292-296], produces a
functional GTPase that displays an increased intrinsic guanine nucleotide exchange rate [294] and
a decreased intrinsic GTPase activity [294, 297] compared with those of RAC1.
2.3.5 Ras-related C3 botulinum toxin substrate 1 (RAC1)
Among the Rho GTPases, the subfamily of proteins named Rac (Ras-related C3 botulinum toxin
substrate) has been most studied. There are four known Rac proteins (RAC1, 2, 3, and 1b), which
display ~ 90% identity in their coding sequences and overall structure [250, 261, 268, 280], but
24
have different expression patterns. Whereas Rac1 is widely distributed in many cells [250, 251],
Rac2 expression is mostly restricted to cells of the hematopoietic lineage [250, 290, 298], and
Rac3 expression is in the brain, liver, and lung [268, 299]. Rac1b, the splice variant of exon 3 of
Rac1, is detected primarily in cancerous cells [292-296]. Available protein sequences indicate that
RAC1 is highly conserved among eukaryotes [241, 259], with high homology between mouse and
human (Figure 2.8).
RAC1 is known to play a fundamental role in a wide variety of cellular processes, which include
actin cytoskeletal reorganization, cell transformation, angiogenesis, axonal guidance, and cell
migration. More recently, researchers have begun to uncover new roles for RAC1 in a variety of
physiological settings [7, 26, 255, 261, 266, 300-302]. Some of the physiologic functions that have
been attributed to RAC1, include regulation of cell growth, vesicle trafficking, and generation of
reactive oxygen species.
Like other members of the Rho family of GTPases, RAC1 transduces extracellular signals from
seven-transmembrane G-coupled protein receptors, integrins, and growth factor receptors to
effector molecules that modulate multiple signaling pathways [260]. RAC1 can promote the
activation of MAP kinase pathways and gene activation, however, its most important functions
include the regulation of cytoskeletal structure and shape, resulting in important cell functions
related to cell attachment, movement, and vesicle trafficking [248, 257, 260, 303-308]. Indeed,
one of the primary functions of RAC1 is to promote the formation of the lamellopodium, which
are sheet-like projections on the leading edge of a motile cell that propel the cell across a substrate
[247, 248]. Furthermore, the action of RAC1 on actin fibers plays critical roles in vesicle transport.
In dendritic cells, RAC1 induces actin-rich membrane protrusions during pinocytosis [309], which
allows for antigen presentation, nutrient uptake, and sampling of the environment. RAC1 is also
involved in phagocytosis by mediating localized polymerization of actin at the membrane to
promote the internalization of attached particles and microorganisms [260]. In other cell types,
RAC1 has been shown to regulate endocytic and exocytic trafficking pathways [247, 308]. Unlike
CDC42, RAC1 is not involved in the early secretory pathway or membrane trafficking from the
rER to the Golgi [308, 310]. However it does play a role in the later stages of secretion. RAC1
regulates clathrin-dependent endocytosis [311] by modulating phosphatidylinositol-3 kinase
25
(PI3K) and synaptojanin 2, a novel RAC1 effector [312]. Phosphatidylinositol 4,5-bisphosphate is
phosphorylated by PI3K to generate inositol triphosphate, which induces the activation of RAC1-
GEFs to regulate actin rearrangement and vesicular trafficking in various cell types [313].
Recently, PREX1, a PI3K-dependent Rac exchange factor, has been identified as a novel regulator
of the glucose transporter 4 (SLC2A4/GLUT4) trafficking in adipocytes. PREX1 can activate
RAC1, and this in turn stimulates actin polymerization and facilitates SLC2A4/GLUT4 transport
to the plasma membrane [314]. Moreover, RAC1 can modulate clathrin-independent endocytosis
by interacting with phosphatidyl-dependentinositol-4-phosphate5-kinase (PIP5K), via the
polybasic region (PBR binding domain of RAC1). Calmodulin can also interact with RAC1 via
the PBR and disrupt the interaction between RAC1 and PIP5K through steric hindrance [315].
RAC1 has also been reported to regulate calcium-dependent exocytosis in some cells. In neurons,
a late step of Ca2+-dependent neurotransmitter release is regulated by RAC1 [316]. Activation of
the RAC1 pathway in bovine chromaffin cells, transfected with human chorionic gonadotropin,
led to an increase in secretion [317]. Similarly, in acinar cells of the pancreas, RAC1 can increase
the secretion of amylase in response to cholecystokinin [318]. However, the precise mechanisms
by which RAC1 pathways regulates exocytosis remain unknown.
Another function of RAC1 that has received attention is its involvement in angiogenesis [203, 275,
277, 319-326]. Hypoxia occurs in many cellular environments that are undergoing growth or
differentiation. Oxygen homeostasis is critical for cell survival and in response to hypoxic
conditions, cells activate the transcription of Hif1α to restore homeostasis [327-334]. Activation
of HIF proteins stimulates the expression and production of Vegf, which drives angiogenesis and
restores oxygen homeostasis [199, 205, 327, 329, 332, 333, 335]. Several lines of evidence have
suggested that RAC1 regulates endothelial cell function by responding to VEGF [277, 323, 326].
Indeed, activation of the RAC1 pathway has been shown to enhance VEGF-induced
neovascularization, whereas inhibition of this pathway prevents neovascularization in vitro [326].
Furthermore, activation of the RAC1 pathway in embryonic endothelial cells is critical for
embryonic angiogenesis, and failure to do so, results in embryo death [277]. Additionally, in other
cell systems such as pulmonary artery muscle cells, RAC1 can also stimulate Hif transcription
[202-204, 280, 325] in a PI3K-dependent pathway [202-204, 280], and indeed Rac1 promoter
26
appears to contain HIF1α response element [325]. Collectively these studies raise the interesting
possibility that RAC1 may play a role in uterine angiogenesis during early pregnancy by regulating
the expression of VEGF, via a HIF-mediated manner.
Although the cellular functions of RAC1 have been described in the literature, very little is known
about their function in the endometrium, especially during pregnancy. Recent studies have
suggested that RAC1 pathway is activated in human endometrial cells and may play a role in
embryo attachment and invasion into the endometrium in the human [7, 26]. Furthermore, in the
above studies, the authors concluded that the activation of the RAC1 pathway in the maternal
decidual cells is necessary to allow invasion of the trophoblasts [7, 26]. However, due to the
obvious ethical limitations, these studies were conducted in vitro and to the best of our knowledge,
the role of RAC1 has not been explored in vivo.
2.3.6 Available mouse models to study RAC1 function
Despite the availability of mice carrying a global knockout mutation of Rac1 gene, these mice are
not suitable to study the functional role of RAC1 in the uterus during early pregnancy. Global
deletion of Rac1 leads to embryonic lethality around the time of during gastrulation [271].
Therefore, to overcome this limitation, we have developed a mouse model system that has
provided us with the unique opportunity to study the functional and physiological role of RAC1
pathway in the uterus. We have made use of the Cre-LoxP technology, to specifically target the
deletion of Rac1 to specific tissues, thereby circumventing the embryonic lethality associated with
the global knockout mutations of Rac1 [271]. This lethality is not seen in global knockouts for
Rac2 [290] and Rac3 [299]. It should be noted that RAC2 and RAC3 mutants are fertile, and have
not shown any indications of infertility or any other reproductive impairments [290, 299]. Our
studies indicate that in contrast to RAC2 and RAC3, RAC1 plays a critical role in maintenance of
pregnancy.
27
2.4 The aims of the dissertation
To establish the functional role of uterine RAC1 during early events of pregnancy, I have used the
PgrCre/+ Rac1f/f mouse model to specifically target Rac1 deletion in any tissue expressing Pgr,
which includes the uterus. The following aims were established and are further discussed in this
dissertation:
Aim 1. Analyze the expression of RAC1 in the uterus during early pregnancy.
1a. Determine the uterine cellular compartment that expresses RAC1 during early
pregnancy.
1b. Determine whether Rac2 and Rac3, the other members of the Rac subfamily, are
expressed in the murine uterus during early pregnancy.
Aim 2. Characterize the defects in fertility and identify the biological processes that prevent
pregnancy maintenance in Rac1-null female mice.
2a. Determine the timing of embryo demise in Rac1-ablated uteri during early pregnancy.
2b. Perform an in depth analysis to determine whether conditional deletion of Rac1 in the
murine uterus leads to improper stromal cell differentiation.
2c. Investigate whether the deletion of uterine Rac1 impairs neovascularization in the
endometrium during early pregnancy.
2d. Determine whether conditional deletion of Rac1 prevents trophoblast migration and
formation of placenta.
Aim 3. Identify the pathways that mediate RAC1 function during decidualization.
3. Perform gene expression profiling using endometrial RNA from control and Rac1-null
uteri on day 8 of gestation to identify the RAC1-regulated pathways involved in
angiogenesis during decidualization
28
2.5 Chapter Figures
FIGURE 2.1: HORMONE PROFILE DURING PREGNANCY IN MICE. Schematic representation of the critical events of pregnancy in mice and the
hormone profile that accompany and regulate these events. The red line shows the hormone level for E2, whereas the blue line shows the hormone
levels of P4. The inset picture, shows an embryo during the time of implantation attached to the luminal epithelium of the uterus.
29
FIGURE 2.2: COMPARISON OF THE EARLY EVENTS OF PREGNANCY IN HUMANS AND MICE. After fertilization, the zygote makes its way to the
uterus, which takes about four days in mice and five to seven days in humans, prior to embryo implantation. For pregnancy timing in mice, our
laboratory defines day 1 of pregnancy as the day of fertilization (others refer to it as E0.5). Around day 5 of pregnancy (in mice), and day 8 of
pregnancy (in humans), the embryo begins the implantation process into the endometrium. Image adapted with permission from © 2001 Terese
Winslow [336].
30
FIGURE 2.3: MORPHOLOGICAL CHANGES DURING STROMAL CELL DIFFERENTIATION. (A) Diagrammatic representation of the early and mid-
stages of pregnancy in mice. Image adapted with permission from [337]. AM = antimesometrial side; M = mesometrial side; E = embryo; PDZ =
primary decidual zone (highlighted by dash-line); secondary decidual zone; DS = deep stromal cells. Images are at the same magnification.
31
FIGURE 2.3: MORPHOLOGICAL CHANGES DURING STROMAL CELL DIFFERENTIATION (CONTINUED). (B) Shown here are cross-sections of
control uteri stained with hematoxylin and eosin at the beginning of embryo implantation and early stromal cells differentiation (day 4 midnight)
and during the later stages of stromal cell differentiation (day 7 of pregnancy). In mice, embryo attachment and invasion occurs in the antimesometrial
side of the uterus, i.e. the side opposite to the mesometrial ligament of the uterus, triggers the fibroblastic stromal cells to differentiate into an
epitheloid and highly secretory decidual cells. Of note, decidualization does not occur at inter-implantation sites. AM = antimesometrial side; M =
mesometrial side; E = embryo; PDZ = primary decidual zone (highlighted by dash-line); secondary decidual zone; DS = deep stromal cells. Images
are at the same magnification.
32
FIGURE 2.4: MAJOR EVENTS DURING IMPLANTATION AND DECIDUALIZATION. Shown here are cross-sections of control uteri, on day 8 of
pregnancy, stained for ALPL activity (blue staining), PRL8A2/dPRP (red staining enclosed by dash-line), and PECAM1/CD31 (red staining), which
are well-known markers of the process of decidualization and vascular remodeling, respectively. AM = antimesometrial decidua; M = mesometrial
decidua; E = embryo; LS = lateral blood sinusoid. Images are at the same magnification.
33
FIGURE 2.5: DEVELOPMENTAL STAGES OF THE MOUSE PLACENTA. Lineage specification and differentiation of the trophectodermal
(extraembryonic) cells begin at on day 4 of pregnancy [embryonic day (E) 3.5] with the formation of the blastocyst. Around day 9 (E8.5) of
pregnancy, chorion and allantois attach and this is followed by formation of the placental labyrinth, via branching morphogenesis. The mature
(definitive) placenta is formed by day 15 (E14.5) of pregnancy, and it consists of three layers: the labyrinth, the spongiotrophoblast, and the maternal
decidua. E0.5 = day 1 of pregnancy. AM = antimesometrial decidua; M = mesometrial decidua, Image adapted and modified with permission from
[28].
34
FIGURE 2.6: STRUCTURAL COMPARISON BETWEEN MEMBERS OF THE HUMAN RHO FAMILY OF GTPASES. The Rho family of GTPases (RHOA
[338], RAC1 [339], and CDC42 [340]) are structurally similar to the prototype GTPase, RAS GTPase [341]. Three-dimensional images based on
X-ray diffraction crystallography for these molecules were extracted, modified, and adapted from the NCBI Molecular Modeling Database [342].
35
FIGURE 2.7: REGULATORY MECHANISMS OF THE RHO FAMILY OF GTPASES. 1) In the resting state cells, the non-activated GTPase is complexed
with a guanine nucleotide dissociation inhibitor (GDI), which maintains the protein inactive in the cytosol by preventing the translocation to the
plasma membrane. 2) Upon a signal, the GTPase is released from GTPase-GDI complex and the membrane association of the protein is promoted
due to the hydrophobic tail in C-terminus. 3) At the same time, cycling between an inactive GDP-bound and an active GTP-bound mediated by
interactions with a guanine nucleotide exchange factors (GEF). The activated membrane-associated proteins interact with downstream effectors to
activate signal transduction pathways that lead to diverse cellular functions. 4) Association with GTPase activating proteins (GAPs) accelerates the
hydrolysis of GTP, attenuating the activity of the GTPase. Once the cycle is complete and the protein is no longer needed, it will again form a
complex with a GDI.
36
FIGURE 2.8: PROTEIN SEQUENCE COMPARISON BETWEEN HUMAN AND MOUSE RAC1. (A) Shown here is the comparison between the 192 amino
acid protein sequence for human (GI 8574038) and mouse (GI 403115559) RAC1. As shown all 192 amino acid in the sequence match.
37
FIGURE 2.8: PROTEIN SEQUENCE COMPARISON BETWEEN HUMAN AND MOUSE RAC1 (CONTINUED). (B) The dot plot shows the graphical
comparison between the two amino acid sequences. The “x”-axis represent each amino acids of the human RAC1 sequence and the “y”-axis shows
those of the mouse RAC1 sequence. The protein sequences for human and mouse RAC1 were obtained from NCBI Protein and aligned using NCBI
BLAST. The alignment and analysis were done using the onboard tool in BLAST [343, 344].
38
CHAPTER 3: GENERATION AND PHENOTYPIC
ANALYSIS OF RAC1D/D MUTANT
FEMALE MICE
39
Abstract
Members of the Rho GTPase family are pleiotropic signaling molecules that regulate a wide range
of cellular processes, including cell cycle, cell differentiation, cell motility (through actin
cytoskeleton remodeling), and angiogenesis. Recent in vitro studies suggested that one of the
members of this superfamily such as Ras-related C3 botulinum toxin substrate 1 (RAC1),
expressed by decidual cells, might be capable of influencing embryo implantation in the human.
Although these studies suggested that RAC1 activity is critical for trophoblast invasion in vitro, it
is not yet known whether RAC1 exhibits similar functions in vivo. Using a conditional deletion
approach (Cre-loxP system), we generated transgenic mice that lack Rac1 gene expression and
protein in uterine cells that are positive for progesterone receptor (PgrCre/+ Rac1f/f or Rac1d/d). Our
results showed that conditional deletion of uterine Rac1 led to a severe subfertility phenotype. We
observed more than 90% reduction in birth rate in Rac1d/d mice when compared to control Pgr+/+
Rac1f/f (Rac1f/f) female mice. Initial assessment of implantation nodules during early pregnancy
(day 6 to 8) revealed that embryo attachment and gross morphology of implantation nodules were
similar in the two genotypes. However, in depth histological analysis of uterine sections on day 8
of gestation revealed dysregulation of stromal differentiation in the uteri of Rac1d/d mice. In
response to aberrant stromal differentiation, we observed that between day 8 and 10 of pregnancy,
a significant number of the implanted embryos became hemorrhagic and had begun to undergo
resorption. By day 15 of pregnancy, most embryos had been lost and the few that survived,
developed abnormal placentas and were surrounded by hemorrhagic maternal tissue. It should be
noted that the fertility defect in Rac1d/d females is due to maternal shortcomings rather than an
embryonic defect. Crossing of PgrCre/Cre Rac1f/f males x Pgr+/+ Rac1f/f females, which produced
Rac1d/d embryos, resulted in viable offspring without pregnancy complications. Whereas a number
of previous studies have shown that a defect in the embryo severely affects placentation and
pregnancy outcome, we are reporting for the first time that a perturbation in the maternal
differentiation program of Rac1d/d uteri adversely affects trophoblast function and maintenance of
pregnancy. Future studies will address an in depth analysis of this new mouse model to improve
our understanding of the cause and consequence of pregnancy complications in the human.
40
Introduction
Implantation of the embryo to the uterine wall followed by placentation are pivotal events for
establishment and maintenance of pregnancy [1-3, 110]. These processes involve steroid-
dependent complex crosstalk between the embryo and the maternal endometrium, which in turn
supports the growing conceptus, and ensure reproductive success [2, 4, 5, 8, 107, 115]. Failure in
any of the events can compromise maintenance of pregnancy and lead to miscarriage [5-7, 26, 32,
143, 345]. In fact, clinically, implantation failure and spontaneous abortion following implantation
are among the leading causes of infertility in women of reproductive age and reveals a major
challenge for assisted reproductive technology [6, 7, 38, 39]. In many instances the reasons for
implantation failure are unknown.
In rodents, as the embryo breaches the luminal epithelium following attachment, the underlying
stromal cells undergo a dramatic transformation to form a specialized tissue, known as the decidua
[5, 23, 25, 29, 117, 154, 346]. During decidualization, the fibroblastic stromal cells proliferate and
then differentiate into mature secretory decidual cells that support embryo growth and survival
until placentation [23-25, 29, 121-123]. In addition, the decidual tissue undergoes extensive
remodeling to accommodate the growing embryo. Concomitant with the differentiation process,
as the decidua forms around the implanted embryo, endothelial cells in the uterine stroma
proliferate to create an extensive vascular network, which is embedded in the decidua and is critical
for embryo development [5, 23, 25, 29, 32, 117, 154, 346]. It is clear that decidualization is a key
event in establishment of early pregnancy, however, the signaling molecules that participate in the
formation and function of this tissue remain poorly understood.
Microarrays are powerful molecular tools that allow rapid screening of gene expression changes
under different experimental conditions. Using this tool, we compared differentially expressed
genes in the uterus during decidualization and identified Ras-related C3 botulinum toxin substrate
1 (RAC1) as one of the genes whose expression is markedly elevated at the time decidualization.
While a previous study performed in vitro, implicated a potential role of RAC1 in embryo invasion,
its role in vivo during implantation remains unknown [7, 26]. We, therefore, decided to explore
the in vivo function of RAC1 in the uterus during early pregnancy.
41
RAC1, a pleiotropic signaling molecule and a member of the Rho family of GTPases, has been
shown to regulate many cellular processes. These include cell cycle, cell-cell adhesion, cell
differentiation, cell motility through actin cytoskeleton remodeling, endothelial cell function, as
well as angiogenesis [113, 242, 250, 254, 261, 262, 269, 271-280, 301]. Because decidualization
encompasses many of these physiological events, it is possible that RAC1 regulates one or more
of these processes during early pregnancy. In support of this notion, Grewal et al. [7, 26] has shown
recently that invasion of the human embryo into endometrial stroma, in vitro, involves the motility
of endometrial stromal cells and this movement is dependent on the activity of Rho GTPases,
especially RAC1. Here, we show that RAC1 is necessary for early pregnancy, as uterine loss of
this molecule led to severe subfertility. However, contrary to the previous in vitro observations,
we now show that RAC1 activity is not necessary for embryo attachment or invasion into the
uterine stroma to initiate embryo implantation.
42
Materials and Methods
Animals and Tissue Collection
All animal breeding, housing, procedures, experiments, and euthanasia were approved and
conducted according to the guidelines of the Institutional Animal Care and Use Committee of
University of Illinois at Urbana-Champaign. The health and care of the animals was provided by
the veterinarians and staff of Division of Animal Resources. Rac1 conditional null mice were
generated using Cre-LoxP strategy [347] (Figure 3.1A). Mice expressing homozygous copies of
the Rac1 gene flanked by loxP sites [Pgr+/+ Rac1f/f (official gene symbol Rac1tm1Djk/J), or Rac1f/f
from now onwards] [267] were obtained from Dr. Ann Sutherland (University of Georgia) in a
C57BL/6-129SV background. Mice carrying the Cre Recombinase under the control of the
progesterone receptor (Pgr) promoter [348] [PgrCre/+ Rac1+/+ (official gene symbol Pgrtm2(cre)Lyd/+),
or PgrCre from now onward], were obtained from Drs. John Lyndon and Francesco De Mayo
(Baylor College of Medicine) in a C57BL/6 background. To establish the mouse colony for this
study, initial breeding between the two mouse lines was done to obtain the F1 progeny, which are
heterozygous for Pgr and Rac1 (Appendix A). Female mice used for this study were generated by
breeding male mice of specific genotypes from the F2 progeny (see Figure 3.1B) with Rac1f/f
female mice. Genotyping of the animals was performed following the protocols established by the
respective laboratories that generated the transgenic mice [267, 348] (Appendix A). Conditional
knockout female mice (PgrCre/+ Rac1f/f) will be referred to as Rac1d/d from now onwards.
Rac1f/f or Rac1d/d females were euthanized by CO2 asphyxiation, followed by a blood draw via
cardiac puncture, and cervical dislocation. Uteri and ovaries from pregnant mice or from mice
subjected to experimentally-induced decidualization (see below) were collected at different time
points during pregnancy or following the differentiation stimulus. The harvested organs were
either fixed for histological evaluation [immersion-fixed in 10% neutral-buffered formalin (NBF)],
flash frozen in liquid N2 for RNA or frozen sectioning, or the decidual mass was harvested for
gene expression studies. For samples collected at different time points during pregnancy,
identification of a copulatory plug following mating was used as a reference and for our
experimental purposes, to indicate day 1 of pregnancy.
43
Some of the frozen implantation nodules, collected on day 8 of pregnancy, were thawed by
submerging them in 1x phosphate-buffer saline (PBS) at room temperature for morphological
evaluation of the decidua. The decidual mass was exposed by mechanical removal of the
myometrium. Pictures were taken using a Leica M165 FC dissection scope connected to a Leica
450 C camera with c-mount interface containing a 5 Megapixel CCD sensor. Similarly, for
morphological evaluation of embryos on day 15 of pregnancy, mechanical removal of the
myometrium was done on samples that had been fixed in 10% NBF under the dissecting scope, as
mentioned above.
Fertility testing
To determine whether conditional mutation of Rac1 in uterine tissues had any effect in female
fertility, Rac1f/f and Rac1d/d mice of reproductive age (8 weeks or more) were paired with proven
wild-type males for a period of six months (Appendix A). The total number of pups born in each
litter as well as the number of pregnancies during this period were recorded.
Experimentally-induced decidualization
To test whether RAC1 plays any role during decidualization, 8 week old female mice were
subjected to experimentally induced decidualization protocol as described previously [32, 154,
349-352]. Briefly, adult, non-pregnant, Rac1f/f and Rac1d/d female mice were ovariectomized under
general anesthesia and rested for two weeks following ovariectomy to remove any circulating
hormones. After two weeks of rest, the animals were injected subcutaneously (s.c.) with 100 ng of
17β-estradiol (E2) in 100 µL corn oil daily for three days, then rested for two days. Following
resting period, mice were injected with a solution containing 6.7 ng E2 and 1 mg P4 per 100 µL
(E2 + P4) corn oil s.c. for three days. On the third day of E2 + P4 injection, decidualization was
initiated, under general anesthesia, in one horn by injection of 50 μL oil, whereas the other horn
was left unstimulated to serve as an internal control. Daily injections of E2 + P4 were continued
for up to an additional 96 h post-stimulation. Mice were euthanized and uterine horns were
collected and weighed at different time points following the decidualization stimulus.
44
Hormonal treatment
To test whether Rac1 gene expression is affected by hormonal treatment, Rac1f/f mice were
subjected to different doses of steroid hormones as described previously [353]. Briefly, female
mice were subjected to bilateral ovariectomy. Two weeks later, the mice were injected
subcutaneously with 100 µL of vehicle (oil), E2 (100 ng), P4 (1 mg) or combination of E2 + P4.
The animals were then euthanized at 6 h and 24 h following E2 alone; after 2 doses daily of P4; or,
in case of the combination, 2 daily doses were given, followed by E2, then tissue was collected 6h
and 24 h after the last injection.
Alkaline Phosphatase Activity Assay
The protocol for detecting alkaline phosphatase (ALPL) activity was followed as previously
published [352, 354], with modifications. Briefly, frozen uterine sections were incubated in 10%
NBF for 10 min at room temperature then washed in 1x PBS three times for 5 min each (3x 5 min.)
The slides were then incubated with a solution containing 0.1 M Tris-HCl pH 8.5, 0.5mM naphthol
AS-MX phosphate disodium salt (ALPL substrate), and 1.5 mM fast blue RR (4-benzoylamino-
2,5-dimethoxyaniline diazonium) salt at 37 oC for 30 min. The uterine alkaline phosphatase
activity releases orthophosphate and naphthol derivatives from the substrate. The naphthol
derivatives are simultaneously coupled with diazonium salt present in the incubating medium to
form a dark blue dye, which marks the site of enzyme action. To terminate the enzymatic reaction,
the tissue sections were rinsed in tap water, mounted using crystal mount medium, and visualized
using an Olympus BX51 microscope equipped for light microscopy connected to a Jenoptik
ProgRes C14 digital camera with c-mount interface containing a 1.4 Megapixel CCD sensor.
Immunohistochemistry and Immunofluorescence
Paraffin-embedded or frozen uterine sections were subjected to immunohistochemistry as
described previously [32, 353, 355, 356]. Briefly, paraffin-embedded sections were deparaffinized
in xylene and rehydrated in a series of graded alcohols followed by rinsing in tap water. When
frozen tissues were used, the sections were thawed at room temperature for 5 min, then fixed for
45
another 5 min in 10% NBF. Sections were then rinsed in 1x PBS, followed by blocking in 10%
non-immunized serum for 1 h at room temperature. The slides were incubated with a solution
containing the diluted antibody (see Appendix E for details) and 1% serum in 1x PBS overnight at
4 oC in a humidified chamber. Following overnight incubation, slides were washed 3x 5 min with
1x PBS, then incubated with diluted secondary antibody (see Appendix E for details) for 1 h at
room temperature in a humidified chamber. When a chromogenic labeling was done, sections were
incubated in a solution containing 3% H2O2 in methanol to quench endogenous peroxidase. Slides
were then rinsed in 1x PBS, followed by a 30-45 min incubation with horseradish peroxidase-
conjugated streptavidin (Invitrogen). Slides were then exposed to chromogens (3, 3’-
diaminobenzidine or 3-amino-9-ethylcarbazole) until optimal signal was obtained. Following the
chromogenic labeling, the slides were counterstained with hematoxylin, and mounted. Slides that
were incubated with a fluorescent secondary antibody were mounted with a medium containing
DAPI. Pictures were taken using the Olympus BX51 microscope equipped for light and fluorescent
imaging and connected to a Jenoptik ProgRes C14 digital camera with c-mount interface
containing a 1.4 Megapixel CCD sensor. Fluorescent images were merged and processed using
CS4 Adobe Photoshop Extend (Adobe Systems).
RNA Isolation and Real-time quantitative PCR
Total RNA was isolated from tissues or cells using TRIzol reagent (Invitrogen) according to the
manufacturer’s instructions. Oligonucleotides specific for genes of interest were developed (see
Appendix F for details) and real-time quantitative PCR (qPCR) reactions were carried out using
SYBR-green master mix (Applied Biosystems) on a 7500 Applied Biosystems Real-time PCR
machine. Gene expression data analysis were done using the 2-ΔΔCt method [357] and 36b4 gene –
which is conserved among many species and encodes an acidic ribosomal phosphoprotein P0 that
interacts with 40S ribosomal subunit – was used as a housekeeping gene and loading control [358].
Endometrial Stromal Cell Isolation
Endometrial stromal cells were isolated from day 1 and 4 pregnancy samples as previously
described [164, 352]. Uteri were removed from mice after euthanasia with CO2. The uteri were cut
46
open and digested with a solution containing 6 g/L dispase (Gibco) and 25 g/L pancreatin (Sigma)
in 1x HBSS (Gibco) for 45min at room temperature, followed by 15 min at 37 oC, with occasional
mixing. Cells were washed and then incubated in 0.5 g/L collagenase (Sigma) in HBSS for 1 h at
37 oC. After incubation, the tubes were vortexed for 10-12 seconds until the supernatant became
turbid with dispersed cells. The contents of the tube were then passed through an 80-μm gauze
filter (Millipore) into a new tube. Cells were resuspended in Dulbeccos modified Eagle's medium-
F12 medium (Gibco) supplemented with 2% heat-inactivated fetal calf serum, 100 units/L
penicillin, 0.1 g/L streptomycin (Gibco), 1.25 mg/L fungizone, 10 nM E2, and 1 µM P4. The
numbers of live cells were assessed by trypan blue staining using a hemocytometer.
Western Blots
Mouse endometrial stromal cells or decidual masses were isolated from female mice at different
days of pregnancy. The cells from day 1 and 4 pregnant uteri were lysed in ice-cold lysis buffer
containing 1% NP40, 50 mM Tris-HCl pH 7.6, 125 mM NaCl, 1 mM EDTA, 1x protease inhibitor
cocktail. Decidual mass on D8 of pregnancy was lysed with lysis buffer aided by maceration using
a mortar and pestle. The cell debris was removed by centrifugation, and the protein concentration
of the lysate was determined by bicinchoninic acid protein assay according the manufacturer's
instruction (Pierce/Thermo/Fisher Scientific). The cell lysates were analyzed by standard Western
blotting, using antibodies against RAC1 (1:1000) and β-TUBULIN (1:5000, as loading control,
see Appendix E for antibody details).
Hormone assay
Serum P4 concentrations were measured for at least 3 animals per time point by radioimmunoassay
at the core facility of University of Virginia at Charlottesville.
Statistical analysis
Experimental data were collected from a minimum of three independent animals (not from the
same litter). Uterine weights, serum hormones, embryo perimeter, placental weight, and qPCR
47
results were expressed as mean ± S.E.M. Litter size were expressed as means. Statistical analysis
was done using Student’s t-test (for single comparison), one-way analysis of variance with a
Dunnett’s post-test (for multiple comparison between treatments or time points) or two-way
ANOVA with a Bonferroni post-test (for multiple comparison between different genotypes and
time points). An analysis of equal variances was done on all numerical data to determine whether
a parametric or non-parametric hypothesis test was appropriate. Data were considered statistically
significant at p ≤ 0.05, and is indicated by an asterisk in the figures. All data was analyzed and
plotted using GraphPad Prism 4.0 (GraphPad Software).
48
Results and Discussion
Rac1 is expressed in uterine stroma during decidualization, but its expression is independent of
steroid hormones
To gain insight into the molecular pathways underlying decidualization, we performed gene
expression profiling using RNA from uterine horns at different times after exposure to decidual
stimulation. Our studies revealed upregulation of Rac1 in response to decidual stimulation. To
confirm the results of the microarray analysis, we performed qPCR using RNA isolated from the
non-stimulated and stimulated horns at different times after decidual stimulation. As shown in
Figure 3.2A, Rac1 expression increased in response to decidual stimulation. A marked increase in
Rac1 expression was observed at 72 h post decidual stimulation. Consistent with this expression
profile, we observed a significant upregulation in Rac1 expression during normal pregnancy on
day 7 and day 8 of gestation (Figure 3.2B).
Because steroid hormones, estrogen (E2) and progesterone (P4) are known to influence
decidualization, we investigated whether Rac1 gene expression is regulated by steroid hormones.
Administration of E2, P4, or E2 + P4 to ovariectomized mice failed to induce Rac1 expression in
the uterus, indicating that it is not regulated by steroid hormones (Figure 3.2C). Interestingly,
previous studies have indicated the presence of response elements for several transcription factors,
including HIFs, in the Rac1 promoter [285]. It is possible that HIFs, which are induced during
stromal differentiation, are involved in the regulation of Rac1 in the uterus during early pregnancy.
However, further analysis of the Rac1 promoter during stromal cell differentiation will be needed
to validate this possibility.
We next monitored by western blot analysis the expression profile of RAC1 protein in the uterus
at different days of gestation. We observed a band corresponding to RAC1 protein on day 4 of
gestation. But consistent with the RNA profile, we observed a marked enhancement in RAC1
protein expression in uterine stromal cells on day 8 of pregnancy, overlapping the window of
decidualization (Figure 3.2D). Interestingly on day 8, we also noted the presence of a band
49
corresponding to phosphorylated RAC1 (pRAC1) in the western blot. The functional significance
of pRAC1 in the uterus remains unknown.
The Rac1d/d mouse model
To address the role of RAC1 in the uterus, we generated a mouse line where the Rac1 gene is
conditionally deleted using a Cre recombinase that is under the control of one copy of the
progesterone receptor promoter (termed PgrCre/+), whereas the other copy expresses functional
Pgr, which is sufficient to preserve fertility of these animals [348]. The conditional deletion
approach was used because the Rac1 global knockout is embryonic lethal [271]. Further, PgrCre
mouse line has been extensively used to ablate genes in specific cells lineages within the body [32,
345, 348, 353, 359-361]. Rac1 gene is conditionally excised in only those tissues that are positive
for Pgr, which includes the uterus. The term Rac1f/f and Rac1d/d will be used to refer to the control
and conditional null mice, respectively.
Ablation of Rac1 in the Rac1d/d mice was assayed by qPCR and our studies indicate efficient
ablation of Rac1 in the uteri on day 4 to day 10 of pregnancy (Figure 3.3A). Consistent with the
RNA profile, we observed a marked decline in the levels of RAC1 protein in Rac1d/d uteri on day
4 or day 8 of gestation (Figure 3.3B). Our results also indicate that the gene expression of other
members of the Rho family of GTPases, such as Rac2, Rac3, Rhoa, and Cdc42, were unaffected
upon conditional deletion of Rac1 (Figure 3.3C).
Conditional deletion of uterine Rac1 leads to severe subfertility
To determine the role of RAC1 in the regulation of fertility, a six-month breeding study was
performed by crossing Rac1d/d or Rac1f/f mice with proven wild-type males (see Appendix A for
details). At the completion of the study, we noted a severe subfertility phenotype in Rac1d/d female
mice compared to Rac1f/f controls. More than 90% of the pups were lost during pregnancy
indicating a drastic reduction in the litter size (Table 2.1). In addition, most Rac1d/d females
exhibited a drastic reduction in their fertility rate after the first pregnancy (data not shown), and
many females did not produce any litters after the second or third pregnancy. Although these
50
results are of interest to us, we have not investigated this issue any further as part of this dissertation
project.
Conditional deletion of Rac1 has no effect on ovarian function
Because progesterone receptor is transiently expressed in the granulosa cells of the ovary around
the time of ovulation [362-366], and given that RAC1 is ubiquitously expressed in many cell types,
it is conceivable Rac1 may be deleted from the ovary, resulting in insufficient ovarian function.
Therefore, to ensure that the severe subfertility phenotype, and pregnancy loss, observed in Rac1d/d
mice was not due to an ovarian defect, ovaries from days 4, 8, 10 (data not shown), 12, and 15 of
pregnancy were collected and evaluated histologically for signs of anovulation or lack of corpora
lutea (CL) (Figure 3.4). Ovaries collected from Rac1d/d females displayed follicles at all stages of
development and CL with normal appearance (Figure 3.4A). Moreover, we monitored Rac1 gene
expression in the ovary on day 4 of pregnancy, and consistent with the ovarian histology data,
there was no significant difference in Rac1 mRNA expression in the ovaries from Rac1f/f and
Rac1d/d mice (Figure 3.4B). We also determined the serum progesterone concentrations in the two
genotypes to ensure that the fertility defect was not due to a deficiency in circulating P4. Indeed,
no significant difference was found in serum P4 concentration between Rac1d/d and Rac1f/f mice
(Figure 3.4C). Moreover, we did not detect any significant difference in the average number of
blastocysts recovered from the uteri of Rac1f/f and Rac1d/d mice on day 4 of pregnancy (data not
shown).
We next investigated the effect of deletion of uterine Rac1 on embryo implantation. Previous
studies by Grewal et al. [7, 26] suggested that activation of the RAC1 pathway in the decidual cells
is required for implantation and trophoblast invasion and expansion. Interestingly, our studies
revealed that uterine deletion of Rac1 gene did not prevent attachment of the embryos into the
maternal endometrium (Figure 3.5A). We noted the presence of implanted embryos in the uterus
beginning on day 4 of pregnancy and, up to day 8 of pregnancy, we did not detect any overt
abnormality of the implantation nodules in the two genotypes. Interestingly, a closer histological
examination of uterine sections on different days of gestation revealed that starting on day 7, the
embryos in Rac1d/d mice started to display abnormality at the implantation site and this became
51
more pronounced on day 8 of pregnancy. Staining of uterine sections on day 8 with cytokeratin,
which marks the trophoblast cells showed aberrant overexpansion of trophoblast cells in maternal
stroma (Figure 3.5B and Appendix C). At this time, we have not fully characterized the
overexpansion phenotype of the embryos. However, it is possible that the dysregulated trophoblast
expansion could be a result of improper differentiation of uterine stroma due to ablation of RAC1.
A number of previous studies have implicated RAC1 as a regulator of critical cellular processes,
including cell proliferation and differentiation [242, 261, 269, 273, 276, 280, 301, 367]. Because
stromal cell differentiation is a critical event for establishment of pregnancy, we determined
whether conditional deletion of uterine Rac1 affected this process by employing a well-established
differentiation model that is independent of the embryo [154]. We observed that the gross decidual
response in Rac1d/d uteri was comparable to that of control Rac1f/f uteri (Figure 3.6, lower panel).
During decidualization, stromal cells proliferate, exit the cell cycle, and terminally differentiate
into mature decidual cells [346, 368-372]. The progression of this differentiation program can be
traced by monitoring the expression of biochemical markers in the uterus. As cells enter the
differentiation program, they express liver/bone/kidney alkaline phosphatase (ALPL) (Figure 2.3).
We further investigated the decidual response in Rac1f/f and Rac1d/d uteri by monitoring the
expression of ALPL. As expected, uterine sections from Rac1f/f mice displayed prominent ALPL
activity at 48 h after the administration of decidual stimulus (Figure 3.6B, upper panel).
Interestingly, we noted a dramatic induction of ALPL activity in Rac1d/d uteri at similar time point,
indicating dysregulated stromal differentiation program in the absence of uterine RAC1 (Figure
3.6B, lower panel).
We next examined the role of RAC1 in stromal decidualization during normal pregnancy. We did
not observe any difference in cell proliferation on day 8 of pregnancy, as indicated by MKI67
staining (Figure 3.7C). However, consistent with the observation that the stromal differentiation
program is dysregulated by the loss of uterine RAC1, we noted a marked upregulation in Alpl gene
expression (Figure 3.7A) as well as activity (Figure 3.7B) in Rac1d/d uteri in comparison to Rac1f/f
controls. We also examined the expression of other markers of differentiation including Wnt4 and
Prl8a2/dPrp during decidualization (Figures 3.7 and Appendix B). While the expression of Wnt4
52
was upregulated (Figure 3.7A) in Rac1d/d uteri, we observed a slight downregulation in the
expression of PRL8A2/dPRP protein (Figure 3.7D) in uterine sections of Rac1d/d mice compared
to Rac1f/f controls on day 8 of pregnancy. A reduction in Prl3c1/Plpj gene expression was also
noted (Figure 3.7A). PRL3C1/PLPJ has been reported to be produced by the decidual cells
immediately surrounding the embryo [373] and may be implicated in decidual cell development
and the endometrial vasculature [374] around day 8 of pregnancy. We also monitored the
expression of Alpl and Wnt4 in cultured stromal cells collected from Rac1f/f and Rac1d/d uteri on
day 4 of pregnancy and subjecting them to in vitro decidualization. Again, similar to the pregnancy
samples, our results revealed a marked enhancement in Alpl and Wnt4 expression in stromal cells
from Rac1d/d uteri (Figures 3.8). Collectively, these results indicate that in the absence of uterine
RAC1, the stromal differentiation program during pregnancy goes awry.
The severe subfertility in Rac1d/d is due to maternal but not embryonic defect
How do we rule out the possibility that the subfertility in Rac1d/d mice is not due to an inherent
defect in the embryo? A number of previous studies have shown that a defect in the embryo
severely affects trophoblast migration and placentation [9, 59, 129, 130, 132, 300]. However, to
the best of our knowledge, only a limited number of mouse models exist in the literature in which
a perturbation in the maternal decidua, and not the embryo, adversely affects trophoblast function
and placentation [32, 359, 360, 375-378]. In that regard, the Rac1d/d mice are unique. Thus, to
avoid any complication of potential non-specific deletion of Rac1 gene in the embryo, we bred the
Rac1d/d and Rac1f/f females to wild-type male mice of proven fertility. This breeding scheme
generated heterozygous embryos for Rac1 gene (see Appendix A for details), therefore any
problems associated with pregnancy are likely due to a defect in the maternal decidua, and not in
the embryo or its membranes. Our breeding studies further indicated that embryo loss occurred
around day 10 to 12 of pregnancy, and appear as hemorrhagic implantation nodules (Figures 3.9
and 3.10A). It should be noted, however, that not all embryos died at this time point. A small
percentage of the embryos managed to survive to later stages of gestation (Figure 3.10C), and even
to be born (Table 3.1). It is intriguing that some embryos managed to survive, whereas others die.
One observation we have consistently made is that there is a cleft that remains in the uteri of
Rac1d/d mice (Figure 3.10B), possibly from defective migration of decidual cells. In Rac1d/d uteri,
53
this area is highly hemorrhagic and many neutrophils infiltrate the luminal space of the cleft, while
a thin decidua surrounds the embryo in the mesometrial side (data not shown). An interesting
possibility is that in Rac1d/d uteri, the decidua fails to form an immune barrier from the allogenic
embryo [23, 29, 149-151], resulting in embryo resorption and termination of pregnancy.
Studies performed in the early 1990s showed that the mouse blastocyst expresses detectable Pgr
mRNA expression [379]. However, we ruled out any possibility that a potential deletion of Rac1
in the embryonic tissues adversely influenced the outcome of pregnancy because of the following
reasons. First, heterozygous females for both Rac1 and PgrCre (PgrCre/+ Rac1f/+) are fertile (Table
3.2), and when bred to either PgrCre/+ Rac1f/f or PgrCre/Cre Rac1f/f males they 1) produced litters
with the expected genotypes (based on genetic expectancy of the crossing; see Appendix A for
details); 2) they produced viable offspring, which were later used for breeding purposes; and 3)
pups with a PgrCre/Cre Rac1f/f genotype were generated in this crossing. In addition, crossing of
Rac1f/f females with either PgrCre/+ Rac1f/f, PgrCre/Cre Rac1f/f, or Pgr+/+ Rac1+/+ males of proven
fertility, showed that Rac1f/f females carrying Rac1d/d embryos were fertile and produced viable
litters (Figure 3.11), strongly suggesting that the fertility defect observed in the mutant females is
due to a maternal rather than an embryonic defect. Collectively, these results indicate that the
maternal environment due to dysregulated stromal differentiation in Rac1d/d uteri is not conducive
to embryo growth. In the next chapter, we have investigated how impaired stromal differentiation
is affecting maternal environment leading to embryo demise in uteri lacking RAC1.
54
Chapter Figures and Tables
FIGURE 3.1: BREEDING SCHEME TO GENERATE THE CONDITIONAL MUTANT FOR RAC1 USING CRE-LOXP STRATEGY. (A) The gene construct
for Rac1 gene and the primer position for genotyping strategy are shown. Floxed and wild-type bands are detected with the primer combination
P91/P33, whereas the conditional knockout band is detected with the primer combination P45/P33 [347]. Triangles indicate the LoxP sites.
55
FIGURE 3.1: BREEDING SCHEME TO GENERATE THE CONDITIONAL MUTANT FOR RAC1 USING CRE-LOXP STRATEGY (CONTINUED). (B) The
breeding scheme for the generation of the experimental animals is shown. Briefly, male mice, with specific genotypes, were bred to Rac1f/f females
to generate the conditional mutant. In these animals, exon 1, which contains the translation initiation site and part of the GTPase activity of RAC1,
will be conditionally excised in all cells that express Pgr gene.
56
FIGURE 3.2: EXPRESSION PROFILE FOR RAC1 GENE AND PROTEIN IN MICE. (A – C) Whole uterus gene expression profile for Rac1 after
experimentally-induced decidualization (panel A), hormonal stimulation (panel B), and during pregnancy (panel C). (D) RAC1 protein expression
profile from isolated endometrial stromal cells on day 4 of pregnancy or decidual cells on day 8 of pregnancy; day 1 shows a similar profile as day
4 of pregnancy (data not shown). As expected, both gene and protein increase in expression during cell differentiation, but are unaffected by hormonal
treatment alone. n ≥ 3 per time point. Asterisks indicate significant differences from 0 h on panel A or day 1 of pregnancy in panel B (p < 0.01).
57
FIGURE 3.2: EXPRESSION PROFILE FOR RAC1 GENE AND PROTEIN IN MICE (CONTINUED). (A – C) Whole uterus gene expression profile for Rac1
after experimentally-induced decidualization (panel A), hormonal stimulation (panel B), and during pregnancy (panel C). (D) RAC1 protein
expression profile from isolated endometrial stromal cells on day 4 of pregnancy or decidual cells on day 8 of pregnancy; day 1 shows a similar
profile as day 4 of pregnancy (data not shown). As expected, both gene and protein increase in expression during cell differentiation, but are
unaffected by hormonal treatment alone. n ≥ 3 per time point. Asterisks indicate significant differences from 0 h on panel A or day 1 of pregnancy
in panel B (p < 0.01).
58
FIGURE 3.3: RAC1 IS EFFICIENTLY DELETED FROM THE UTERUS USING PGRCRE. (A) Whole uterus gene expression profile demonstrates an
efficient knockdown of Rac1 gene in Rac1d/d mice in samples obtained at different times during pregnancy. (B) RAC1 is efficiently ablated in
endometrial stromal cells on day 4 of pregnancy, as well as on decidual cells on day 8 of pregnancy. Residual RAC1 protein may come from
contamination from other cell populations (like uterine natural killer cells), which migrate into the uterus around day 8 of pregnancy.
59
FIGURE 3.3: RAC1 IS EFFICIENTLY DELETED FROM THE UTERUS USING PGRCRE (CONTINUED). (C) Whole uterus gene expression profile on day
8 of pregnancy shows that Rac1 is the only member that is affected by the conditional deletion; other members of the Rho family of GTPases are
not affected by the conditional deletion. n ≥ 3 per time point. Asterisks indicate significant differences from controls (**, p < 0.01, ***p < 0.001).
60
TABLE 3.1: IMPACT OF RAC1 DELETION ON FEMALE FERTILITY
Genotype # Females † # Pups # Litters
Average
Pups/Litter ‡
Pgr+/+
Rac1f/f
7 315 31 10
PgrCre/+
Rac1f/f
7 19 11 2
† One Rac1d/d
female did not produce any litters during or after the breeding study ‡ p < 0.0001
61
FIGURE 3.4: CONDITIONAL DELETION OF RAC1 DOES NOT AFFECT OVARIAN FUNCTION. (A) Representative histological samples of ovaries from
Rac1f/f and Rac1d/d on days of pregnancy (D) 4, 8, 12, and 15 stained with H&E. No differences in the ovarian morphology were observed at these
time points. Bars = 200 µm.
62
FIGURE 3.4: CONDITIONAL DELETION OF RAC1 DOES NOT AFFECT OVARIAN FUNCTION (CONTINUED). (B) Consistent with the observation in
Panel A, no significant difference ovarian was observed in Rac1 gene expression by D4. (C) Serum progesterone (P4) concentration was unaffected
by the conditional deletion of Rac1. n = 3 per time point. (p > 0.05).
63
FIGURE 3.5: CONDITIONAL DELETION OF RAC1 HAS DOES NOT PREVENT EMBRYO ATTACHMENT AND INVASION INTO THE ENDOMETRIUM. (A
and B) Shows representative samples from Rac1f/f and Rac1d/d on day 8 of pregnancy. No gross morphological difference was observed between
conditional null and control mice (panel A).
64
FIGURE 3.5: CONDITIONAL DELETION OF RAC1 HAS DOES NOT PREVENT EMBRYO ATTACHMENT AND INVASION INTO THE ENDOMETRIUM
(CONTINUED). (A and B) Also, uterine deletion of Rac1 gene does not prevent embryo implantation (panel B). CYTOKERATIN 8 was used as a
general marker of trophoblast cells to highlight the implanted embryo. Bars = 500 µm. n ≥ 5.
65
FIGURE 3.6: CONDITIONAL DELETION OF UTERINE RAC1 DOES NOT AFFECT THE FORMATION OF THE DECIDUOMA, BUT DISPLAYS
DYSREGULATION IN ALPL ACTIVITY. (A) No difference was detected in the wet weight of the deciduoma induced experimentally in conditional
Rac1-null mice compared to controls (p > 0.05). Shown here are representative sample after 96 h post stimulus uteri. The right horn was stimulated,
whereas the left horn served as an internal control and was not stimulated. (B) Conditional deletion of uterine RAC1 led to a dysregulated increase
in the activity of the early marker of decidualization, kidney/liver/bone alkaline phosphatase (ALPL) activity at all the observed time points. Shown
here is uterine ALPL activity 48 h post stimulus. n = 3 per time point.
66
FIGURE 3.7: CONDITIONAL DELETION OF RAC1 LEADS TO A DYSREGULATED STROMAL CELL DIFFERENTIATION. (A) Gene expression assay
was done on day 8 of pregnancy to access the expression of well-known makers that are differentially expressed during stromal cell differentiation.
Critical genes that are known to be expressed during the early stages of differentiation, i.e. Alpl and Wnt4, show a dysregulated gene expression.
Prl3c1/Plpj a putative marker of late stromal cell differentiation was reduced (p = 0.14), as compared to control uteri. Other genes involved in the
differentiation of stromal cells, were not significantly changed at this time point of pregnancy. Asterisks indicate significant differences from controls
(**, p < 0.01, ***p < 0.001).
67
FIGURE 3.7: CONDITIONAL DELETION OF RAC1 LEADS TO A DYSREGULATED STROMAL CELL DIFFERENTIATION (CONTINUED). (B) Alkaline
phosphatase (ALPL) activity is a well-known indication of early stromal cell differentiation, and persist during the differentiation of stromal cells.
Our data show that there is noticeable elevation in both Alpl gene expression (panel A) and activity in day 8 pregnancy samples. Similar observations
were made on day 7 of pregnancy (data not shown). Bars = 500 µm.
68
FIGURE 3.7: CONDITIONAL DELETION OF RAC1 LEADS TO A DYSREGULATED STROMAL CELL DIFFERENTIATION (CONTINUED). (C & D) It is
well established that during the differentiation process, decidualizing stromal cells rapidly proliferate and quickly enter the differentiation program
[346, 368-372]. Our data indicate that stromal cell from Rac1d/d uteri are capable of initiating the differentiation into decidual cells, as no difference
in MKI67 (panel C), a maker of cell proliferation. However, despite no change in gene expression, uteri deficient for Rac1 display a reduced
expression of PRL8A2/dPRP (panel D), a marker of late stromal cell differentiation, when compared to control uteri. The area below the dash mark
indicate the area negative for MKI67, which positive cells have a reddish/brown nuclear staining. Bars = 200 µm (panel C and D).
69
FIGURE 3.7: CONDITIONAL DELETION OF RAC1 LEADS TO A DYSREGULATED STROMAL CELL DIFFERENTIATION (CONTINUED). (C & D) It is
well established that during the differentiation process, decidualizing stromal cells rapidly proliferate and quickly enter the differentiation program
[346, 368-372]. Our data indicate that stromal cell from Rac1d/d uteri are capable of initiating the differentiation into decidual cells, as no difference
in MKI67 (panel C), a maker of cell proliferation. However, despite no change in gene expression, uteri deficient for Rac1 display a reduced
expression of PRL8A2/dPRP (panel D), a marker of late stromal cell differentiation, when compared to control uteri. PRL8A2/dPRP is highlighted
by the cells with a red cytoplasmic stain under the dash mark. Bars = 200 µm (panel C and D). n ≥ 4.
70
FIGURE 3.8: ACTIVATION OF THE DECIDUAL CELL RAC1 PATHWAY IS NECESSARY FOR STROMAL CELL MORPHOLOGY AND NORMAL
DIFFERENTIATION. Shown here are cultured uterine decidual cells, under differentiation conditions for 72 h. As shown here, cells, where Rac1 gene
has been deleted (Rac1d/d), show abnormal cell morphology, compared to control cells (Rac1f/f). Similarly, during the differentiation of decidual
cells, gene expression of factors, likes alkaline phosphatase (Alpl) and Wnt4 are known to be expressed, but shown here is a dysregulated gene
expression of these factors in the mutant decidual cells; other factors, like decidual prolactin-related protein (Prl8a2/dPRP) gene expression remain
unchanged. Blue = DAPI, which stains the nucleus, and green = Phalloidin, which stains actin filaments. n = 3. Asterisks indicate significant
differences from controls (p < 0.001).
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FIGURE 3.9: REPRESENTATIVE UTERI SAMPLES AT DIFFERENT STAGES OF PREGNANCY. Uteri samples at different times of pregnancy were
collected from Rac1f/f and Rac1d/d on days 8 through 15 of pregnancy. Black arrows point hemorrhagic implantation nodules, whereas blue arrow
head highlight lateral hemorrhage, which commonly seen in hemochorial placentation. n ≥ 3.
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FIGURE 3.10: SEVERE HEMORRHAGE IS OBSERVED IN IMPLANTATION NODULES FROM RAC1D/D UTERI. (A) The graph shows a survival curve
analysis of the onset of hemorrhagic uteri collected from day 4 of pregnancy. Hemorrhagic implantation nodules are typically noticed as early as
day 7 of pregnancy.
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FIGURE 3.10: SEVERE HEMORRHAGE IS OBSERVED IN IMPLANTATION NODULES FROM RAC1D/D UTERI (CONTINUED). (B) Shown here are
representative samples of pregnancy nodules on day 8 of pregnancy from control, conditional knockout, and heterozygous uteri after the nodules
were liberated from the myometrium to expose the decidua. Severe hemorrhage is typically observed in the conditional knockout decidua, whereas
the control and heterozygous uteri appear do not display this severe bleeding. Bars = 10 mm.
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FIGURE 3.10: SEVERE HEMORRHAGE IS OBSERVED IN IMPLANTATION NODULES FROM RAC1D/D UTERI (CONTINUED). (C) Representative samples
implantation nodules on day 15 of pregnancy from control and conditional knockout females after the nodules were liberated from the rest of the
uterine horn to expose each embryo and the placenta (P). Severe hemorrhage are observed in nodules obtained from conditional knockout uteri,
whereas the control uteri appear do not show signs of hemorrhaging. Also, note that the placenta of the conditional knockout female have many
focal hematoma (H), and this is typically observed in these females. Of note, the hemorrhage observed in the pregnancy nodule originates from small
hemorrhages in the maternal tissues (M) surrounding the placenta and chorioallantoic membranes (CA) of the embryo. Once the maternal layers are
removed and the fetal chorioallantoic membranes are exposed, no hemorrhage is observed. Bars = 5 mm. n ≥ 3.
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TABLE 3.2: SINGLE OR EPITHELIAL-SPECIFIC RAC1 ALLELE DELETION DOES NOT IMPACT FERTILITY
Genotype # Females # Pups # Litters Average
Pups/Litter ‡
Pgr+/+
Rac1f/f
7 307 31 10
PgrCre/+
Rac1f/f
7 19 11 2
PgrCre/+
Rac1f/+
2†
76 12 6
Wnt7aCre/+
Rac1f/f
3 141 15 9
† The data shown represent the breeding performance over a six-month period. Different from the Rac1f/f
, Rac1
d/d, and Wnt7a
Cre/+ Rac1
f/f females, the
data collected for PgrCre/+
Rac1f/f
(heterozygous) females was from the current breeding colony, where these females are paired to PgrCre/Cre
Rac1f/f
or
PgrCre/Cre
Rac1f/+
males, not wild-type males.
‡
This table is for comparison purposes only. Because of the limitation in samples of heterozygous, appropriate statistical analysis could not be
performed. Data collected from Wnt7aCre females were collected at a time PgrCre and control females. Data from heterozygous females was
collected from the available data from the breeding colony, which at the time was experiencing breeding problems. Nonetheless, the data here show
that heterozygous females are fertile and capable of producing viable litters.
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FIGURE 3.11: PREGNANCY DEFECT OBSERVED IN RAC1 CONDITIONAL-NULL UTERI IS NOT DUE TO AN EMBRYONIC DEFECT. (A) To determine
if the potential expression of Pgr in the blastocyst is sufficient to produce any of the defects that are seen in Rac1d/d females, uteri were collected
from control (Pgr+/+ Rac1f/f) female mice on day 8 of pregnancy after they were mated with either homozygous Cre (PgrCre/Cre Rac1f/f), floxed (Pgr+/+
Rac1f/f), or wild-type (Pgr+/+ Rac1+/+) male mice. Homozygous Cre males bred to control females produce offspring that have the same genotype as
the conditional null mice (PgrCre/+ Rac1f/f), while wild-type males bred to control females, produce offspring that are similar to the experimental
conditions of the mating for this study (Pgr+/+ Rac1f/+). In all, the gestation nodules collected were normal, without any alteration to the expected
uterine gross morphology.
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FIGURE 3.11: PREGNANCY DEFECT OBSERVED IN RAC1 CONDITIONAL-NULL UTERI IS NOT DUE TO AN EMBRYONIC DEFECT (CONTINUED). (B)
Uteri in panel A were further dissected and representative implantation nodules, and the correspondent decidual masses and embryos are shown. No
morphological perturbations were observed. Bars = 1 mm. n ≥ 2 per genotype
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CHAPTER 4: RAC1 IS A CRITICAL REGULATOR OF
UTERINE ANGIOGENESIS DURING
EARLY PREGNANCY IN THE MOUSE
79
Abstract
In mice, following implantation, the uterine stromal cells differentiate into specialized decidual
cells, which support the growth of the embryo before placentation. Concomitantly, new blood
vessels begin to form in the maternal decidua from pre-existing vasculature through angiogenesis,
an absolutely critical event for the establishment of placenta and maintenance of pregnancy. It is
clear that decidualization and angiogenesis play crucial roles during early gestation, however, the
complex molecular pathways underlying these processes remain unknown. Our recent studies
revealed that Rac1 (Ras-related C3 botulinum toxin substrate 1), a pleiotropic signaling factor, is
induced in uterine stroma during decidualization and may play a role in regulating this process. To
address the role of RAC1 during pregnancy, we generated a conditional knockout of the Rac1 gene
in the uterus by crossing mice carrying floxed Rac1 with PgrCre mice. A six-month breeding study
indicated a severe defect in fertility of the Rac1-ablated (Rac1d/d) females. Further analysis showed
hemorrhagic uteri starting on day 7 of pregnancy and by day 10 of gestation, resorption of embryos
was clearly evident. An in depth analysis showed dysregulation of the stromal differentiation
program during decidualization in Rac1d/d uteri. Interestingly, the loss of Rac1 expression in
uterine stromal cells also resulted in a marked impairment in the development of maternal blood
vessels as indicated by the expression of PECAM1/CD31, Hif2α, Angpt2, and NRP1. This uterine
angiogenic defect is, presumably, one of the main contributors to pregnancy loss seen in Rac1d/d
mice at mid-gestation. Based on these results, we propose that RAC1 controls the differentiation
of stromal cells to decidual cells, which produce critical local factors that impact on blood vessel
formation at the maternal-fetal interface during early pregnancy.
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Introduction
Decidual neovascularization, the process by which new blood vessels originate from pre-existing
vessels, during the post-implantation period is an absolute requirement for pregnancy maintenance
in humans and rodents [73]. Indeed, it is thought that the newly formed uterine vasculature during
decidualization functions as an early site of nutrient and gas exchange for the developing embryo,
until the placenta becomes functionally competent [57, 74]. Although the details underlying the
process of decidualization and angiogenesis are slightly different between humans and rodents, the
basic sequence of events and many of the molecular pathways that regulate these processes are
similar between the two species.
In the pregnant uterus, angiogenesis is a critical process that precedes placentation, and requires
signals that originate from the embryo and the endometrium [1, 5, 9, 10, 29, 32, 75-77, 360].
Among the many signaling molecules, hypoxia-inducible transcription factor (HIF), vascular
endothelial growth factor (VEGF), and angiopoietins (ANGPTs) play major roles in endometrial
vascular remodeling during decidualization. Interestingly, hypoxia is a detectable feature in the
uterine environment during embryonic development [199-201]. Oxygen homeostasis is critical for
cell survival and in response to hypoxic conditions, cells activate the transcription of Hif1α to
restore oxygen homeostasis [206, 207, 327-334]. Activation of HIF stimulates Vegf expression,
which promotes angiogenesis [203, 205, 327, 329, 332, 333, 335]. Several lines of evidence
suggest that under hypoxic conditions, RAC1 regulates endothelial cell function by responding to
VEGF signaling [323]. Activation of the RAC1 pathway, using a dominant active form of RAC1,
has been shown to enhance VEGF-induced neovascularization, whereas inhibition of RAC1
activity prevents neovascularization in vitro [326]. Further, activation of RAC1 in embryonic
endothelial cells is critical for embryonic angiogenesis, and global mutations of RAC1 lead to
defective vascularization and embryonic death [277]. Interestingly, in other cell systems such as
pulmonary artery muscle cells, RAC1 has been shown to stimulate Hif transcription [202-204, 280,
325] via the PI3K-dependent pathway [202-204, 280]. Collectively, these studies raise the
interesting possibility that RAC1 may play a role in angiogenesis in decidual tissues by regulating
VEGF expression.
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To gain an insight into the pathways that are affected by the ablation of Rac1 in uterine stroma and
lead to termination of pregnancy, we performed gene expression profiling of decidual RNA
collected from Rac1-intact and Rac1-null uteri on day 8 of gestation. We focused on day 8 because
severe hemorrhage and embryo loss occur by day 10 of pregnancy, suggesting that the processes
that are leading to embryo demise are most likely arising on day 8 of pregnancy. Consistent with
RAC1’s role in angiogenesis, our studies revealed that RAC1 is a critical regulator of this process
in the uterus. Ablation of uterine RAC1 led to a dysregulation in vascular network formation in
the maternal decidual bed due to defective expression and secretion of pro-angiogenic factors.
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Materials and Methods
Animals and Tissue Collection
All animal breeding, housing, procedures, experiments, and euthanasia were approved and
conducted according to the guidelines of the Institutional Animal Care and Use Committee of
University of Illinois at Urbana-Champaign. The health and care of the animals was provided by
the veterinarians and staff of Division of Animal Resources. Rac1 conditional null mice were
generated using Cre-LoxP strategy [347] (Figure 3.1A). Mice expressing homozygous copies of
the Rac1 gene flanked by loxP sites [Pgr+/+ Rac1f/f (official gene symbol Rac1tm1Djk/J), or Rac1f/f
from now onwards] [267] were obtained from Dr. Ann Sutherland (University of Georgia) in a
C57BL/6-129SV background. Mice carrying the Cre Recombinase under the control of the
progesterone receptor (Pgr) promoter [348] [PgrCre/+ Rac1+/+ (official gene symbol Pgrtm2(cre)Lyd/+),
or PgrCre from now onward], were obtained from Drs. John Lyndon and Francesco De Mayo
(Baylor College of Medicine) in a C57BL/6 background. To establish the mouse colony for this
study, initial breeding between the two mouse lines was done to obtain the F1 progeny, which are
heterozygous for Pgr and Rac1 (Appendix A). Female mice used for this study were generated by
breeding male mice of specific genotypes from the F2 progeny (see Figure 3.1B) with Rac1f/f
female mice. Genotyping of the animals was performed following the protocols established by the
respective laboratories that generated the transgenic mice [267, 348] (Appendix A). Conditional
knockout female mice (PgrCre/+ Rac1f/f) will be referred to as Rac1d/d from now onwards.
Rac1f/f or Rac1d/d females were euthanized by CO2 asphyxiation, followed by a blood draw via
cardiac puncture, and cervical dislocation. Uteri from pregnant mice were collected at different
time points during pregnancy. The harvested organs were either fixed for histological evaluation
[immersion-fixed in 10% neutral-buffered formalin (NBF)], flash frozen in liquid N2 for RNA or
frozen sectioning, stromal cell isolation, or decidual mass was harvested for gene expression
studies. For samples collected at different time points during pregnancy, identification of a
copulatory plug following mating was used as a reference and for our experimental purposes, to
indicate day 1 of pregnancy.
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For microarray analysis, decidual nodules from day 8 of pregnancy were isolated following blood
perfusion, as previously described [380-382]. Briefly, day 8 pregnant mice were anesthetized with
ketamine-xylazine cocktail (87 mg ketamine; 15 mg xylazine / kg body weight). After the mice
were unconscious, a V-shape incision expanding radially to the chest was made so that the uterus,
liver, and ribs were visible. The chest cavity was opened and the heart was exposed. Immediately,
the mouse was perfused through the left ventricle of the heart, while the blood drained through the
right atrium, with at approximately 20 mL of Hank’s balanced salt solution containing heparin
(HBSS+H , 30 USP Units / mL) until the liver and uterine implantation nodules were visually
cleared of blood. Following perfusion, the uterine horns were dissected and placed in HBSS+H
for separation of the decidual mass. With the use of a dissection microscope (Leica M165 FC), the
uterine myometrium was mechanically removed with jeweler forceps and the decidual mass
containing the embryo was excised. Once all the masses were separated from the muscle, they
were transferred into a new 35 mm dish with fresh HBSS+H to remove any leftover blood and the
embryo. To remove the embryo, the isolated decidual mass was rotated so that one of the two
mesometrial indentations was visible. Then, using the indentation as a guide, the decidua was
carefully opened and the embryo was removed from antimesometrial side by carefully lifting it
out of the decidual mass. The decidual masses, freed from the embryos, were transferred to a new
35 mm dish with fresh HBSS+H for a final rinse before collected and flash frozen for further RNA
isolation. Four decidual masses/animal were collected for microarray analysis.
Microarray and expression analysis
Total RNA was extracted from the isolated and perfused decidual masses with TRIzol (Invitrogen)
following the manufacturer's protocol. The RNA preparations were then purified with the RNeasy
kit (Qiagen). Samples were stored in RNase-free water until further use. To evaluate the purity and
quality of the isolated RNA, the samples were submitted to the Roy J. Carver Biotechnology
Center (at the University of Illinois) for assessment of RNA quality and chip hybridization for
microarray analysis. Quality of the RNA samples were assessed using an Agilent Bioanalyzer
System and samples with a RNA integrity number of 10 were used for microarray analysis using
a GeneChip Mouse Genome 430A 2.0 Array (Affymetrix), containing approximately 14,000
annotated gene sequences. Subsequently, the chips were scanned and the data were extracted using
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Gene-Chip Operating Software version 1.3 (Affymetrix), which later were exported to Microsoft
Excel 2013 (Microsoft Corporation). Expression values for each gene below 50 was considered as
background. Relative gene expression fold value was determined by the ratio of gene expression
of Rac1d/d decidua to Rac1f/f decidua. A list of genes that had a relative fold change of +/- 1.5, were
further analyzed for gene ontology and functional classification using the Database for Annotation,
Visualization and Integrated Discovery (DAVID, National Institute of Allergy and Infectious
Diseases, National Institutes of Health) [383].
Quantitative PCR gene validation and RNA isolation
Total RNA was isolated from whole uterine samples, as reported above. RNA was converted to
cDNA using a kit (Applied Biosystems), according to the manufacturer’s instructions.
Oligonucleotides specific for genes of interest were developed (see Appendix F for details) and
real-time quantitative PCR (qPCR) reactions were carried out using SYBR-green master mix
(Applied Biosystems) on a 7500 Applied Biosystems Real-time PCR machine (Applied
Biosystems). Gene expression data analysis were done using the 2-ΔΔCt method [357] and 36b4
gene – which is conserved among many species and encodes an acidic ribosomal phosphoprotein
P0 that interacts with 40S ribosomal subunit – was used as a housekeeping gene and loading
control [358].
Immunohistochemistry, Immunocytochemistry, and Immunofluorescence
Cultured cells, paraffin-embedded or frozen-sectioned uterine sections were subjected to
immunohistochemistry or immunocytochemistry as described previously [32, 353, 355, 356].
Briefly, paraffin-embedded sections were deparaffinized in xylene and rehydrated in a series of
graded alcohols followed by rinsing in tap water. When frozen tissues were used, the sections were
thawed at room temperature for 5 min, then fixed for 5 min in 10% NBF at room temperature. If
cells were used, they were fixed for 5 min in 10% NBF at room temperature. Sections or cells were
then rinsed in 1x phosphate-buffer saline (PBS), followed by serum blocking using 10% non-
immunized serum for 1 h at room temperature. Following serum block, slides were incubated with
a solution containing the diluted antibody (see Appendix E for details) and 1% serum in 1x PBS
85
overnight at 4 oC in a humidified chamber (for slides) or in the culture plate (for cells). Following
overnight incubation, slides were washed 3 times, 5 min each, with 1x PBS, then incubated with
diluted secondary antibody (see Appendix E for details) for 1 h at room temperature. When
chromogenic labeling was done, the samples were incubated in a solution containing 3% H2O2 in
methanol to quench endogenous peroxidase. Samples were then rinsed in 1x PBS, followed by a
30-45 min incubation with horseradish peroxidase-conjugated streptavidin (Invitrogen). The
samples were then exposed to chromogens (3, 3’-diaminobenzidine or 3-amino-9-ethylcarbazole)
until optimal signal was obtained. Following the chromogenic labeling, the samples were
counterstained with hematoxylin and mounted. When immunofluorescence was done, the samples
were incubated with a fluorescent secondary antibody were mounted with a medium containing
DAPI. Pictures were taken using the Olympus BX51 microscope equipped for light and fluorescent
imaging and was adapted to a Jenoptik ProgRes C14 digital camera with c-mount interface
containing a 1.4 Megapixel CCD sensor or an Olympus Ix70 inverted microscope adapted to
Diagnostic Instrument digital camera containing a 2.0 Megapixel CCD sensor. Fluorescent images
were merged and processed using CS4 Adobe Photoshop Extend (Adobe Systems).
Primary endometrial stromal cell isolation and culture
Endometrial stromal cells were isolated, from three independent mice per genotype on day 4 of
pregnancy as previously described [164, 352]. Briefly, uteri were removed from mice after
euthanasia with CO2. The uteri were flushed with 200 – 300 µL of HBSS to remove embryos and
to confirm pregnancy. Then, the uteri were cut open and digested with a solution containing 6 g/L
dispase (Gibco) and 25 g/L pancreatin (Sigma) in 1x HBSS (Gibco) for 45min at room
temperature, followed by 15 min at 37 oC, with occasional mixing. The enzyme activities were
quenched by adding equal volumes of 10% FBS solution, then cells were washed three times with
1x HBSS (Gibco). Cells were then incubated in 0.5 g/L collagenase (Sigma) in HBSS for 1 h at
37 oC. After incubation, the tubes were vortexed for 10-12 seconds until the supernatant became
turbid with dispersed cells. The contents of the tube were then passed through an 80-μm gauze
filter (Millipore) into a new tube. Cells were resuspended and plated for cell culture in Dulbeccos
modified Eagle's medium-F12 medium (Gibco) supplemented with 2% heat-inactivated fetal calf
serum, 100 units/L penicillin, 0.1 g/L streptomycin (Gibco), 1.25 mg/L fungizone, 10 nM E2, and
86
1 µM P4. The numbers of live cells were assessed by trypan blue staining using a hemocytometer.
Cells were plated for 2 hours at a density of 5 x 105 cell/well in 6-well culture plates to allow
stromal cells, but not epithelial cells, to attach. After the 2 hour incubation (0 h of culture), the
medium was aspirated and the cells were rinsed with two to three changes of fresh media, until
most of the unattached cells were removed. Media were again changed at 24 h (and stored for a
baseline reading) and 48 h after the initial change (0 h). Following the 48 h media change, the cells
were cultured for up to 96 h from the initial plating (0 h), and media were collected in 24 h
intervals. The media from three-wells were pooled and collected at 24, 72, and 96 h time point,
and the cellular debris was removed via centrifugation, and stored for ELISA. In addition, the cells
were collected by rinsing with HBSS followed by incubation with 0.25% trypsin at 37 oC until the
cells detached. The cells and media were stored at -80 oC until needed. The numbers of live cells
were determined using trypan blue staining and a hemocytometer, then, the cells were placed in
TRIzol reagent (Invitrogen) for further RNA isolation.
Measurement of vascular-endothelial growth factor A (VEGFA) in culture medium
Measurements of VEGFA production in vitro were done from pooled media from three wells (from
a 6-well plate, Corning) from at least three independent cell cultures (one culture = one animal),
plated at an initial density of 5x105 cell/well. Enzyme-linked immuno sorbent assay (ELISA) for
mouse VEGF were done according to the manufacturer recommendations (R&D Systems).
Statistical analysis
Experimental data were collected from a minimum of three independent animals (not from the
same litter), which were subjected to the same experimental conditions. ELISA and qPCR results
were expressed as mean ± S.E.M. Statistical analysis was done using Student’s t-test. An analysis
of equal variances was done in all numerical data to determine whether a parametric or non-
parametric hypothesis test was appropriate. Data were considered statistically significant if p ≤
0.05, and is indicated by an asterisks in the figures. All data were analyzed and plotted using
GraphPad Prism 4.0 (GraphPad Software) or Microsoft Excel 2013 (Microsoft Corporation).
87
Results and Discussion
Microarray analysis reveals impaired angiogenic network in Rac1-ablated uteri
We previously established that the severe subfertility phenotype observed in the Rac1d/d female
mice was due to a defect in maternal environment. Our studies also revealed that embryo loss
occurred between days 10 to 12 of pregnancy in Rac1d/d females, suggesting that the disruption of
events preceding these dates are critical for maintenance of gestation. During this window, several
biological processes are known to occur in the uterus. These include ongoing differentiation of the
decidual cells, extensive vascular remodeling and angiogenesis [23, 31, 57, 166, 167], trophoblast
proliferation and invasion into the endometrium, as well as formation of the placenta [5, 9, 28, 78,
112, 126, 128, 129, 131-133] (Figures 1.4 and 1.5). We employed the microarray approach to
identify the genes and the regulatory pathways that might be affected by Rac1 deletion in the
endometrium. The microarray analysis was performed by isolating RNA from decidua of Rac1-
intact and Rac1-null uteri on day 8 of pregnancy.
Out of the approximately 14,000 well-characterized genes that were analyzed in the 430A
microarray chip, 1345 genes were differentially regulated (± 1.5 fold). Out of the differentially
regulated genes, 796 were down-regulated in Rac1-null decidua compared to Rac1-intact controls.
Gene ontology was performed by using NIH DAVID for analysis [383]. We identified multiple
biological processes, including angiogenesis, which appeared to be regulated by RAC1 in the
decidua during pregnancy (Figure 4.1).
The expression of several genes that are known to play major roles in neovascularization in the
endometrium during early pregnancy were affected by the loss of RAC1 [32, 194, 384]. Validation
of the microarray data showed that expression of these genes including hypoxia-inducible factor 2
alpha (Epas1/Hif2α) [207, 385], angiopoietin 2 (Angpt2), and Angpt4 were markedly reduced in
the absence of uterine expression of Rac1 (Figure 4.2). The expression of other genes involved in
angiogenesis, including sphingosine kinase 1 (Sphk1), neuropilin 1 (Nrp1), and the clotting factor
XIII subtype A1 (F13a1) were also attenuated by the loss of uterine Rac1 (Figure 4.2).
Intriguingly, however, the expression of hypoxia-inducible factor 1 alpha (Hif1α) and vascular-
88
endothelial growth factor A (Vegfa), which are well known regulators of angiogenesis (Figure 4.3),
remained unchanged. Nonetheless, the significant alterations in transcription of genes associated
with uterine angiogenesis led to a marked reduction in vascular network formation, as noted by
the reduced expression of platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) on day
8 (Figure 4.4A) or day 10 of pregnancy (Figure 4.4B).
Whereas the expression of Vegf was unaltered, we observed a marked downregulation of
angiopoietin 2 in the absence of RAC1 in the uterine stroma. Angiopoietins, acting on the
angiopoietin receptor, TEK/TIE2, work along with VEGF to promote new blood vessel formation
from pre-existing ones [30, 58, 63, 64, 72, 180, 184, 197]. Interestingly, decidual tissues of women
with recurrent miscarriages show imbalance in the expression ratio of Angpt2 and Angpt1, to favor
Angpt1 rather than Angpt2 [63], which is predominant during uterine angiogenesis. ANGPT2 plays
a role in branching angiogenesis, which may be involved in the development of complex vascular
network seen in the uterus during pregnancy [58, 63, 64]. Although the precise role of
angiopoietins in uterine angiogenesis remains unknown, previous studies have shown that genetic
deletion of Angpt2 causes capillary dysmorphogenesis in the kidney [386], as well as abnormal
patterning of the blood vessels of the retina in mice [387].
The actions of VEGF are mediated through several receptors, primarily VEGFR1/FLT1 and
VEGFR2/ FLK1. VEGFs can also activate neuropilin receptors including NRP1 [168, 178, 184,
185]. Our studies revealed that along with Angpt2, neuropilin 1 gene and protein expression was
severely compromised in the absence of uterine Rac1 (Figures 4.2 and 4.5). Previous studies have
shown that in response to VEGF stimulation, NRP1 plays a role in guidance of neuronal and
endothelial cells during the process of neurogenesis, which is usually accompanied by
angiogenesis [388, 389]. Studies have also shown that mutant embryos, deficient for Nrp1, display
disorganization of the vasculature, without affecting the formation of primary vascular network or
differentiation of endothelial cells [390]. The fact that Nrp1 is significantly downregulated in the
decidua (Figure 4.5), suggests that migration and proliferation of endothelial cells might be
affected in the uteri of Rac1d/d mice. It is possible that during decidualization, RAC1-dependent
pathways in the maternal stroma are involved in the regulation of endothelial cell reorganization.
89
Ablation of Rac1 in the uterus leads to a decreased secretion of pro-angiogenic factors
To gain insights into the underlying mechanisms of the angiogenic defect observed in Rac1d/d
females, we used a well-established in vitro cell culture system of decidualization [164, 352].
Stromal cells isolated from Rac1-intact and Rac1-null uteri cultured in vitro showed distinct
differences in morphology and architecture as indicated by staining for F-actin using phalloidin
(Figure 3.7). It is known that actin cytoskeletons regulate certain aspects of vesicle trafficking and
protein secretion [248, 255, 257, 260, 308, 314, 316, 391, 392]. Indeed, one of the described
functions of RAC1 is to promote the formation polymerization of actin fibers to regulate vesicle
transport [247, 255, 263, 308, 314, 391]. We, therefore, explored the possibility that RAC1 may
be involved in secretion of proteins such as VEGFA in decidual cells. We analyzed the protein
expression of VEGFA by immunocytochemistry in cultured mouse endometrial stromal cells that had
been subjected to decidualization in vitro. Our results show that Rac1-null cells display an impaired
vesicular transport of VEGFA. Stromal cells from conditional knockout uteri displayed an
accumulation of VEGFA in the cellular compartment where Golgi/rER are normally located within the
cell (Figure 4.6A). Consistent with this observation, measurement of VEGFA in the supernatant of
mature decidual cells revealed a significant reduction in VEGFA production and secretion in Rac1-
null cells (Figure 4.6). Vegfa gene expression, however, was similar in Rac1-intact and Rac1-null
stromal cells. The precise stage at which RAC1 regulates the secretory activity of decidual cells
warrants further investigation. However, previous data has implicated RAC1 in trafficking secretory
vesicles from the Golgi to the plasma membrane [308, 310, 314]. Thus, it is highly possible that
RAC1’s actions are at the final stages of vesicular transport.
It is believed that RAC1 is not involved in the early stages of secretory pathway (i.e. rER to Golgi)
[310], nor has it been localized to the rER membrane. However, RAC does play a role in the later
stages of secretion. Recently, PREX1, a PI3K-dependent Rac exchange factor, has been identified
as a novel regulator of the glucose transporter 4 (SLC2A4/GLUT4) trafficking in adipocytes.
PREX1 can activate RAC1, and this in turn stimulate actin polymerization and facilitate
SLC2A4/GLUT4 transport to the plasma membrane [314]. Additionally, RAC1 has also been
reported to regulate exocytosis in a calcium-dependent manner [316, 318, 392]. The precise
mechanisms and the cellular compartments in which RAC1 elicits its effect to regulate exocytosis
90
is still controversial. However, it is clear that actin polymerization plays a critical role in this
process.
Conditional deletion of Rac1 in the uteri leads to severe hemorrhage
Despite a severe reduction of the vascular network on day 8 of pregnancy, we consistently observed
an increase in blood hemorrhage in Rac1-null decidua, especially around the lateral sinusoids (Figures
2.6 and 3.7). Using eosin-Y with phloxine to labels red blood cells, we noticed that the cells were
extravascular, indicative of vascular leakage and hemorrhage (Figure 4.7). Whereas Rac1f/f decidual
blood was mostly cleared following blood perfusion, Rac1d/d decidua consistently failed to clear,
indicative of hemorrhage (Figure 4.8). Interestingly, sphingolipid signaling has been shown to prevent
vascular leakage during uterine angiogenesis [377]. The fact that members of the sphingolipid pathway
such as Sphk1 are down regulated in the Rac1d/d uteri, suggests that attenuation of sphingolipid
pathways functioning downstream of RAC1 may be involved in the vascular leakiness observed in
Rac1d/d uteri (Figure 4.2). In addition to factors in the sphingolipid pathway, tight junction proteins in
endothelial cells are known to regulate vascular permeability [277]. Interestingly, our microarray
analysis identified a tight junction molecule such as claudin 3, whose expression was significantly
downregulated in the absence of uterine Rac1 (Figure 4.2). Claudin 3 (CLDN3) has been shown to be
present in the blood-brain barrier and loss of Cldn3 in the endothelial cells compromises the integrity
of the blood-brain barrier [393]. Exactly how stromal RAC1 regulates claudins in the endothelial cells
remains to be determined, but it is known that decidual cells secrete factors that affect endothelial cell
function [30, 57, 170, 177, 178, 194, 198, 207].
It is also possible that the severe hemorrhage observed in Rac1-null uteri is because of the reduction
in the expression of F13a1 – the gene coding for coagulation factor XIII subtype A1, which is the last
zymogen to become activated in the blood coagulation cascade and is the factor responsible for
crosslinking fibrin molecules that stabilize the blood clot. F13a1 global knockout female mice are
available and are able to get pregnant. However, breeding studies of these mice show that a severe
uterine hemorrhage develops around day 10 to 12 of pregnancy, which often leads to maternal death
[384]. Interestingly, there are also loss-of-function mutations in the human isoforms of F13 (A and B
subunits) [394]. It has been reported that certain women with recurrent miscarriage possess one or both
of these mutations. The fact that in Rac1d/d mice, there is a significant reduction in this enzyme,
91
suggests that RAC1 signaling may be crucial for F13A1 production and/or secretion in the uterus
during early pregnancy.
92
Chapter Figures
FIGURE 4.1: BIOLOGICAL PROCESS LIST OF GENES THAT WERE DOWN-REGULATED LESS THAN 1.5 FOLD ON MICROARRAY. Gene list shown
here is a condensed list of genes that were clustered by biological by NIH DAVID. The gene list is based on 576 unique gene probes out of which
259 gene probes were charted based on their known function within a biological process. The size of the pie slices are representative of the number
genes within each biological process. Some gene probes are contain within more than one biological process. Not all biological process are listed in
this pie chart.
93
FIGURE 4.2: CONDITIONAL DELETION OF RAC1 IN THE UTERUS LEADS TO DOWN-REGULATION OF SEVERAL GENES THAT ARE CRITICAL FOR
UTERINE ANGIOGENESIS. Gene expression analysis of day 8 of pregnancy uterine samples confirms the microarray analysis, which showed that
critical genes involved in uterine vascularization and angiogenesis during pregnancy are critically reduced in Rac1d/d compared to Rac1f/f uteri. Genes
such as Epas1/Hif2α, Angtp2, and Nrp1 are typically expressed during vascular remodeling in the pregnant uterus. n ≥ 3. Asterisks indicate significant
differences from controls (* p < 0.05, ** p < 0.01, *** p < 0.001).
Vegfr1/Flt1
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Vegfr2/Flk1
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Pecam1/Cd31
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Epas1/Hif2
Rac1f/f
Rac1d/d
0
1
2
*
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Angpt1
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Angpt2
Rac1f/f
Rac1d/d
0
1
2
**R
ela
tiv
e G
ene
Ex
pre
ssio
n
(2-
Ct )
Angpt4
Rac1f/f
Rac1d/d
0
1
2
***
Rela
tive G
en
e E
xp
ress
ion
(2-
Ct )
Sphk1
Rac1f/f
Rac1d/d
0
1
2
*
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Nrp1
Rac1f/f
Rac1d/d
0
1
2
*
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Cldn3
Rac1f/f
Rac1d/d
0
1
2
*
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
F13a1
Rac1f/f
Rac1d/d
0
1
2
*R
ela
tiv
e G
ene
Ex
pre
ssio
n
(2-
Ct )
Mmp9
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
94
FIGURE 4.3: EXPRESSION OF TWO WELL-KNOWN GENE TARGETS OF RAC1 ARE UNAFFECTED. Under hypoxic conditions, RAC1 has been shown
previously to regulate the expression of Hif1α, indirectly leading to an increased production of Vegfa gene expression, via HIF1α. Interestingly, in
the uterus, conditional deletion of Rac1 does not affect the expression of these two genes on day 8 of pregnancy. n ≥ 3. (p > 0.05).
Hif1
Rac1f/f
Rac1d/d
0
1
2
Rela
tiv
e G
en
e E
xp
ress
ion
(2-
Ct )
Vegfa
Rac1f/f
Rac1d/d
0
1
2
Rela
tiv
e G
en
e E
xp
ress
ion
(2-
Ct )
95
FIGURE 4.4: SUPPRESSION OF THE RAC1 PATHWAY COMPROMISES THE FORMATION OF THE DECIDUAL VASCULAR PLEXUS. (A and B) The
extent of the angiogenic defect was evaluated using PECAM1/CD31, which is a general marker of endothelial cells and is labeled by the red staining.
Our results show a dramatic reduction on blood vessel density in Rac1d/d uteri on days of pregnancy 8 (panel A) and 10 (panel B). A diagrammatic
representation of the vascular network of the implantation nodules is shown to aid in the visualization (adapted and modified with permission form
[155]). E = embryo, AM = anti-mesometrial decidua, M = mesometrial decidua, LS = lateral blood sinusoids, BZ = basal zone, CS = circular smooth
muscle zone, CZ = compact zone, DC = decidua capsularis, P = Placenta, L = luminal epithelium, LDZ = lateral decidual zone, MY = myometrium,
MG = metrial gland, MT = mesometrial triangle.
96
FIGURE 4.4: SUPPRESSION OF THE RAC1 PATHWAY COMPROMISES THE FORMATION OF THE DECIDUAL VASCULAR PLEXUS (CONTINUED). (A
and B) The extent of the angiogenic defect was evaluated using PECAM1/CD31, which is a general marker of endothelial cells and is labeled by
the red staining. Our results show a dramatic reduction on blood vessel density in Rac1d/d uteri on days of pregnancy 8 (panel A) and 10 (panel B).
A diagrammatic representation of the vascular network of the implantation nodules is shown to aid in the visualization (adapted and modified with
permission form [155]). E = embryo, AM = anti-mesometrial decidua, M = mesometrial decidua, LS = lateral blood sinusoids, BZ = basal zone, CS
= circular smooth muscle zone, CZ = compact zone, DC = decidua capsularis, P = Placenta, L = luminal epithelium, LDZ = lateral decidual zone,
MY = myometrium, MG = metrial gland, MT = mesometrial triangle. n ≥ 3.
97
FIGURE 4.5: CONDITIONAL DELETION OF UTERINE RAC1 IMPAIRS ANGIOGENESIS. Expression of neuropilins in the uterus has been implicated in
the regulation of angiogenesis. Here, we show that neuropilin 1 (NRP1) expression is dramatically reduced in Rac1d/d decidua on day 8 of pregnancy.
PECAM1, a marker for endothelial cells, is used to highlight the vascular tree. Lower left inset on the panel shows no primary antibody control. In
panel B, high magnification images of the vascular plexus (boxed areas 1 and 2) are shown to the right of each image. E = embryo, AM = anti-
mesometrial decidua, M = mesometrial decidua, LS = lateral blood sinuses (also known as vascular web). Bars = 500 µm. n ≥ 3.
98
FIGURE 4.6: CONDITIONAL DELETION OF RAC1 IMPAIRS VEGFA TRANSPORT AND SECRETION OF UTERINE ENDOMETRIAL CELLS. VEGFA
immunocytochemistry done in decidual cells that were cultured for 72 h in differentiation media are shown here. In all instances, we stained 72 h
cultured cells, VEGF accumulated in the perinuclear region of the cell, whereas in the control cells VEGF vesicles were either uniformly distributed
in the cytoplasm or the number was reduced, presumably, due to secretion. Gene expression analysis of the differentiation marker Prl8a2/dPrp, as
well as Vegfa, shows no change in expression levels between the experimental and control groups. n = 3. Asterisks indicate significant differences
from controls (*** p < 0.001).
99
FIGURE 4.7: RAC1D/D UTERI DISPLAY EXTRAVASATION OF RED BLOOD CELLS. Extravasation and vascular leakage was confirmed in Rac1d/d mice
by staining uterine sections on day 8 of pregnancy with eosin-Y containing phloxine. Here, we show that despite a severe reduction in blood
vasculature in the conditional null uteri, blood hemorrhage, and blood vessel leakiness is observed (inset box). Dotted lines demarcate the blood
sinuses. Upper left inset on panel shows high magnification images of the boxed area near the lateral sinuses. E = embryo, AM = anti-mesometrial
decidua, M = mesometrial decidua, LS = lateral blood sinuses (also known as vascular web). Bars = 500 µm. n = 4.
100
FIGURE 4.8: DECIDUA FROM RAC1D/D MICE SHOW SEVERE BLOOD HEMORRHAGE IN THE LATERAL BLOOD SINUSES. Representative images of
uterine samples following blood perfusion, as compared to a non-perfused Rac1f/f control uterus. The upper images in the panel show three isolated
implantation nodules, including myometrium. The lower images in the panel show the isolated decidua (rotated 90o from the position observed in
the upper images of the panel). Here, we show that Rac1-null decidua fail to clear the vascular sinuses, as control decidua does, following vascular
perfusion with saline containing heparin. All samples shown here were evaluated on day 8 of pregnancy. n = 3.
.
101
CHAPTER 5: CONCLUSIONS AND WORKING
MODEL
102
The work presented as part of this dissertation reveals novel roles for RAC1 in the uterus during
pregnancy (Figure 5.1). Our studies have shown that in uteri lacking RAC1, the embryos are able
to attach to the epithelium to initiate implantation. However, a six-month breeding study showed
that female mice with a null mutation of Rac1 in the uterine tissues became severely subfertile,
and towards the end of the study became sterile. Although we have not unraveled the complete
repertoire of the pathways affected by this mutation, we were able to identify certain critical events
in pregnancy that were severely compromised in response to this mutation. For example, we found
that mutation of uterine Rac1 led to dysregulation of genes that are involved in endometrial stromal
cell differentiation. Because decidualization involves stromal cell proliferation followed by
differentiation and RAC1 has been reported previously to regulate proliferation in other systems
[367], it is of interest to determine the impact of RAC1 ablation on proliferation of stromal cells.
Our studies revealed no significant difference in cell proliferation of stromal cells in Rac1f/f and
Rac1d/d uteri. However, a marked upregulation of Alpl and Wnt4 and a downregulation of
Prl3c1/Plpj gene expression were observed in Rac1-ablated uteri, indicating dysregulated
differentiation program in Rac1d/d mice during decidualization. The expression of certain other
genes known to be critical for stromal differentiation such as Bmp2, Gjp1/Cx43, or Vegfa were not
affected by the deletion of Rac1. It is possible that these genes are functioning independent of
RAC1 pathway in the uterus. It is also possible that RAC1 regulates the post-transcriptional events
and is involved in the secretion of some of these factors, including VEGFA.
A consequence of impaired stromal differentiation in Rac1-null uteri is a defect in uterine
angiogenesis and blood vessel stability. Gene expression profiling of the decidual tissues showed
that expression of many genes that are necessary for uterine angiogenesis, such as Hif2α, Anpt2,
Angpt4, and Nrp1, were significantly downregulated in Rac1-ablated uteri. Interestingly, however,
Hif1α, a known target of RAC1 during hypoxic conditions [319-322, 324, 395-399], was not
affected upon uterine deletion of Rac1. It is possible that in decidual uteri, Hif2α rather than Hif1α
is functioning downstream of the RAC1 pathway to regulate angiogenesis. Consistent with these
observations, we noted impaired vascular plexus formation in Rac1d/d uteri.
Immunostaining with PECAM1/CD31 showed a noticeable reduction in the vascular tree in
Rac1d/d uteri. Absence of blood clearance following perfusion of the blood vasculature clearly
indicated unstable and leaky blood vessels in uterine tissues of Rac1d/d females. Interestingly,
103
disruption of endothelial cell network, vascular breakage, and hemorrhage during early pregnancy
has been reported previously in female mice deficient for Sphk1 gene. Consistent with this
observation, we noted a marked reduction in the expression of Sphk1 in the Rac1d/d uteri. Because
the suppression of the sphingolipid pathway was sufficient to cause significant alterations in the
blood vasculature in the Sphk1 mutant mice [377], it is possible that similar events could be
occurring in the Rac1d/d uteri. Furthermore, we noted with interest that the expression of F13a1, a
gene involved in clotting and hemorrhage, was reduced in the Rac1d/d uteri, further supporting the
notion of a compromised vascular tree in these mutant animals.
Interestingly, the expression of Vegfa was not altered in the uteri of Rac1d/d females. VEGFA,
acting through VEGF receptors (VEGFR1, VEGFR2, and NRP1) and in conjunction with the
ANGPT system, coordinates many important processes that ultimately lead to vascular remodeling
[178, 184, 390, 400]. Gene expression analysis on day 8 of pregnancy showed a marked reduction
in the expression of Angpt2 and Nrp1 in the uterine tissue. Both ANGPT2 and NRP1 have been
implicated in uterine angiogenesis [30, 57, 72, 178, 195, 198, 400, 401]. The fact that these two
molecules are downregulated in the Rac1d/d uteri further supports the angiogenic defect observed
in these animals. Additionally, VEGF, in the absence of the ANGPT system can lead to abnormal
vascular leakage and blood vessel instability [184]. It should be noted, however, that we made one
critical observation with regard to VEGFA secretion in Rac1-deficient decidual cells. These cells
displayed a marked reduction in VEGFA secretion compared to Rac1-intact stromal cells.
However, besides decidual cells, trophoblasts [57, 62, 69, 186, 402] and uterine natural killer cells
[360, 403, 404] can also contribute to the production and secretion of VEGFA, which can lead to
dysregulated VEGFA signaling. Taken together, we are of the view that severe vascularization
defect observed in the Rac1d/d female mice is the primary cause of embryo loss by mid-gestation.
It has been recognized for a long time that the differentiated decidual cells secrete a variety of
factors that are important for the growth and development of the implanted embryo [23-25, 29, 91,
121-123]. However, the molecular mechanisms by which the decidual cells undergo secretory
activity remain largely unknown. In this study, we show for the first time that primary decidual
cells from Rac1d/d uteri display reduced VEGFA secretion, despite no impairment in VEGFA
synthesis and initial packaging into secretory vesicles. It is possible that decidual cells utilize
RAC1-dependent actin polymerization for vesicle transport. CDC42, like RAC1, can induce actin
104
polymerization and has been involved in vesicle secretion. However, CDC42 is known to regulate
the earlier events of vesicle trafficking (i.e. rER to Golgi), whereas RAC1 seems to regulate the
later stages of trafficking (i.e. Golgi to plasma membrane) [310]. Nonetheless, it is clear that a
RAC1-dependent secretory function of decidual cells is important for embryo survival and
maintenance of pregnancy.
Studies aiming to understand the underlying reasons for failures in implantation and placental
development are clinically important. Indeed, about one-third of normal human pregnancies end
in spontaneous abortion, and a fair percentage of these pregnancies will be lost before they are
detected clinically [12-14]. Similarly, failure in embryonic development, due to defects in the
embryo or the maternal environment, account for almost 80% of the embryonic loss that occurs in
farm animal species [405-407]. In humans, abnormalities in the vascular connections result in
preeclampsia and/or IUGR, diseases that account for significant morbidity and mortality to both
the mother and fetus [12, 44, 51, 59, 62]. Therefore, research aimed to understand the physiological
and pathological events that lead to pregnancy complications are required to further our
understanding of this complicated process. Studies described in this thesis suggest that
disturbances in RAC1-dependent pathways may be relevant to humans with recurrent pregnancy
failures.
105
Chapter Figure
FIGURE 5.1: WORKING MODEL FOR THE ROLE OF RAC1 IN THE UTERUS DURING EARLY EVENTS OF PREGNANCY. Activation of RAC1 in the
endometrium during early events of pregnancy results in a series of signaling cascades that ultimately: 1) regulate gene expression of factors
responsible for stromal cell differentiation, as well as angiogenesis and vascular stability; 2) induce changes in actin dynamics to promote vesicle
transport from the rough endoplasmic reticulum (rER) to the plasma membrane, as well as the secretion of the protein content to the extracellular
matrix. Finally changes in actin dynamics also promote 3) cell migration to establish an intimate relationship with the embryo. All these events are
necessary to ensure embryo survival and pregnancy maintenance.
106
CHAPTER 6: FUTURE DIRECTIONS
107
The work described in this dissertation uncovered new roles for RAC1 in uterine tissue and raised
interesting questions related to maternal-fetal interactions during pregnancy. RAC1, a pleotropic
signaling molecule, is known to be involved in various biological processes. We have observed
some of the critical functions displayed by RAC1 in the uterus during early pregnancy.
During the exploration of the Rac1d/d mouse model, we made a few critical observations that might
be of clinical importance. We noted that implanted embryos displayed a dysregulated trophoblast
expansion by day 8 of pregnancy (Appendix C, Figure C3.1). Although we do not know what
drives trophoblast cells to over expand in the uterus of the conditional null mice, it is our hypothesis
that the overexpansion of the trophoblast cells is most likely linked to the inherent impairment of
Rac1-deficent stromal cells. Our preliminary wound-healing assay (data not shown) showed that
Rac1-deficient stromal cells exhibit migratory impairments. Furthermore, our studies have
revealed that the Rac1-null decidual cells have an overall secretion defect. Therefore, it is possible
to assume that the dysregulated trophoblast expansion could be the result of a combinatorial effect
arising from maternal deficiencies in migration and secretion.
In an attempt to gain insights into the potential pathways that might be dysregulated in the decidua,
we performed gene expression analysis of known molecules that have been implicated in
regulating trophoblast migration (Appendix C, Figure C3.2). Whereas IGF2 is secreted from the
blastocyst to promote trophoblast migration, IGFBPs, secreted from the decidual cells, are thought
to interfere with this process. Our preliminary data show an upregulated expression of Igbp4, while
gene expression of adrenomedullin (Adm) and Igfbp3 is not changed. Both Igfbp4 and Adm have
been suggested to regulate trophoblast migration. In particular, high expression of IGBP4 has been
found in women with recurrent miscarriage. Because decidual cells deficient for RAC1 exhibit a
defect in secretory activity, it is possible that certain factors critical for proper functioning of the
mother and the fetus are absent in Rac1-null uteri. Thus, it would be important to determine if
other factors besides VEGFA exhibit impaired secretory activity. It is clear that the secretory
functions of the decidual cells are crucial to maintain the embryo under control in the proper
cellular compartment. Any perturbation in this process and a dysregulation in the migratory
potential of the trophoblast cells can have serious consequences to the outcome of pregnancy.
108
Thus, elucidating how decidual cells precisely regulate trophoblast invasion has great clinical
significance.
Intrauterine fetal growth restriction (IUGR) is another area of great clinical importance. It is now
widely accepted that the exposure of developing embryo to the environment can have severe
consequences on the metabolic phenotype of an organism without alterations in the genomic
sequence. Epigenetic modifications caused by the nutritional environment of the developing
embryo have gained great interest recently because of the obesity epidemic that is affecting the
USA. Interestingly, our preliminary observations of implantation nodules obtained on day 15 of
pregnancy, suggest that the Rac1-deficient uterine environment may not be fully conducive to
proper embryonic growth. When implantation nodules were dissected, we noticed growth
retardation in the embryos we were able to collect (Appendix C, Figure C3.3B). Although we have
a small sample size, we did notice a strong trend towards IUGR, as assessed by the weight of the
embryos from Rac1f/f and Rac1d/d mice. Additionally, the placentas obtained from these growth-
retarded embryos displayed abnormal shapes, and were significantly heavier than the control
placenta (Appendix C, Figures C3.3B to C3.3D). Consistent with the phenotype associated with
IUGR, implantation nodules collected on day 12 of pregnancy displayed abnormalities in blood
vasculature. Further, uterine samples from Rac1d/d mice showed intermediate to no modifications
of spiral artery, which is detrimental to maintenance of pregnancy (Appendix C, Figure C3.4).
Further studies should establish whether the lack of spiral artery modification combined with the
vascular defect is driving embryonic loss at mid-gestation in the Rac1d/d mouse. We anticipate that
further studies in each of these areas will provide an in depth knowledge of function of RAC1 in
the uterus at distinct stages of early pregnancy. Ultimately this knowledge will be useful in
developing treatment options for women with pathologies associated with pregnancy, including
recurrent miscarriage and IUGR.
109
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APPENDIXES
135
APPENDIX A: BREEDING STRATEGIES FOR THE FOUNDER AND EXPERIMENTAL
COLONY
FIGURE A.1: BREEDING STRATEGY FOR GENERATING THE RAC BREEDING COLONY. Transgenic mice
carrying the PgrCre knockin mutation were generously provided by Drs. Franco De Mayo and John Lyndon,
Baylor College of Medicine, TX. Mice carrying the knockin floxed mutation for Rac1 gene were generously
provided by Dr. Ann Sutherland, University of Georgia, GA. To establish the breeding colony, animals
from F1 were bred to each other. Only a select group of animals from the resulting F2 progeny (shown in
the figure above) were used to generate the experimental animal.
136
FIGURE A.2: EXPECTANCY FREQUENCIES OF ANIMALS/GENOTYPE BASED ON THE BREEDING
STRATEGY FOR GENERATING THE FOUNDING ANIMAL COLONY. The table shows the expected frequency
of animals in the F2 progeny, by genotype and sex, resulting from the mating of F1 male with F1 female.
Expectancy
Expectancy
by Sex
PgrCre/Cre
Rac1f/f 1/16 1/32
PgrCre/Cre
Rac1f/+ 1/8 1/16
PgrCre/+
Rac1f/f 1/8 1/16
PgrCre/+
Rac1f/+ 1/4 1/8
PgrCre/Cre
Rac1+/+ 1/16 1/32
PgrCre/+
Rac1+/+ 1/8 1/16
Pgr+/+
Rac1f/f 1/16 1/32
Pgr+/+
Rac1f/+ 1/8 1/16
Pgr+/+
Rac1+/+ 1/16 1/32
f
Cre/Cre
f/+
Cre/+
f/+
Cre/Cre
f/f
Cre/Cre
f/+
Cre/+
f/f
Cre/+
f/+Cre f
+
f
+
Cre Cre + +
Mal
e A
llel
es
PgrC
re/+
Rac1
f/+
Female Alleles
PgrCre/+
Rac1f/+
Pgr
Cre/+
+/+
+/+
f/+
+/+
+/+
Rac
Cre
+
+
Cre/Cre
+/+
Cre/+
f/+
Cre/+
+/+
Cre/+
f/f
Cre/+
f/+
+/+
f/f
+/+
f/+
+ f
Frequency
+
137
FIGURE A.3: BREEDING STRATEGY FOR GENERATING THE EXPERIMENTAL ANIMALS. The experimental
animals for the project animals were obtained by mating mice from the F2 progeny using the scheme above.
The tables on the next page show the expected frequency of animals, by genotype and sex, resulting from
any F2 breeding combinations.
138
FIGURE A.4: EXPECTANCY FREQUENCIES OF ANIMALS/GENOTYPE BASED ON THE BREEDING
STRATEGY FOR GENERATING THE BREEDING COLONY. The table shows the expected frequency of
experimental animals, by genotype and sex, resulting from the mating of F2 male with F2 female.
Expectancy
Expectancy
by Sex
PgrCre/+
Rac1f/f 1 1/2
Expectancy
Expectancy
by Sex
Pgr+/+
Rac1f/f 1 1/2
Expectancy
Expectancy
by Sex
PgrCre/+
Rac1f/f 1/2 1/4
Pgr+/+
Rac1f/f 1/2 1/4
Rac f f f f
Female Alleles
Pgr+/+
Rac1f/f
Pgr + + + +
Mal
e A
llel
es
Pg
rCre
/Cre
Ra
c1f/
f
Cre fCre/+
f/f
Cre/+
f/f
Cre/+
f/f
Cre fCre/+
f/f
Cre/+
f/f
Cre fCre/+
f/f
Cre/+
f/f
Cre/+
f/f
Cre/+
f/f
Cre/+
f/f
Cre fCre/+
f/f
Cre/+
f/f
Cre/+
f/f
Cre/+
f/f
Rac f f f f
Female Alleles
Pgr+/+
Rac1f/f
Pgr + + + +
Mal
e A
llel
es
Pg
r+/+
Ra
c1f/
f
+ f+/+
f/f
+/+
f/f
+/+
f/f
+ f+/+
f/f
+/+
f/f
+ f+/+
f/f
+/+
f/f
+/+
f/f
+/+
f/f
+/+
f/f
+ f+/+
f/f
+/+
f/f
+/+
f/f
+/+
f/f
Rac f f f f
Female Alleles
PgrCre/+
Rac1f/f
Pgr + + + +
Mal
e A
llel
es
Pg
rCre
/+ R
ac1
f/f
Cre fCre/+
f/f
Cre/+
f/f
Cre/+
f/f
+ f+/+
f/f
+/+
f/f
+ f+/+
f/f
+/+
f/f
+/+
f/f
+/+
f/f
Cre/+
f/f
Cre fCre/+
f/f
Cre/+
f/f
Cre/+
f/f
Cre/+
f/f
Frequency
Frequency
Frequency
+/+
f/f
+/+
f/f
+/+
f/f
+/+
f/f
Cre/+
f/f
Cre/+
f/f
139
Fertility Studies
FIGURE A.5: BREEDING STRATEGY FOR THE SIX-MONTH BREEDING STUDY. To rule out the possibility
that the subfertility in Rac1d/d mice is due to an inherent defect in the embryo, we bred the Rac1d/d and
Rac1f/f females to wild-type male mice of proven fertility. This breeding scheme generated pups that were
heterozygous for Rac1 gene. Therefore, any problems associated with pregnancy are likely due to a defect
in the maternal decidua, and not in the embryo or its membranes.
140
FIGURE A.6: EXPECTANCY FREQUENCIES OF ANIMALS/GENOTYPE BASED ON THE SIX-MONTH
BREEDING STUDY. Fertility testing was done using the breeding scheme above. The tables show the
expected frequency of animals, by genotype and sex, resulting from the breeding combinations.
Expectancy
Expectancy
by Sex
PgrCre/+
Rac1f/+ 1/2 1/4
Pgr+/+
Rac1f/+ 1/2 1/4
Expectancy
Expectancy
by Sex
Pgr+/+
Rac1f/+ 1 1/2
Rac f f f f
Female Alleles
PgrCre/+
Rac1f/f
Pgr Cre Cre + +
Mal
e A
llel
es
Pg
r+/+
Ra
c1+
/+
+ +Cre/+
f/+
Cre/+
f/+
+/+
f/+
+ +Cre/+
f/+
Cre/+
f/+
+ +Cre/+
f/+
Cre/+
f/+
+/+
f/+
+/+
f/+
+/+
f/+
+ +Cre/+
f/+
Cre/+
f/+
+/+
f/+
+/+
f/+
Rac f f f f
Female Alleles
Pgr+/+
Rac1f/f
Pgr + + + +
Mal
e A
llel
es
Pg
r+/+
Ra
c1+
/+
+ ++/+
f/+
+/+
f/+
+/+
f/+
+ ++/+
f/+
+/+
f/+
+ ++/+
f/+
+/+
f/+
+/+
f/+
+/+
f/+
+/+
f/+
+ ++/+
f/+
+/+
f/+
+/+
f/+
+/+
f/+
Frequency
Frequency
+/+
f/+
+/+
f/+
+/+
f/+
+/+
f/+
141
APPENDIX B: GENE EXPRESSION VALIDATION OF OTHERS MOLECULES INVOLVED IN STROMAL CELL
DECIDUALIZATION AND ANGIOGENESIS
FIGURE B.1: GENE EXPRESSION OF OTHER CRITICAL MOLECULES, IMPLICATED IN STROMAL CELL DIFFERENTIATION, WERE NOT AFFECTED
BY THE CONDITIONAL RAC1 MUTATION.
Pgr
Rac1f/f
Rac1d/d
0
1
2
3
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Gjp1/Cx43
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Mmp9
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Hif1
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Vegfa
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Tek/Tie2
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Vegfr1/Flt1
Rac1f/f
Rac1d/d
0
1
2
Rel
ati
ve
Gen
e E
xp
ress
ion
(2-
Ct )
Vegfr2/Flk1
Rac1f/f
Rac1d/d
0
1
2
Rel
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e E
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FIGURE B.2: GENE EXPRESSION OF OTHER MOLECULES IMPLICATED IN UTERINE ANGIOGENESIS.
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APPENDIX C: PRELIMINARY DATA FOR FUTURE DIRECTIONS
C1. Background information and the rationale
Embryo implantation into the maternal endometrium is crucial for embryo survival, and therefore
sustains pregnancy. It well establish that the invading embryo promotes well-orchestrated vascular
remodeling in the uterus to provide access to nutrient-rich maternal blood. Decidual cells are
known to actively regulate this process through complex crosstalk between the embryonic and
maternal tissues. In some pathological conditions associated with pregnancy, this crosstalk is
disrupted. In cases of shallow invasion of the trophoblast into the endometrium, the fetal stress
that arises causes hypertensive crisis condition in the mother, known as preeclampsia. In the US
alone, about 5% of pregnancies will develop preeclampsia and, so far, the only effective solution
is to deliver the baby, which often is premature. In contrast, in about 5% of pregnancies,
trophoblasts will migrate beyond their limits and placenta accreta will occur. In 70% of patients
with placenta accreta, trophoblast cells will only invade past the functionalis decidual layer (where
the trophoblast usually stops) into the basalis decidua. However, in more severe cases, placenta
increta or placenta percreta occurs, resulting in excessive invasion of the trophoblast deep into the
myometrium or into the peritoneum, respectively. In either case, surgical removal of the placenta
is necessary, which can put the mother and baby at risk. In both the instances, under- or over-
nutrition of the fetus can have long lasting effects on the conceptus’ metabolomics that can be
exacerbated into metabolic disturbances later in adulthood, like early onset diabetes, obesity, or
any other metabolic-related disease.
Early in vitro studies have suggested that RAC1 is present in the decidual cells and seems to play
a role in embryo invasion and placentation. However, our work has clearly showed that RAC1
activity is not necessary for embryo attachment and implantation into the endometrium (Chapter
3). However, uterine deletion of the Rac1 gene led to an apparent overgrowth of the trophoblasts
in the uterus of Rac1d/d females on day 8 of pregnancy (Figures 3.6 and C3.1). Therefore, we
decided to begin exploring how conditional deletion of Rac1 in the uterine tissues leads to
overgrowth of the trophoblast, and whether this overexpansion of the trophoblast leads to placental
abnormalities during mid-gestation. Because mice with conditional mutation for RAC1 have
144
severe angiogenic defects (Chapter 4), we only focused on those implantation nodules that were
as morphologically similar as possible to the control implantation nodules.
145
C2. Materials and Methods
Animals and Tissue Collection
All animal breeding, housing, procedures, experiments, and euthanasia were approved and
conducted according to the guidelines of the Institutional Animal Care and Use Committee of
University of Illinois at Urbana-Champaign. The health and care of the animals were provided by
the veterinarians and staff of Division of Animal Resources. Rac1 conditional null mice were
generated using Cre-LoxP strategy [347] (Figure 3.1A). Mice expressing homozygous copies of
the Rac1 gene flanked by loxP sites [Pgr+/+ Rac1f/f (official gene symbol Rac1tm1Djk/J), or Rac1f/f
from now onwards] [267] were obtained from Dr. Ann Sutherland (University of Georgia) in a
C57BL/6-129SV background. Mice carrying the Cre Recombinase under the control of the
progesterone receptor (Pgr) promoter [348] [PgrCre/+ Rac1+/+ (official gene symbol Pgrtm2(cre)Lyd/+),
or PgrCre from now onward], were obtained from Drs. John Lyndon and Francesco De Mayo
(Baylor College of Medicine) in a C57BL/6 background. To establish the mouse colony for this
study, initial breeding between the two mouse lines was done to obtain the F1 progeny, which are
heterozygous for Pgr and Rac1 (Appendix A). Female mice used for this study were generated by
breeding male mice of specific genotypes from the F2 progeny (see Figure 3.1B) with Rac1f/f
female mice. Genotyping of the animals was performed following the protocols established by the
respective laboratories that generated the transgenic mice [267, 348] (Appendix A). Conditional
knockout female mice (PgrCre/+ Rac1f/f) will be referred to as Rac1d/d from now onwards.
Rac1f/f or Rac1d/d females were euthanized by CO2 asphyxiation, followed by a blood draw via
cardiac puncture, and cervical dislocation. Uteri from pregnant mice were collected at different
time points during pregnancy. The harvested organs were either fixed for histological evaluation
[immersion-fixed in 10% neutral-buffered formalin (NBF)], flash frozen in liquid N2 for RNA or
frozen sectioning. For samples collected at different time points during pregnancy, identification
of a copulatory plug following mating was used as a reference and for our experimental purposes,
to indicate day 1 of pregnancy.
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Some of the implantation nodules collected during pregnancy were used for morphological
evaluation of the decidua or placenta. The decidual mass was exposed by mechanical removal of
the myometrium. Pictures were taken using a Leica M165 FC dissection scope connected to a
Leica 450 C camera with c-mount interface containing a 5 Megapixel CCD sensor. Similarly, for
morphological evaluation of embryos on day 15 of pregnancy, mechanical removal of the
myometrium was done under the dissecting scope, as mentioned above. At least two implantation
nodules/female were analyzed on day 15 of pregnancy.
Quantitative real-time PCR and RNA Isolation
Total RNA was isolated from tissues or cells using TRIzol reagent (Invitrogen) followed by RNA
cleanup kit (Qiagen), according to the manufacturer’s instructions. RNA was converted to cDNA
using a kit (Applied Biosystems), according to the manufacturer’s instructions. Oligonucleotides
specific for genes of interest were developed (see Appendix F for details) and qPCR reactions were
carried out using SYBR-green master mix (Applied Biosystems) on a 7500 Applied Biosystems
real-time PCR machine (Applied Biosystems). Gene expression data analysis were done using the
2-ΔΔCt method [357] and 36b4 gene – which is conserved among many species and encodes an
acidic ribosomal phosphoprotein P0 that interacts with 40S ribosomal subunit – was used as a
housekeeping gene and loading control [358].
Immunohistochemistry, Immunocytochemistry, and Immunofluorescence
Cultured cells or paraffin-embedded uterine sections were subjected to immunohistochemistry or
immunocytochemistry as described previously [32, 353, 355, 356]. Briefly, paraffin-embedded
sections were deparaffinized in xylene and rehydrated in a series of graded alcohols followed by
rinsing in tap water. When frozen tissue were used, the sections were thawed at room temperature
for 5 min, then fixed for another 5 min in 10% NBF. When cells were used, they were fixed for
another 5 min in 10% NBF. Sections or cells then were rinsed in 1x phosphate-buffer saline (PBS),
followed by serum blocking using 10% non-immunized serum for 1 h at room temperature.
Following serum block, slides were incubated with a solution containing the diluted antibody (see
Appendix E for details) and 1% serum in 1x PBS overnight at 4 oC in a humidified chamber (for
147
slides) or in the culture plates (for cells). Following overnight incubation, slides were washed 3x
5 min with 1x PBS, then incubated with diluted fluorescent-labeled secondary antibody (see
Appendix E for details) for 1 h at room temperature. Samples then were rinsed in 1x PBS and
mounted with a medium containing DAPI. Pictures were taken using the Olympus BX51
microscope equipped for light and fluorescent imaging and was connected to a Jenoptik ProgRes
C14 digital camera with c-mount interface containing a 1.4 Megapixel CCD sensor. Fluorescent
images were merged and processed using CS4 Adobe Photoshop Extend (Adobe Systems).
Embryo and placental perimeter measurement
Java-based image analysis software NIH ImageJ (http://rsb.info.nih.gov/nih-image/) was utilized
for the outlining and making measurements in embryonic tissues. The perimeter of embryos on
day 8 of pregnancy, which were previously stained with hematoxylin and eosin, and the placenta
on day 15 of pregnancy were also measured using this software.
Statistical analysis
Experimental data were collected from a minimum of three independent animals (not from the
same litter), which were subjected to the same experimental conditions. Embryo weights and qPCR
results are expressed as LSMeans and mean ± S.E.M, respectively. Statistical analysis was done
using Student’s t-test. An analysis of equal variances was done on all numerical data to determine
whether a parametric or non-parametric hypothesis test was appropriate. Data were considered
statistically significant if p ≤ 0.05, and is indicated by an asterisks in the figures. All data were
analyzed and plotted using GraphPad Prism 4.0 (GraphPad Software) or Microsoft Excel 2013
(Microsoft Corporation).
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C3. Preliminary data
FIGURE C3.1: CONDITIONAL DELETION OF RAC1 IN UTERINE DECIDUAL CELLS, DOES NOT PREVENT EMBRYO IMPLANTATION, BUT LEADS TO
TROPHOBLAST OVERGROWTH IN VIVO. (A) Implantation nodules collected on day 8 of pregnancy were serially sectioned and every 10th section
was stained with hematoxylin and eosin. The section with the maximum embryo size was used to measure the total perimeter covered by the
trophoblast (outlined by the dotted lines). The data show a close trend toward trophoblast outgrowth (p = 0.08).
149
FIGURE C3.1: CONDITIONAL DELETION OF RAC1 IN UTERINE DECIDUAL CELLS, DOES NOT PREVENT EMBRYO IMPLANTATION, BUT LEADS TO
TROPHOBLAST OVERGROWTH IN VIVO (CONTINUED). (B) Isolated embryos on day 8 of pregnancy, showed outgrowth of the trophoblast cells.
Bars = 1 mm. n ≥ 5.
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FIGURE C3.2: GENE EXPRESSION PROFILE OF REGULATORS OF TROPHOBLAST EXPANSION. IGFBPs are known to interfere with embryo-
produced IGF2, which promotes forward migration of trophoblast cells into the maternal endometrium. Of the known IFGBPs that could be found
in the mouse uterus, IGFBP4 is only expressed by the uterine decidua [90, 92], while IGFBP3 can be expressed by both the decidua and the
myometrium. Adrenomedullin has been shown to be secreted by both the maternal tissues, as well as the embryonic tissues. Our data show that on
day 8 of pregnancy, only IGFBP4 is differentially regulated by the conditional deletion of RAC1 in the uterus. n = 4. Asterisks indicate significant
differences from controls (** p < 0.01).
Igfbp3
Rac1f/f
Rac1d/d
0
1
2
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Igfbp4
Rac1f/f
Rac1d/d
0
1
2
3
**
Rel
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ene
Exp
ress
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(2-
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Igf2
Rac1f/f
Rac1d/d
0
1
2
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ene
Exp
ress
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(2-
Ct )
Adm
Rac1f/f
Rac1d/d
0
1
2
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Exp
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(2-
Ct )
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FIGURE C3.3: CONDITIONAL DELETION OF RAC1 IN UTERINE TISSUES LEADS TO PLACENTAL ABNORMALITIES AND INDICATIONS OF
INTRAUTERINE GROWTH RESTRICTION. (A and B) Samples collected on day 15 of pregnancy show a wide range of variation on the placental and
embryo outcome. However, consistently all the implantation nodules that were dissected from Rac1d/d females showed placental shape abnormalities
and wide-range in the level of hemorrhage that was observed in the placental tissues.
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FIGURE C3.3: CONDITIONAL DELETION OF RAC1 IN UTERINE TISSUES LEADS TO PLACENTAL ABNORMALITIES AND INDICATIONS OF
INTRAUTERINE GROWTH RESTRICTION (CONTINUED). (C) Furthermore, some of the placentas had adopted a convex shape in the embryo side
of the placenta, instead of the normal concave shape. (D) Although these placentas did not extend inwards towards the uterus, the placentas obtained
from Rac1d/d females were expanded sideways and displayed abnormal shapes, as indicated by the increased placental perimeter. Similarly, these
placentas were significantly heavier than control counterparts, suggesting placental modifications, perhaps, as a result of uterine RAC1 mutation or
an adaptation to the hemorrhagic environment, which led to growth restrictions as observed in some of the more severe embryos. Bars = 1 cm. n =
3. Asterisks indicate significant differences from controls (* p < 0.05, ** p < 0.01).
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FIGURE C3.4: CONDITIONAL DELETION OF RAC1 IN UTERINE TISSUES LEADS TO PLACENTAL ABNORMALITIES THAT DISPLAY IMPAIRED
VASCULAR REMODELING. (A) This section show the expected placental structures on day 12 of pregnancy. Bars: low power images = 500 µm and
high power images = 200 µm. White circles highlight blood vessels in the decidua. E = embryo, P = placenta, and M = mesometrial decidua. Area
1 = placenta (Red = CYTOKERATIN 8 positive cells); Area 2 = feto-maternal interphase; Area 3 = deep decidua and myometrium. All sections
shown here were stained with alpha smooth muscle actin (αSMA = green fluorescent stain) to assess the extent of vascular remodeling.
154
FIGURE C3.4: CONDITIONAL DELETION OF RAC1 IN UTERINE TISSUES LEADS TO PLACENTAL ABNORMALITIES THAT DISPLAY IMPAIRED
VASCULAR REMODELING (CONTINUED). (B and C) Samples collected on day 12 of pregnancy show a wide range of variation on the placental
outcome as shown here these implantation nodules from Rac1d/d females. Whereas one of the samples displays a placenta with normal histological
features as those seen in the controls (panel B), the lower most panel shows a disorganized, bifurcated (panel C, below), placenta (red fluorescent
stain = CYTOKERATIN 8). All sections shown here were stained with alpha smooth muscle actin (αSMA = green fluorescent stain) to assess the
extent of vascular remodeling. Our results show, that although there is some degree of vascular remodeling that can occur in Rac1d/d uteri, this seems
to be partial as there were some areas in the decidua that stained positive for αSMA, which were not positive in the control samples (areas 2 and 3).
Furthermore, it seems that in the severe cases, little vascular remodeling occurs, and whether this is a consequence of improper decidual function or
the fact that the embryo might be dying, remains to be elucidated. Bars: low power images = 500 µm and high power images = 200 µm. White
circles highlight blood vessels in the decidua. E = embryo, P = placenta, and M = mesometrial decidua. Area 1 = placenta (Red = CYTOKERATIN
8 positive cells); Area 2 = feto-maternal interphase; Area 3 = deep decidua and myometrium.
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FIGURE C3.4: CONDITIONAL DELETION OF RAC1 IN UTERINE TISSUES LEADS TO PLACENTAL ABNORMALITIES THAT DISPLAY IMPAIRED
VASCULAR REMODELING (CONTINUED). (B and C) Samples collected on day 12 of pregnancy show a wide range of variation on the placental
outcome as shown here these implantation nodules from Rac1d/d females. Whereas one of the samples displays a placenta with normal histological
features as those seen in the controls (panel B), the lower most panel shows a disorganized, bifurcated (panel C), placenta (red fluorescent stain =
CYTOKERATIN 8). All sections shown here were stained with alpha smooth muscle actin (αSMA = green fluorescent stain) to assess the extent of
vascular remodeling. Our results show, that although there is some degree of vascular remodeling that can occur in Rac1d/d uteri, this seems to be
partial as there were some areas in the decidua that stained positive for αSMA, which were not positive in the control samples (areas 2 and 3).
Furthermore, it seems that in the severe cases, little vascular remodeling occurs, and whether this is a consequence of improper decidual function or
the fact that the embryo might be dying, remains to be elucidated. Bars: low power images = 500 µm and high power images = 200 µm. White
circles highlight blood vessels in the decidua. E = embryo, P = placenta, and M = mesometrial decidua. Area 1 = placenta (Red = CYTOKERATIN
8 positive cells); Area 2 = feto-maternal interphase; Area 3 = deep decidua and myometrium. n = 3
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APPENDIX D: GENOTYPING PRIMERS AND PROTOCOL
PCR Cycling conditions for genotyping
Rac1 Genotyping
Rac1 Primers:
P91: 5'-GAT GCT TCT AGG GGT GAG CC-3'
P45: 5'-CAG AGC TCG AAT CCA GAA ACT AGT A-3'
P33: 5'-TCC AAT CTG TGC TGC CCA TC-3'
Cycling Program for Rac1:
1. 9 minutes @ 92 °C
2. 30 sec @ 92 °C
3. 30 sec @ 65 °C
4. 1 minute @ 72 °C
5. Cycle 35 times steps 2 to 4
6. 3 minutes @ 72 °C
7. ∞ @ 4 oC
8. End
This reaction condition gives 3 potential bands:
Rac1 Wild-type band 115 bp
Rac1 knockout band 140 bp
Rac1 floxed band 242 bp
Run in 2-3% Agarose Gel in TBE Buffer for at least 45 minutes @ 120 Vconstant
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PgrCre Genotyping
PgrCre Primers:
R1: 5'-TAT ACC GAT CTC CCT GGA CG-3'
F1: 5'-ATG TTT AGC TGG CCC AAA TG-3'
F2: 5'-CCC AAA GAG ACA CCA GGA AG-3'
Cycling Program for PgrCre:
1. 5 minutes @ 94 °C
2. 1 minute @ 94 °C
3. 1 minute @ 60 °C
4. 2 minute @ 72 °C
5. Cycle 39 times steps 2 to 4
6. 7 minutes @ 72 °C
7. ∞ @ 4 oC
8. End
This reaction condition gives 2 bands:
Pgr Wild-type band 250 bp
PgrCre band 600 bp
Run in 1.5-2% Agarose Gel in TBE Buffer for at least 30 minutes @ 120 Vconstant
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APPENDIX E: ANTIBODY DILUTIONS
Immunohistochemistry
For immunostaining, the blocking serum (donkey or goat) used depended on the secondary
Primary antibodies
Antibody Dilution used Company Catalog Host Species
Anti-Human MKI67 1:750 BD Parmigen 550609 Mouse
Anti-Mouse
PRL8A2/dPRP 1:750 Serum obtained
from Dr. Soares ----- Rabbit
Anti-Mouse PECAM1/
CD31 1:200 BD Parmigen 557355 Rat
Anti-Mouse α-SMA 1:200 Abcam Ab5694 Rabbit
Anti-Mouse Pan VEGFA
(A-20) 1:100 Santa Cruz
Biotechnology SC-152 Rabbit
Anti-Mouse
CYTOKERATIN 8
(Troma-I)
1:50 Hybridoma Bank ----- Rat
Anti-Mouse NRP1
(EPR3113) 1:200 Abcam Ab81321 Rabbit
Secondary antibodies
Biotinylated Anti-Rabbit
IgG 1:250 Invitrogen 95-6543B Goat
Biotinylated Anti-Mouse
IgG 1:250 Invitrogen 95-6143B Goat
Cy3-Conjugated Anti-Rat
IgG 1:250 Jackson
ImmunoResearch
712-165-
150 Donkey
Cy3-Conjugated Anti-
Rabbit IgG 1:250 Jackson
ImmunoResearch
711-165-
152 Donkey
Alexa 488-Conjugated
Anti-Mouse IgG 1:250 Jackson
ImmunoResearch
715-545-
151 Donkey
Fluorophores
Texas Red Avidin 1:500 Vector A-2006 -----
Fluorescein Avidin 1:500 Vector A-2001 -----
159
Western Blots
Antibody Dilution used Company Catalog Host Species
Anti-Mouse RAC1
(0.T.127) 1:1000 Abcam Ab33186 Mouse
Anti-β-Tubulin 1:2000 Sigma T5293 Mouse
HRP-Conjugated Anti-
Mouse IgG 1:5000 Sigma A9044 Rabbit
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APPENDIX F: PRIMER SEQUENCES FOR GENE EXPRESSION ASSAYS
The primers for gene expression assays were generated using the Custom Primers - OligoPerfect™
Designer software (Invitrogen), based on the known mRNA sequence of the genes of interest. The
obtained oligonucleotide sequence were then submitted to NCBI BLAST tool to confirm their
specificity to the gene of interest. Sequences with 100% max match identity were used for gene
expression analysis. For all the primer sequences below, a single dissociation curve was obtained
during qPCR analysis.
Parameters used with the Invitrogen software:
Primer size 18 – 27 (min – max) ok, with opt (optimal) 20
Primer temp 57 – 63, with 60 opt
% GC 45 – 55, 50 opt
Product size 100 – 150 bp
Salt and primer concentration 50 nM
The 3’ nucleotide must be a perfect match to the target cDNA sequence
List of primers
Primers were purchased from either Sigma or Invitrogen.
36b4 Primers: F: 5’-CATCACCACGAAAATCTCCA-3’
R: 5’-TTGTCAAACACCTGCTGGAT-3’
Rac1 Primers: F: 5’-CTGAAGTGCGACACCACTGT-3’
R: 5’-CTTGAGTCCTCGCTGTGTGA-3’
Rac2 Primers: F: 5’-CAATGCAGGCCATCAAGTGT-3’
R: 5’-TGGGGATGTATTCTCCAGGG-3’
Rac3 Primers: F: 5’-ATCAAGTGCGTGGTGGTTG-3’
R: 5’-CGTTGGCTGAGTAGTTGTCG-3’
Cdc42 Primers: F: 5’-TGCTGCTATGAACGCATCTC-3’
R: 5’-CATGAGTGCATGTGGGTAGG-3’
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RhoA Primers: F: 5’-TGGTGATGGAGCTTGTGGTA-3’
R: 5’-TCTACCTGCTTCCCATCCAC-3’
Hif1α Primers: F: 5’-TCTCCAAGCCCTCCAAGTATG-3’
R: 5’-CTACAGTGGTGGCAGTTGTG-3’
Epas1/Hif2α Primers: F: 5’-CAGGCAGTATGCCTGGCTAATTCCAGTT-3’
R: 5’-CTTCTTCCATCATCTGGGATCTGGGACT-3’
Cldn3 Primers: F: 5’-CCACTACCAGCAGTCGATGA-3’
R: 5’-AGCCTGTCTGTCCTCTTCCA-3’
F13a1 Primers: F: 5’-CAAGGATTCGGTGTGGAACT-3’
R: 5’-GCTTAACGGCTTGGACAGAG-3’
Wnt4 Primers: F: 5’-TTCTCACAGTCCTTTGTGGACG-3’
R: 5’-TCTGTATGTGGCTTGAACTGTG-3’
Wnt5a Primers: F: 5’-TCTTGGTGGTCTCTAGGTATG-3’
R: 5’-CACTGTGCTGCAGTTCCATCT-3’
Alpl Primers: F: 5’-TGGATGTGACCTCATTGC-3’
R: 5’-CATATAACACCAACGCTCAG-3’
Gpj1/Cx43 Primers: F: 5’- CTATCGTGGATCAGCGACCTTC-3’
R: 5’- CACGGGAACGAAATGAACACC-3’
Prl8a2/dPRP Primers: F: 5’-CTCACTTCTCAGGGGCACTC-3’
R: 5’-GGATCTGAGCAGCCATTCTC-3’
Runx1 Primers: F: 5’-AGATTCAACGACCTCAGGTTT-3’
R: 5’-CGGATTTGTAAAGACGGTGA-3’
Pgr Primers: F: 5’-CTAAATGAGCAGAGGATGAAGGAG-3’
R: 5’-TGGGCAACTGGGCAGCAATAAC-3’
Vegfa Primers: F: 5’-CAGGCTGCTGTAACGATGAA-3’
R: 5’-TTTCTTGCGCTTTCGTTTTT-3’
Tek/Tie2 Primers: F: 5’-TCCGAGCTAGAGTCAACACC-3’
R: 5’-GCCATCCACTATTGTCCAAG-3’
Vegfr1/Flt1 Primers: F: 5’-TGGCCAGAGGCATGGAGT-3’
R: 5’-TCGCAAATCTTCACCACATTG-3’
Vegfr2/Flk1 Primers: F: 5’-TCTGTGGTTCTGGTGGAGA-3’
R: 5’-GTATCATTTCCAACCACCCT-3’
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Pecam1/Cd31 Primers: F: 5’-GAGGCCCAATCACGTTTCAGTT-3’
R: 5’-TCCTTCCTGCTTCTTGCTAGCT-3’
Angpt1 Primers: F: 5’-GGGGCTGGAAGGAGTATAAA-3’
R: 5’-CCTCAGCATGTACTGCCTCT-3’
Angpt2 Primers: F: 5’-TCCAAGAGCTCGGTTGCTAT-3’
R: 5’-AGTTGGGGAAGGTCAGTGTG-3’
Angpt4 Primers: F: 5’-AGCAGCCAACTGACGGAGTTT-3’
R: 5’-CTCTGCACAGTCCTGGAACA-3’
Sphk1 Primers: F: 5’-CATGCATGAGGTGGTGATG-3’
R: 5’-TGCTCGGTACCCAGCATAGTG-3’
Nrp1 Primers: F: 5’-TGTGGGTACACTGAGGGTCA-3’
R: 5’-CAGCAATTCCACCAAGGTTT-3’
Mmp9 Primers: F: 5’-TCTTCCCCTTCGTCTTCCT-3’
R: 5’-TATACAGCGGGTACATGAGCG-3’
Adm Primers: F: 5’-GGGTTCACTCGCTTTCCTAGGGTTCACTCGCTTTTCTA-3’
R: 5’-GCTGCTGGATGCTTGTAGTTGCTGGCTGGATGCTTGTAGTT-3’
Igf2 Primers: F: 5’-ATGACACCTGGAGACAGTCC-3’
R: 5’-TGGCCTCTCTGAACTCTTTG-3’
Igfbp3 Primers: F: 5’-GGTCCTTATTGTTGCCATCTC-3’
R: 5’-AGATGCCTGACAAAGAAAGG-3’
Igfbp4 Primers: F: 5’-TGCAGACCTCTGACAAGGAT-3’
R: 5’-GTCCCCACGATCTTCATCCTT-3’
Prl3c1/Plpj Primers: F: 5’-GTTTTTGATTTTGCCATGCT-3’
R: 5’-TCGCAAATCTTCACCACATTG-3’
