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CORTICOTROPIN-RELEASING HORMONE RECEPTOR SUBTYPE 1 AND SUBTYPE 2 mRNA EXPRESSION AND PROTEIN LOCALIZATION IN THE
MYOMETRIUM IN PREGNANCY
Mata Yvette Stevens
A thesis submitted in c o n f o d t y with the requirements for the degree of Masters of Science Graduate Department of Physiology
University of Toronto
8 Copflght by Miata Yvette Stevens 1998
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Corticotropin-Releasing Hormone Reeeptor Sobtype 1 And Subtype 2 mRNA
Expression And Protein Locaiization in The Myometrium In Pregnnncy
Masters of Science 1998
Miata Yvette Stevens
Department o f Physiology, University of Toronto
ABSTRACT
The human placenta secretes increasing concentrations of corticotropin-
releasing hormone (CRH) in late pregnancy and in labour. CRH has been implicated in
the regulation of myometnal contractility. We hypothesized that CRH receptoa, CRH-
R1 and CRH-R2, mRNA in the rnyometrium would be upregulated in labour.
Myometrial samples were collected from nonpregnant, pregnant and laboring patients
nom the upper and lower uterine segment. CM-RI mRNA and protein were
downregulated with pregnancy and significantly upregulated at labour. This rise
appeared to be exclusive to the lower segment. Cm-W mRNA did not change.
Rats have been used extensively to shidy the regulation of CRH receptoa. We
examined CRH receptor mRNA in rat myomeûium. CRH-RI rnRNA was
undetectable. CRH-R2 mRNA was significantly increased at labour concomitant with a
nse in cornexin 43 mRNA, a gap junction protein associated with labour. In
conclusion, at the time of labour CRH-R1 mRNA is upregulated in the human but in
the rat CRH-EU mRNA is upregulated.
Dedicated to my mother, my inspiration.
Let earth and sky be your yardstick and eterniîy your nceasuremenL There is no height that you cannot accomplisi by &g the active intelligence of your mind
The Hon. Marcus Mosaih Garvey
iii
1 am forever indebted to my s u p e ~ s o r Dr. John Challis for giving me the
opportunity to experience research. Research, with al1 its highs and lows. I thank John for
his guidance, his support and his understanding throughout the time I have known him.
1 would like to thank my Advisory Cornmittee members Dr. Steve Lye, Dr. Steve
Matthews and Dr Neil MacLusky for their helpful cooperation throughout my degree and
for their invaluable assistance in editing and revising this thesis.
I thank al1 my lab members for the fond mernories of early morning meetings and
late night research always interlaced with good partying techniques. 1 would particularly
like to thank Dr. Mhoyra Fraser, Dr. Alison Holloway, Dr. Wendy Whittle, Dr. Vicky
Clifion, Debbie Sioboda and Fa1 Patel for their constant source of technical and
emotional support. A very special thanks to the "Lye Lab" especially to Dr. Isabella
Caniggia, Dr. Daniel MacPhee, Dr. Ryan Ou and Lindsay McWhirter for their readiness
to teach me and help me with invaluable techniques.
Special thanks to Boris, Rebecca and Alex. Your love, your support and the
wondemil strong bond we share makes me remember, every day, how blessed I am to
have siblings. Extreme gratitude to rny "twinie", Yvonne, completing a Masters of
Science concornitantly with you has made us share laughter, tears and a world of
understanding. My heartfelt thanks to my Mom who taught me everything 1 need to know
about being a powerful , intelligent and motivated woman. Infinite love and thanks to
Toke Petersen- my love, my heart, my best fiiend. You make life, love and success worth
living for.
TABLE OF CONTENTS
TITLE PAGE ..............................-.....o..a....................a........*..o...-.*..........a........................ i
1.1 Overview ................................................................................................ 1
1.2 The Placenta And Fetai Membranes ....................... ... ................................ 3
1 -3 The Uterus ..... ......... .... ......... ...... ., . . . ................ . ............ . . . . . . 5
1 -4 Stimulation Of Myometnal Activity .......... .. .................. .. .--..a.-... ..... ... ............ .... .. -6
1.5 Progesterone And Estrogen In Labour ............................... ... ..--..............-.... ......... - 8
1.6 Corticotropin Releasing Hormone ..... . .. ... .. .. . .. ... .... ...... .. . . . . . . . . . . . . . . . . . . 1 O
1.6.1 CRH-Related Peptides ... .... .... ................ .. ........... . . . . . . . . . ....... 12
1 -7 Human Placental C M ...... .. .. . ... . ... .. . .. . ..... . . .. ..... ....... . . . ....--.-.. .. . .. .. . .. ... .-. .. . . . . 12
1.8 CM-Binding Protein ... . . .. . ... . . .. .. .. . ... ... .. ... ... ......... . . . . . . . . . . . . . . . . . . . . . . 1 6
1.9 ûther Regdators Of CRH.. . .... .. .. .. . . .. .. ......... .. .. ... .... ..... . . . .. .. .... . ..... ...... ... .. ... . . ... 1 7
1 .10 Myometrial Contractility .......... ....... . ...... .. ....... .. ............................ . . 17
1.1 1 Other Roles For CRH ................................................................................ 18
1.12 CRH Receptors .................................. ................................................... 19
CHAPTER TWO: RATIONALE A , ! EYPOTHESIS .......................................... 44
CHAPTER THREE: CRE-Rl mRNA IS SIGNIFIC-Y UPREGULATED IN TEE HUMAN MYUMETRI[UM AT THE OF LABO UR .................... .47
3.1. Introduction ................................................................................................. .. 47
. ............................. 3 -2 Matends And Methods ... 4 8 3 .2.1 Biopsy Samples ............................................................................................. 48
....................... ...................................*............. 3 2.2 Total RNA E.xtraction .,,. 5 0 3.2.3 Reverse Transcription Polymerase Chain Reaction (RT-PCR) .................... 51 . . 3.2.4 Semi-Quantitative PCR ............................. .... ............................. 5 3 3.2.5 Immunohistochemistry ................................................................................ 5 4 3.2.6 Protein Extraction ............................... ., ................................................ 56 3.2.7 Western Blots ............................................................................................... 57 3.2.8 Data Anaiysis ............................. ... ..................................................... 5 8
...................................................................................... ................... 3.3 Results ... 5 9 3.3.1 CRH Receptor Expression in the Myometrium of Nonpregnant Patients .... 59
3.3.2 CRH-RI mRNA and CRH-R2 mRNA Expression in Myornetrium Pregnant
................................................................................................................... Patients 60
3.3 -3 Regional Expression of CRH-R 1 in Myometriurn From Nonpregnanf
Pregnant, Pregnant in Labour and Post-Pamim Patients ....................................... 61
3.3.4 CRH-RI mRNA Expression in the Fetai Membranes and Decidua ............. 62
.................................. 3.4 Discussion .-. ............................................................... 62
CHAI'TF,R 1: THE RAT- A SUITABLE MODEL TO STUDY THE REGULATION OF CRH RECEPTOR EXPRESSION IN THE
....................................................................................................... WOME- ? 95
4.1 Introduction .......................................................................................................... 95
4.2 Materials And Methods ..................................................................................... 9 6 4.2.1 Animais ................... ... ................................................................................. 9 6 4.2.2 Total RNA Extraction ............................................................................... ,... 97 4.2.3 Reverse-transcription-PCR ........................................................................... 98 4.2.4 Semiquantitative-PCR .............................................................................. 100 4.2.5 Data andysis ......................................................................... ................ 100
4.3 Results .............. ,....... ........................................................................................ 101 4.3.1 CRH-RI mRNA And CRH-R2 mRNA Expression in Rat Myometrium On
Day 15. Day 20. Day 2 1. Day 22. At Labour And 1 Day Postpammi ................. 101
4.3.2 Cx 43 Expression In Rat Myomeûium On Day 15. Day 20. Day 2 1. Day 22.
At Labour And 1 Day Postpamim ...................................................................... 102
.......................................................................................................... 4.4 Discussion 102
C W T E R 5: F7PiA.L DISCUSSION... .................................................................... 114
vii
LIST OF TABLES
Table 1.1 DiEerent Species Homologs of CRH-RI ......................................................... 42
......................... Table 1.2 DifZerent Species Hornologs of CM-EU ...................... .. 43
Table 3.1 CRH-R2 mRNA Expression Prior to and at the Onset of Labour in Term and
Preterm Patients -94 .............................................................................................................
viii
LIST OF FIGURES
Figure 1.1 . Schematic Diagram of the Structures W ithin the Uterus ..................................... 33
Figure 1.2. Schematic Diagram Demonstrating the Change in the S ù e and Shape of the
Uterus in Term Pregnancy and in Labour. ...................................................................... 35
Figure 1.3. Schematic Diagrarn Depicting the Upper Active Segment and the Lower Passive
..................................................................................................................... Segment. 37
Figure 1.4. Schematic Diagram Comparing the Amino Protein Sequence of CRH and CRH-
...................................................................................................... Related Peptides. ...... 39
Figure 1 S. Schematic Diagrarn Depicting the Seven Trammembrane Structure and the .
Arnino Acid Sequence Of the CRH Receptor Subtypes ............................ .. ........... 4 1
Figure 3.1. Photograph of a PCR Gel Showing C M - R I cDNA Enzyme Digestion ..........A9
Figure 3.2. Photograph of a PCR Gel Showing the Detection of CRH-RI mRNA and CRH-
R2 mRNA in Myometnum frorn Nonpregnant Women ............................................... 7 1
Figure 3.3. Photograph Showing Irnmunostaining for CRH-RI Protein in Human
Myometrium Smooth Muscle ......................................................................................... -73
Figure 3.4. Photograph Showing Immunostaining for CM-W Protein in Human
................................................................................................. Myometrium Smooth Musc le 75
Figure 3.5. Photograph Showing Immunostaining for CRH-RI Protein in the Smooth
Muscle Layer of the Uterine Vascuiature .............................................................................. -.77
Figure 3.6. Photograph Showing Immunostaining for CRH-R2 Protein in the Smooth
Muscle Layer of the Uterine Vasculature .......................... ... ............................................. -.79
Figure 3 -7. Photograph of CRH-R 1 and CRH-R2 Protein Immunostaining in the
Sheep Pituitary .................... .. ................................................................................................ 8 1
Figure 3.8. Photograph of PCR Gel Showing the Detection of CRH-RI mRNA
in Human Myometrium in Term and Preterm Patients ........................................................... 83
Figure 3.9. Analysis Of Sq-PCR for CRH-RI mRNA In Human Myornetrium in Tem
Patients .......................................................................................................................... 85
Figure 3.10. Analysis Of Sq -PCR for CRH-RI mRNA In Human Myometriurn in
................................................................................................................... Pretenn Patients .-..87
Figure 3.1 1. CRH-RI mRNA Expression in the Human Myometrium from
32 Weeks to 39 Weeks of Gestation and at the Time of Labour
in Preterm and Term Pregnancies ....................................................................................... 89
Figure 3.12. Photograph of a PCR gel and analysis of Sq-PCR showing
the regional detection of CRH-RI mRNA in human myometrium in
nonpregnant, pregnant, labourhg and postpartum patients .................................... .... ........... 9 1
Figure 3.13. Analysis Of Sq-PCR for CRH-R1 mRNA Expression in the Chorion and the
Decidua in Term Pregnancy and at the Time of Labour ............................................... 93
........... Figure 4.1. Photograph of a PCR Gel Showing CRH-EU cDNA Enzyme Digestion 107
Figure 4.2. Photograph Of A Sq-PCR Gel Showing the Detection of CM-R2 mRNA and
........... Cx 43 mRNA in Rat Myometrium in Pregnancy, at Delivery and Post-Partum 109
Figure 4.3. Analysis of Sq-PCR for CRH-R2 mRNA Expression in Rat Myometrium
in Pregnancy, at Delivery and Post-Partum ....................................... .. ............................... 1 1 1
Figure 4.4. Analysis of Sq-PCR for Cx 43 mRNA Expression in Rat Myometrium
in Pregnancy, at Delivery and Post-Partum ...................................... .. ............................... 1 13
ACTH ANOVA
bp BSA
CAMP cDNA Cx CRH CRH-BP C M - R I CRH-R2
DHEAS
MGA mRNA MW
PCR PGs PGE2 PGF2a PGHS
adrenocortico trophin hormone analysis of variance
base pairs bovine serum albumin
cyclic adenosine 3',5-mono phosphate complementary deoxyribonucleic acid connexin corticornopin releasing hormone cortico tropin-releasing hormone binding pro tein corticotropin releasing hormone receptor subtype- 1 corticotropin releasing hormone receptor subtype-2
dehydroepiandrosterone sulphate
estradiol
human placentai CRH human rat CRH
radioiabeled iodine immunoreactive
kilobase dissociation constant kilodaltons
rnean gestational age messenger ribonucleic acid molecuiar weight
oxytocin ovine CRH
polymerase chah reaction prostaglandins prostaglandin E2 prostaglandin F2-alpha prostaglandin H synthase
PKA PKC PLC POMC PTL PVN
RT RT-PCR
SDS sq-PCR
protein kinase A protein kinase C phosphotipase C pro-opiomelanocorlln pre-term labour paraventricular nucleus
reverse transcriptase reverse transcription polymerase chain reaction
sodium dodecyl sulfate semi-quantitative polymerase chah reaction
xii
CELWlXR Ir INTRODUCTION
Human parturition aises as a result of complex interactions within the matemal-
placental-fetal unit Spontaneous term human labour is defined as Zhe onset of reguiar
uterine contractions of increasing intensity and fkquency associated with progressive
effacement and dilatation of the cerWt following the completion of 38 weeks of
gestation" (Cunningham et al., 1993) and is the initial stage in a series of events leading
to birth. An understanding of the mechanisms regulating human parturition is vital for the
prevention and treatrnent of labour complications including dy stocia pre-term labour
(PTL) and post-term pregnancy each of which is associated with increased morbidity,
rnortaiity, health care costs and emotional distress. PTL is the most fiequent complication
of pregnancy and occurs in about 7% of a l i pregnancies; however, despite the advances in
perinatal medicine over the past 20 years littie change has been made in the incidence of
PTL, and delivery (Cunningham et ai., 1993). Given the potential threats of labour
complications on the health and survival of both the mother and the fenis, there exists a
crucial need for scientists to study the mechanisms involved in the onset, the progress and
the termination of human labour.
There are currently several hypotheses regarding the mechanisms involved in the
omet of labour. These include the progesterone withdrawal theory and the myometrium
activation-stimulation theory. The progesterone withdrawal theory
proposes that progesterone maintains uterine quiescence throughout pregnancy and
that a decrease in maternai plasma concentrations of progesterone close to term triggers
uterine contractility. W e this theory applies to the sheep mode1 of parturition
(Thorburn and Chailis, 1979), midies in humans have fded to observe a decrease in
circdating concentrations of progesterone prior to the onset of labour (TulchinsIq et
al., 1972). The myometrium activation-stimulation theory forwarded by Chaliïs and
Lye (1994) proposes that with the approach of labour the myomeaium is activated
(increased muscle excitability), by the synthesis of "contraction -associated proteins",
that renders the myometrium more responsive to stimulation by uterotonic agents.
Severai factors, produced by the placenta and the fetal membranes, such as
corticotroph-releasing hormone (CRW) (Riley et al., 1991), have the ability to
stimulate the myometnum (Quartero et al., 1989) The human placenta synthesizes and
secretes increasing concentrations of placental CRH (hCRH- pl) into the materna1
circulation in the third triinester of pregnancy and throughout labour (Goland et al.,
1986; Okamoto et al., 1989). The physiologicai significance of hCRH-pl in human
pregnancy still rernains to be established CRH exerts its actions via specific G-protein
coupled receptors (Chen et al., 1993; Lovenberg et al., 1995a) of which two subtypes
have been identifie4 CRH-RI and CRH-R2 (Chen et al., 1993; Lovenberg et al.,
1995a). CRH-RI messenger ribonucleic acid (mRNA) and CRH-R2 mRNA are
expressed in the myometnum of both nonpregnant and pregaant women (Rodriguez-
Limes et al., 1998). CRK receptors have been linked to secondary pathways
associated with both relaxation and contraction in pregnanc y (Grammatopoulos ., 1 994)
suggesting that hCRH-pl may potentially have differential effects on the human
myornetrium.
As of yet no studies have examined the mRNA expression and protein levels of
CRH-RI and CRH-R2 at the time of labour. Given the increase in circuiating levels of
hCRH-pl in the m a t e d circulation at that tirne and the presence of CRH recepton in
human myometnum it has been suggested hCW-pl may play a role in prepancy
and/or labour. One of the overall objectives of our lab is to understand the role of
hCRH-pl in the initiation a d o r progression of labour. The specinc aims of the present
study were a) to determine the expression of CRH-RI mRNA and CRH-R2 mRNA in
the myometrium both in pregnancy and at the time of labour and b) to establish a
mammalian mode1 to study the regulation of CRH receptor expression in the
myometrium.
1.2 THE PLACENTA AND FETAL MEMBRANES
The human placenta is classified as a hemochorioendothelial placenta The
materna1 blood does not directly contact the fetal blood and the exchange of gases,
nutrients and wastes occurs via a chorioendotheliai membrane. Shortiy following
fertilization, the ceUs of the early blastocyst differentiate into the inner cell mass and
the trophoblasts. The blastocy st descends into the uterus and the tropho blasts
differentiate into the outer syncytiotrophoblast layer and the inner proliferative
cytotrophoblast. The syncytiotrophoblast layer results from the fusion of
cytotrophoblast cells. The cytotrophoblasts and the synctial layer form the primary
placental villi. The placentai villi are the fûnctional mits of the human placenta and are
classined as prïmary, secondary or tertiary. Prirnary villi become secondary in structure
following the invasion of mesenchymai celis flom the extraembryonic rnesenchyma
Tertiary villi result fiom angiogenesis within the mesenchyme of the secondary vilh
and consist of a centrai core of fetal blood vessels surrounded by the cytotrophoblasts
and sync ytio tropho blast lay er. With advancing gestation, the ratio of cytotropho blasts
to the syncytiotrophoblast component decreases progressively, until the syncytial layer
is the dominant trophobiastic compogent at term (Petraglia et al., 1996a)Xhe
syncytiotrophoblast layer is a major site of protein and steroid hormone synthesis
within the human placenta (Petraglia et al., 1996a).
The amnion and the chorion are the fetal membranes and together form the
amniotic sac which holds the fetus and the amniotic fluid. The amnion is a thin
avascular membrane comprised of a single layer of epithelial cells on a loose
connective matrix. The chorionic membrane is composed of an inner connective tissue
layer and an outer epithelial layer. It is in direct contact with the decidua except at the
site of placenta implantation. The decidua is composed of the decidua basalis; the
region directiy beneath the site of implantation of the embryo, the decidua capsuloris;
the region surroundhg the embryo and the decidua vera which lines the remainder of
the uterus. The three layers of the decidua represent a vascular anatmnical interface
that ailows communication between the fetd membranes and the uterine myometnum
(see Figure 1.1) (Peûaglia et al., l996a).
13 THE UTERUS
The utem is a muscular, pear shaped organ located in the pelvic cavity between
the bladder and the rectum (Pritchard et al., 1985). The size and the shape of the utenis
vary markedly, dependent on age and parity. The u tem is composed of four regions:
the fundus, the corpus, the isthmus, and the cervix. The fundus is the uppermost and
widest region followed by the corpus, the isthmus and the narrow cervix.
The wail of the utenis is composed of three layea: the outer perimetriurn, the rniddle
myometrium and the inner endomemum. The myometrium is comprised of an outer
layer of longitudinal muscle fibers parallel to the long axis of the uterus and an inner
layer of circular muscle fibers perpendicuiar to the long axis of the utem (Pritchard, ef
al., 1 985). The longitudinal and the cucular muscle fibers of the myometrium originate
from the subserosd comective tissue and the paranephrie ducts. respectively (see rev.
Challis and Lye., 1994).
The rnyometrium undergoes sporadic, low pressure minimal intensiq
contractions kaown as Brmton-Hicks connacrions throughout pregnancy. Labour is
charac terized by high-fiequency , high amplitude, intense contractions of the
myometnum. At the time of labour, the human uterus differentiates into an actively
contracthg upper segment and a relatively quiescent lower segment (see Figure 1.2).
Uterine contractions of hi& intensity and long duration are present in the funda1
segment when compared to the activity of the lower segment (see Figure 1.3). The
development of regions of opposing contractile activity within the uterus aiiows for the
effective and forcehi expulsion of the fetus fiom the uterus.
Challis and Lye (1994) hypothesized that the activity of the myometrium, in
pregnancy, was fkely regulated by the synthesis of "contraction-associated proteias"
and the balance of stimuiatory versus inhibitory factors acting on the myornetrium.
Lye coined the term "contraction-associated" proteins to refer to the proteins involved
in increasing the excitability of the myometriai smooth muscle including the gap
j unctions and the uterotonic agonist receptors.
Gap junctions are specialized regions of the ceil membrane through which
direct cell-to-cell contact is maintained. They are formed fiom the aggregation of
hundreds or thousands of hydrophilic protein charnels (connexins) on the plasma
membranes of adjacent cells. Gap junctions provide the structural basis necessary for
coordinated synchronized rnyometriai contraction (Mackenzie and Garfield, 1 98 5 ;
Kilarski et al., 1994). Petroceiii and Lye, (1993) reported that, one of the gap junction
proteins, cornexin 43 (Cx 43) mRNA and protein levels were low in rat myometriurn
in pregnancy and increased markedy prior to labour attaining a peak during delivery.
Oxytocin (OT) is a nonapeptide hormone that stimulates uterine smooth muscle
contraction. This hormone is thought to be vital for the initiation of labour in humans,
but maternal plasma OT concentrations do not rise pnor to labour. In addition, serial
studies showed that the maternal plasma concentrations of oxytocin only increased
during the expulsive phase of labour (Leake et al., 198 1). Fuchs et al., (1982) reported
a signincant conelation between the concentration of OT receptors and the sensitiviw
of the uterus to OT. This led to the proposition that the concentrations of OT receptors
could be a possible regdatory mechanism mediating the effects of OT. However,
despite a reported increase in oxytocin receptor numben with the onset of labour
(Fuchs et al., 1994)- it is not clear whether OT is imporîant for the initiation of labour.
It has been suggested that increased OT receptor nurnbes facilitate the effects of OT
on an already "activatedm myometrium (see rev. Challis and Lye. 1994).
Prostaglandins (PGs) play a central role in stirnulating myometnal contractility
in many species ( see rev. Challis and Lye, 1994). A role for PGs in labour is suggested
by the demonstration that prostaglandin E2 (PGE3 and prostaglandin F2 alpha (PGF2a
stimulate the contraction of myometrium collected fkom pregnant women (Embrey and
Momson, 1970), that the concenaations of PGF2, and PGEz in amniotic fluid and of
their metabolites in matemal plasma and urine increase at labour (see rev. Challis and
Lye, 1994) and that the inhibition of PG synthesis suppresses uterine activity and
prolongs the length of pregnancy (Anderson et al., 1981).
PGs are forrned fkom arachidonic acid, that is liberated fiom cellular
phospholipids by phospholipase C (PLC) and PLAz enzyme activity. Arachidonic acid
is metabolized by the cyclooxygenase activity of prostaglandin H synthase to the
unstable endoperoxide, PGG2, that is reduced to PGH2. PGHz is unstable and
isomerizes readily to PGE,, PGF2, and PGD2. PGs are metabolized by an oxidized
fom of nicotinamide adenine dinucleotide type 1 15-hydroxyprostaglandin
dehydrogenase to form 15-keto compounds. The 15-keto compounds are reduced by
A ~ ~ ' ~ ~ reductase to 13- 14 dihydro- 15 keto derivatives that are oxidized and excreted in
urine.
1.5 PROCESTERONE AND ESTROGEN IN LABOUR
The placenta plays an essential role as an endocrine organ of pregnancy and is
actively involved in the synthesis and secretion of progesterone and estrogen. The
effects of progesterone and estrogen are complimentary, as well as, antagonistic to each
other (Roy and hdkumara, 1991). In addition to other effects , both steroids regulate
the formation of gap junction proteins and the synthesis of OT, and therefore have
critically important roles in regulating the key physiological events vital to the
initiation and progression of labour.
Progesterone inhibits the appearance of gap junctions between rat myometrial
ceIls and reduces cell-to-ce11 coupling (Garfield et al., 1990). Specificaily, Petrocelli
and Lye (1993) have shown that progesterone blocks the expected rise in Cx 43
mRNA at the Mie of labour in rat myomeaium. The myometnum under progesterone
dominance is characterized by its rehctoriness to stimulation by OT and PGF2,
(Neulen and Breckwoldt, 1994). These bioactive effects of progesterone are necessary
for the maintenance of human quiescence (see rev. Challis and Lye, 1994).
Matemd plasma progesterone concentrations increase corn the sixth week of
gestation on through to term (Tulchinslq et al., 1972). Progesterone is formed fkom
circulating low-density lipoproteins (LDL) Iûiked to cholesterol. LDL bind to specific
membrane receptors on the syncytiotropho blast and are internaiized by endocytosis.
Within the ceIl cytoplasm, LDL fuse with lysosomes and are hydrolyzed to amino
acids and cholesterol esten. Cholesterol esters are m e r hydroiyzed to fatty acids and
cholesterol and cholesterol is cleaved by the side chain cleavage (P,,,scc) eiuyme
to pregnenolone (C21) that is hydroxylated by the mitochondrial placental type 1 3L1-
hydroxysteroid dehydrogenase: A 5 4 isomerase e n y m e (30HSD) to progesterone.
Estrogens accelerate the biosynthetic pathways involved in placentai
progesterone production (Pepe and Albecht, 1993). They upregulate LDL receptor
expression and increase the expression of P4SOSCC in the baboon syncytiotrophoblast
layer. However, estradiol (E2) stimulates the development of myometrial gap junctions
in the pregnant rat (Mackenzie and Garfield, 1985) and Lye et al., ( 1993) have shown
that the rise in Cx 43 &A expression in labour is associated with an increase in the
plasma estr0gen:progesterone ratio. Estrogens also increase the responsiveness of the
myometrium to OT and PGFZ, (Leslie et al., 1994), therefore in general estrogens
promote myometrial contractility (see rev. Challis and Lye, L 994).
Matemal plasma concentrations of E2 rise throughout gestation (Tulchinsky et
al., 1972). The synthesis of estrogens in human pregnancy requires an obligatory
interaction between the placenta and the maternai and fetal adrenal glands. The human
placenta lacks the P45Ki7 enzyme and is therefore unable to convert C21 steroids
(progestins) directly to C 19 steroids (androgens). Estmgen (C 1 8) synthesis is
dependent on the production of dehydroepiandrosterone sulphate (DHEAS) in the
maternal and fetal adrenals. DHEAS is extracted fiom the fetal and maternai
circulations by the placenta. It is desuifonated to DHEA that is converted to
androstenedione. Androstenedione is readily aromatized to estrone and E2. The
majority of DHEAS produced in the fetal adrenal is converted in the liver to 16a
hydroxy-DHEAS before entering the placenta 16a-OH-DHEAS is cleaved to 16u-
OH-DHEA which is converted to 16a-OH-mdrostenedione and aromatized to estriol.
1.6 CORTICOTROPIN RELEASING HORMOIW
ln addition to steroid hormone synthesis, the human placenta the decidua and
the f e d membranes produce a large number of hypothalamic-like peptides which
ioclude luteinking hormone releasing hormone and CRH (Khodr and Siler-Khod.,
1978: Riley et al., 1991). These peptides which appear to be stnicturally and
bioactively identical to their hypothalamic counterparts. are thought to modulate
autocrine andlor paracrine mechanisrns within the uterus. This review will initially
discuss the properties of hypothalamic CRH. This will be followed by an in depth
explanation of the synthesis and secretion of hCRH-pl and discussion of the putative
roles of hCRH-pl in human pregnancy and labour.
CRH is a 41 amino acid peptide, initially characterized in the ovine
hypothalamus by Vale er al., (1 981). It is synthesized predominantly in the perikarya
of neurons within the medial pawocelldar region of the paraventricular nucleus (PVN)
and released into the hypophyseal portal system in response to stress (Vde et al., 198 1 ;
Antoni, 1983). The gene for prepro CRH, the biosynthetic precursor of CRH, was
isolated fkom a human genomic DNA library with an ovine CRH (oCRH)
complementary DNA (cDNA) probe (Shibahara et al., 1983). The deduced amino acid
sequence of human CRH exhibits seven amino acid substitutions when compared to the
ovine CRH (Vale et al., 1981): glutamic acid for glutamine (at position 2), alanine for
threorine (22), arginine for lysine (23), methionine for leucine (38), glutamic acid for
aspartic acid (39) and isoleucine for alanine (41). AU of these substitutions represent
changes in chemically sirnilar amino acids resulting fiom single nucleotide amino acid
substitutions except for the isoleucine/alanine substitution in the carboxyl terminus of
CRH (Shibahara et al., 1983: Vaie et al., 1981). The rat CRH (rCRH) gene has a
similar structurai organization to that of the human CRH gene and the deduced amino
acid sequence of human and rat CRH (&RH) appears to be identical (Rivier et al.,
1993). In addition, a well-conserved CRH amino acid sequence has also been isolated
and characterked in the mouse and the dog hypothalamus (Keegan et al., 1994; Mol et
al., 1994).
CRH acts on the corticotrophs of the pituitary to upregulate pro-
opiomelanocortin O M C ) mRNA expression and the release of adrenocoticotropin
hormone (ACTH). ACTH increases the expression of key enzymes involved in
corticosteroid synthesis in the fetal adrend gland (Fujieda et al., 1981) leading to an
increase in plasma glucocorticoid concentrations. Glucorticoids feedback on the
hypothalamus and the pituitary, via specific receptors to decrease ACTH release from
the pituitary (Yang et al., 1990; Lu et al., 1991).
1.6.1 CRB-Related Peptides
Sauvagine and urotensui 1 are 50 % homologous to hir CRH at the amino acid
level with hi& homology particularly in the carboxy tenninus (see Figure 1.4)
(Montecucchi and Henschen, 1981 ; Lederis et al., 1982; Ichikawa et al., 1982).
Sauvagine is a 40 amùio acid peptide, isolated fiom the skin of the frog Phylzomedusa
sauvogei (Montecucchi and Henschen, 198 1) and urotensin I is a 41 amino acid peptide
isolated from the caudal neurosecretory system of both Catostomus mersoni (sucker
fish) and C'yprinus curpio (carp fi&) (Lederis et al., 1 982; Ichikawa et al., 1 982). Both
sauvagine and urotensin I stimulate the release of ACTH from anterior pituitary ce11
cultures (Lederis et al., 1982; Tm. et al., 1990).
Recently, another CRH-related peptide has been isolated and characterized in
the rat midbrain and fiom a human genomic DNA library (Vaughan et al., 1995;
Donalcison et al., 1996). Urocortin is a 40 amino acid peptide that is highly
homologous to urotensin I (63%) and CRH (45%). This peptide also stimulates ACTH
release firom dispersed rat anterior pituitary cells in culture (Donalson et al.? 1996).
Urocortin mRNA and peptide have been identified in the human placenta and fetal
membranes (Petraglia et al., 1996b), corresponding to a site of CRH synthesis and
secretion.
1.7 HUMAN PLACENTAL CRH
Shibasaki et al., (1982) were the £kt to demonstrate CRH-like bioactivity in
human placenta. These investigators showed that CRH exûacted from purified
placental extracts, obtained fiom term spontaneous deliveries, was biologicaily active
in vifro, stimdated the release of ACTH and D-endorphin from cultured rat anterior
pituitary ceils. Later studies by Frim et al., (1 988) reveded that hCRH-pl mRNA was
expressed in human placental tissue in huma. placenta fiom the seventh week of
gestation. Human CRH-pl levels increased more than 20-fold during the last 5 weeks of
pregnancy, concomitantly with an increase in hCRH-pl peptide (Frim et al.. 1988).
These scientists reported that the expression of hCRH-pl mRNA and the leveis of
peptide were low in the nrst trimester of pregnancy, increased during mid-gestation and
increased exponentially during the 5 weeks prior to labour (Frim et al., 1988). Sasaki
et al., (1988) reported the presence of three molecular species with CRH
immunoreactivity in extracts of human terni placenta. While, the major species eiuted
with KRH the other two had apparent molecular weights of 18 kiloclaltons @Da) and 8
kDa, respectively. These two higher molecular weight species did not stimulate ACTH
release fkom cultured rat anterior pihiitary cells (Sasaki ei al., 1988) and are thought to
represent a precursor and a biosynthetic intermediate. The CRH gene is expressed in
human placenta and hCRH-pl mRNA is the same size (1.3 kilobases; kb) as
hypothalamic CRH (Grino et al., 1 987)
Jones et al., (1989b) showed that hCRH-pl was synthesized in human p lacen~
decidua and fetal membrane ce11 cultures. These investigatoa shawed that significantly
more CRH was produced in placental and fetal membrane tissues collected afier
spontaneous term labour than tissues obtained at elective cesarean sections. It is now
generally accepted that CRH immunoreac tivity is present on the syncytiotro phoblast of
term placenta (Riiey et al., 1991). Earlier studies by Saijonmaa et ai., (1988) and
Petraglia et al., (1987) had localized immunoreactive (ir)-hCRH-pl on the
cytotrophoblasts in early and tem human placenta, respectively. However, Riley et al.,
(1991) and showed that ir-hCRH-pl is present on the syncytiotrophoblast layer but that
it was undetectable in the cytotrophoblasts (Cooper et al., 1994; Warren and Silverman,
1 995; Perkins and Linton, 1 995). Ir-hCRH-pl staining on the syncytiotropho blast layer
increased fkom the ninth week of gestation onwards. Positive staining for ir-hCRH-pl
was also present on the intemediate trophoblasts cells, the chorionic tmphoblasts and
the epithelial cells of the amnion in term placenta (Riley et al., 199 1; Warren and
Silverman, 1995; Perkins and Linton, 1995). Ir-hCRH-pl is also present in the decidua
and increases with gestational age (Petmglia et al., 1992).
Human CRH-pl is secreted into the matemal and fetal circulation and released
into the d o t i c tluid throughout human pregnancy (Sasaki et al., 1987; Goland et al.,
1988). Plasma concentrations of ir-hCRH-pl in the matemal and fetal circulation
increase in p d e l to hCRH-pl levels in the placenta (Frim et al., 1987). In the first
trimester of pregnancy plasma concentrations of h-CRH-pl are barely detectable (5.9 f
0.8 pg/mL) (Sasaki et al., 1987; Goland et al., 1986; Maser-Gluth et al., 1985;
Campbell et al., 1987). Plasma ir-hCRH-pl concentrations rise progressively from the
second trimester of pregnancy (35.4 f 5.9 pg/mL) (Sasaki et al., 1988) to term
(- 1 O00 pg/mL) (Camp bel 1 et al., 1 987; Sasaki et al., L 987). Wiîh a significant increase
throughout labour (- 4000 pg/mL) (Okarnoto et al., 1989; Petraglia et al., 1990).
h e d i a t e l y post-partum matemal plasma hCRH-pl concentrations decrease rapidly to
barely detectable levels (Sasaki et al., 1987; Campbell et al., 1987; Schdte and Healy
1987; Goland ef al., 1988; Stalla et a[., 1 989). No information has been reported on the
concentrations of the other CRH mokcular weight species.
Hunan CM-pl is thought to be secreted predominantly into the matemal
circulation. At the time of delivery, mean umbilical cord hCRH-pl concentrations are
1000 fold lower than t h s e reported in the maternal circulation (Sasaki et al., 1988).
The umbilical venous plasma hCRH-pl concentration (50.6 + 6.1 pg/mL ) was
signiticantly greater than that in simultaneouslysbtahed umbilicai arterial plasma
(41.8 t 4.9 pg/mL) (Schulte and Healy, 1987). The decrease in matemal plasma
hCRH-pl concentrations post-partum and the elevated concentrations of hCRH-pl in
the umbilical vein when compared to the urnbilical artery c o n f i the placenta as a
source of hCRH-pl. Human CRH-pl concentrations, in the amniotic fluid increase
progressively throughout gestation (Maser-Gluth et al., 1 987; Campbell et al., 1 987:
Laatikainen et al., 1988). Contrary to plasma hCRH-pl concentrations, however,
amniotic fiuid concentrations of hCRH-pl are not altered during labour (Petraglia et al.,
1990).
M a t e d plasma hCRH-pi concentrations are significantly elevated in PTL
patients in comparison to gestational-age matched controls. This rise is apparent prior
to the onset of labour (Linton et al 1987; Warren et al., 1992; McLean et al., 1 995).
CRH binding protein (CRH-BP) is a 37 kDa peptide that binds hCRH-pl and
inhibits its ACTH-releasing activity in anterior pituitary cultures (Orth and Mount,
1987; Linton et al., 1988). In addition CRH-BP decreases PG release fkom cultured
matemal decidua and dampens the potentiated contractile activity of CRH and PGF2,
on human myometnum in vitro. CRH-BP is predominantly secreted by the matemal
Iiver (Potter et al., 1991) but has also been identified on the syncytiotrophoblast, the
intermediate trophoblast cells, the stroma1 celIs of the decidua and the epithelial celIs of
the amnion (Petmglia et al-, 1993; Rarnirez et al., 1995).
Matemal plasma CRH-BP concentrations (- 5 nrnol/L) fa11 sigrilficantly (- 2
nmoVL) in late pregnancy retuming to basal concentrations 48 hours postpartum
(Linton et al., 1993). In pregnancies complicated with PTL the decrease in plasma
CRH-BP concentrations is more pronounced when compared with gestationai-age
matched controls (Orth, 1992). Mean CRH-BP concentrations, in fetal plasma also
decrease pnor to term (Linton et al., 1993). The mechanisms controlling the decrease
of CRH-BP concentrations, in both pre-term and term pregnancies are d l under
investigation. Woods et al., (1994) suggested that the association of CRH-BP with
CRH in blood may triggers the clearance of CRH-BP h m the circulation. A decrease
in plasma CRH-BP in both the matemal and the fetal circulations, would result in a net
increase in the circulating concentrations of "biologically active" hCRH-pl (Linton et
al., 1988), primarily because the majorïty of hCRH-pl in both the matemal and fetal
circulations is bound to CRH-BP (Salminen-Lappalainen and Laatikainen, 1 990).
1.9 OTHER RIZGULATORS OF CRB
It is well established that glucocorticoids inhibit hypothalamic CRH release in
humans (Vale and Rivier, 1977; Owens and Nemeroff., 1991 ; Orth, 1992). However,
in human pregnancy hCRH-pl concentrations nse paradoxically with plasma
concentrations of cortisol (Fencl et al., 1 980; Cam et al., 1 98 1). Robinson et al., ( 1 988)
and Jones et ai., (1989b) have shown that dexamethasone, a synthetic glucocorticoid,
stimdates hCRH-pl synthesis and secretion from prhary cultures of term human
placen-ta, decidua, amnion and chorion. In addition, the neurotransmitters.
norepinephrine and acetycholine, stimulate hCRH-pl output (Petraglia et al., 1989). in
agreement with the mechanisms regdating hypothalamic CRH secretion interleukin-
la, neuropeptide Y, angiotensin II ( h g II), arginine vasopressin and OT increase the
release of hCRH-pl in vitro from cultured trophoblast cells (Petraglia et ai., 1991). In
contrast, progesterone and nitric oxide (NO) inhibit hCRH-pl output fiom tenn human
placenta, decidual, amnion and chorion (Jones er al ,1989b; Karialis and Mazoub,
1994; Sun et al., 1994).
1.10 MYOMETRIAL CONTRACTILITY
CRH does not have the intrinSic ability to stimulate human myometrial activity.
Quartero and Fry (1 989) have shown that CRH has both a priming and potentiating
effect on the myometrial contractile response to OT. In addition, hCRH-pl stimdates a
3 to 4 fold increase in ir-OT release fiom placental cells in vitro (Florio et a l , 1996).
The rise in circulating concentrations of hCRH-pl in pregnancy coincides with an
increase in the sensitivity of the myometrium to OT (Caldeyro-Barcia and Serono,
196 1 ; Fuchs et al., 1994) and may niggest a role for h-CRH in the regulation of OT
receptor nurnbers. McLean et al., (1994) showed a positive association between
matemal plasma hCRH-pl concentrations and the fkquency of uterine contractions in
women who entered labour following OT infusion.
CRH also enhances PGF, -stimulated myometrial activity (Benedetto et al.,
1994) and stimulates PGF2, and PGk release fiom primary cell cultures of placenta,
chorion and amnion (Jones and Challis, 1989a). A role for hCRH-pl in the initiation of
labour is also suggested by the presence of CRH binding sites in the myomehium
(HilIhouse et al., 1993) and the recent identification of CRH receptor mRNA
(Rodriguez-LinZres et al., 1998). This is central to our study and will be discussed
later.
1.11 OTHER ROLES FOR CRH
McLean et al., (1995) proposed that the materna1 plasma concentrations of
hCRHpl at 16-20 weeks of human pregnancy could be an indicator of the timing of
delivery and a determinator of patients at risk of PTL. The role of CRH as a rnarker of
PTL has been controversial, because of wide interpatient variations in plasma CRH
concentrations and the stability of hCRH-pl in plasma samples (Warren et al., 1992).
CRH-pl is a potent vasodilator in the human placenta vasculature and mediates
its vasoactive effects via NO (Clifton et al., 1994). in addition, it has been
hypothesized that hCRH-pl might contribute to the activation of the fetal pituitary
adrenai axis in late pregnancy, thus indirectiy contributing to prenatal lmg maturation
(Petraglia et al., 199 1 ) . F W y , it has k e n suggested that there is a local CRH-POMC-
ACTH axis within the placenta responsible for the increased concentrations cortisol in
matemal plasma and urine (Cam et al., 198 1 ; Goland et al., 1986; Margioris et al.,
1988). This suggestion was made based on the presence of POMC mRNA and POMC-
denved peptides in the placenta (Margioris et al., 1988).
1.12 CRET RECEPTORS
CRH binding sites have been identified in several tissues including the pihutary
gland (De Souza et al., 1984), the brain (De Souza et al.. 1985), the placenta (Petraglia
et al., 1990; Clifton et al., 1995) and the myomehium (HiIlhouse et al., 1993) by
radioligand bhding, autoradiographicai and in situ hybridization techniques. There are
at Ieast two major classes of mammalian CRH receptos, CRH-R 1 and C W R 2 .
Chen et al., (1993) recently reported the cloning of a cDNA encoding a CRH-
R1 fiom a human corticotropic tumour library. The CRH receptor is comprised of
seven putative membrane-spanning domains and belongs to the calcitonin/vasoactive
intestinal peptidelgrowth hormone releasing hormone subfamily of G-protein coupled
receptors. The cloned CRH-RI cDNA encoded two -apparent spliced forms of the
receptor, CRH-Rla and CM-RI P (Chen et al., 1993). CRH-Rla encodes a 41 5
amino acid peptide and appears to be the predominant form ( see rev. Dieterich et al.,
1997). CRH-RlP encodes a 444 amino acid peptide which has not been identified in
any other species or specific tissue other than the human piniitary. Recently, cDNA's
encoding CRH-RI homologs have been isolated and characterized in the human cortex,
braiostem, hippocampus and testis, mouse pituitary (Vita et al., 1993) and rat brain
(Perrin et al., 1993). Two other spliced variants of CRH-R1 have been reported; a
receptor with a 40 amino acid deletion in the amino terminal domain (Ross et a[.. 1994)
and a truncated receptor that contains a fhmeshift that only encodes the fïrst 185 amino
acids of CRH-Rla (Chang et al., 1993) (see Table 1.1). Al1 species homologs of the
CRH-RI are of comparable size and are 98% homologous to one another.
CM-RI is a glycoproteh (Grigonadis and D e S o w 1989) and contains five
potential N-linked glycosylation sites in the N-terminal extracellular domain (Chen et
al., 1993). While the amino acid sequence of rat brain and rat pituitary CRH-R1 are
97% identical differences have been reported in their molecular weights which appear
to be due to differentiai tissue specinc glycosylation of CRH-R1 (Grigoriadis and De
Souza, 1989a). In addition, five potential protein kinase C (PKC) phosphorylation sites
have also been identified in the first and second intracellula. loops and in the
C-tenninal tail (Chen et al., 1993) Both a casein kinase II and a protein kinase A
(PKA) phosphorylation site are present in the thud intracellular loop (Chen et al., 1993;
Perrin et al., 1993).
CRH-R2 is encoded by a separate and distinct gene This receptor subtype has
been cloned fiom rat brain (Lovenberg et al., 1995a), human (Liaw et al., 1996), mouse
heart and skeletal muscle (see Table 1.2) (Kishimoto et al., 1995; Perrin et al., 1995;
Stenzel et al., 1995). Two alternatively spliced forms of the receptor, CRH-RZa and
CRH-WP, encoding a 41 1 amino acid peptide and a 43 1 amino acid peptide
respectively, have been identified. Their amino acid numbers cliffer in that the fïrst 34
amino acids in the N-tenninal of CRH-R2a are substituted with a unique sequence of
54 amino acids in the CRH-R2B protein (see Figure 1.5). CRH-R2a and CRH-WP
have been identified in distinct anatomical zones. (Lovenberg et al., 1995b). CRH-R2a
mRNA is abundantly expressed in rat brain and CRH-R2P mRNA is predorninantly
expressed in the rat heart and skeletal muscle. In the human CRH-R2a is the prevailing
CRH-R2 subtype in the heart and skeletal muscle (Vaidenaire et al., 1997).
The CRH-R2 variants ais0 have 5 potential N-linked glycosylation sites,
identical to those in CRH-RI, as well as potential PKC phosphorylation sites
(Lovenberg et ai., 1995a). Both rat and human CRH-R2a share 70% sequence identity
to CRH-Rla with the clifference being the insert of amino acids in the first cytoplasmic
loop (Lovenberg et al., 1995a). The receptors show high homology particularly in
respect to the complete identity of the third intracellular domain (see rev. Dieterich et
al., 1997). It has been reported that a major determinant of the coupling of G-proteins
receptors to adenylate cyclase is located in the third intraceIlular loop (Okamoto et al.,
1991). This is consistent with the demonstrated abilities of both CRH-RI and CRH-R2
to increase intracelluiar CAMP (Chen et al., 1993; Lovenberg et al., 1995) and suggests
conservation of second messenger function between the CRH-R1 and the CRH-EU
receptor genes. Nabhan et al., (1 999, however, have reported that CRH-R.2 is not well
coupled to the G-protein in LLCPK-1 ceils and requires elevated levels of CRH to
stimulate intracellular CAMP levels. The CRH-RI gene is present in the 17q12-q22
interval of the long arm of chromosome 17 (Polymeropuoulos et al., 1995) but the
CRH-Rî gene has not yet been locaiized.
CRH binds to its receptor to fom a complex that subsequently binds and
activates a guanosine 4' triphosphate stimulatory binding protein (Gs), causing GDP to
dissociate nom the inactive G protein, and GTP to bind the alpha (a) subunit of the G
protein. The G protein a subunit dissociates fiom the py subunits, binds and stimulates
the adenylate cyclase enzyme, which catalyses the intracelldar CAMP synthesis fiom
ATP. The released CAMP can bind CAMP dependent PEU. It is well documented that
CRH binds its receptor with high affinity and stimulates adenylate cyclase activity
leading to increased intracellular cyclic adenosine 3'5' monop hosphate (CAMP) in the
rat pituitary (Labrie et al., 1982; Aguilera et al., 1983; Bilezikjian and Vale. 1983), the
brain (Battaglia et al., 1987; Chen et al., 1986) and the myometrium (Grammatopodos
et al., 1994). The EC, values in stimulating CAMP production in the above rnentioned
tissues are similar (ES, = 0.2 - O.5nM). In addition, the binding of 125~-radiolabeled
oCRH ('"1-OCRH) to the receptor i s inhibiteci by guanyl aucleotides, confirming that
CRH receptors are coupled to the adenylate cyclase pathway by a guanyl nucleotide
regdatory protein (Aguilera et al., 1987).
It has been suggested that optimal CRH receptor expression requires both the
PKC and the adenylate cyclase pathway (Lutz-Bucher et al., IWO). The PKC pathway
appears to be an important modulator of CRH function. A role for PKC in regulating
CRH function is substantiated by the ability of arginine vasopressin and angiotensin II,
which use PKC as their secondary messenger, to potentiate CRH-stimulated ACTH
from the pituitary (Abou-Sambra et al., 1986) and the placenta (Petraglia et al., 199 l),
and the demonstrated ability of direct activators of the PKC pathway to stimulate
ACTH secretion and CAMP production in comcotrophs (see rev. Dieterich et al.,
1997). Possible cross taik between PKC and adenylate cyclase is of interest as in
certain sites, such as the rat Leydig ceus, CRH receptoa are aot directly coupled to a
G-protein, but mediate their actions via the direct or indirect actions of PKC (Dufau et
al., 1989). Activation of several receptors fkom the 7 transmembrane receptor family
has shown that they are able to couple to PLC and stimulate phosphoinositol hydrolysis
when in the presence of high receptor or Ligand concentrations (Jelinek et al., 1993;
Spengler et al., 1993; Usdin et al., 1993; Nabhan et al., 1995).
The pharmacological properties of CRH binding sites are usually determined by
the relative ability of a varîety of CM-related and unrelated peptides to displace
specifically bound 12*1 oCRH or I2*l ratmuman CRH (rh CRH) ( see rev. Dieterich et
al., 1997). The binding of 1 2 S ~ ~ ~ ~ to specific sites in the CNS and the periphery
appears to be both saturable and reversible (De Souza es al., 1984; 1985; Hillhouse et
al., 1993; Hatzoglou et al., 1996). In addition, CRH and CRH d o g s bind these sites
with high &ty bkding, an apparent dissociation constant (K.,& of 0.2 - 0.8 nM. The
simila. Kd of CRH and CRH fragments suggest that similar structurai ligand
requirernents are shared by CRN receptors in the pituitary, the brain, the placenta and
the myornetrium. Recent work by Liaw et al., (1997) has found three regions in a
chimeric receptor construct of CRH-Rla and CRH-ma, that were required for the
optimal binding of ' 2 S ~ - r h CRH andor receptor activation.
Increased exposure of target tissues to peptide hormones is u d y Linked to the
downregulation and desensitization of the homologous receptors. Desensitization
reflects either a short term, (affinity change, conformational alteration of the receptor
protein, uocoupling fiom the corresponding G-protein) or a long-term (downregulation
of the receptor) adaptation process (Dietench et al., 1997). CRH regulates the
expression of its receptor in a tissue-specific manner, such that exogenous C RH
decreases CRH receptor concentrations in the rat anterior pituitary both in vivo and in
vitro (Wynn et al., 1983; 1988; Hauger and Aguilera., 1993; Lu et PI.. 1994; Sakai et
al 1996; Pozzoli et al., 1996) whereas intracerebroventncular administration of CRH
stimulates a significant increase in CRH-RI mRNA in the PVN (Mansi et al.
1996).The expression of CRH receptors are also regulated by other factors such as
stress, adrenaiectomy and glucocorticoids. Stress leads to a rise in circuiating C M ,
causing a decrease in C M receptor numbers in the hypothalamus (Makino et al.,
1995). Adrenaiectomy resufts in an increase in cuculating hypothalamic C M , due to
withdrawai of adrenal cortisol negative feedback. This increase in plasma CRH causes
a û-ansient decrease in CRH-RI mRNA expression in the anterior piniitary and an
increase in CR,-RI rnRNA expression in the PVN (Luo et al., 1995). This effect was
removed with exogenous glucocorticoid administration (Wynn et al., 1983). High
circuiating concentrations of glucocorticoids are known to decrease the expression of
CRH receptors in the PVN, the anterior pinlltary and the brain (Makino et al., 1992)
and it has been proposed that hi& concentrations of glucocorticoids act synergisticaily
with CRH to decrease CRH receptor mRNA in the anterior pituitary (Makino et al.,
1995). Recent studies by Makino et al., (1997) have shown that CRH-Rla mRNA and
CRH-R2a &A are differentially regulated in the rat hypothalamic PVN after
adrenalectorny and glucocorticoid administration: CRH-Rla mRNA was demeased in
foiIowing these treatments whereas CM-R2 mRNA Ievels were unaltered. This
suggested that the expression of CRH-RI mRNA and CRH-R2 mRNA is differentially
regulated in the rat brain. Ttis may also be reflected at other sites that express both the
receptor subtypes.
CRH binds CRH-R1 with a higher affinty than CRH-R2 (Lovenberg et al..
1995a) whereas urotensin 1 and sauvagine bind CRH-R2a with a higher aanity than
CRH in transfected rnouse Ltk- ceUs and in rat heart and muscle cells (Lovenberg et
al., 1995a; Perrin et al., 1995). It has been suggested that CRH is not the preferential
ligand of CRH-W implying the possible existence of another endogenous ligand for
this receptor subtype. A possible candidate is urocortin which has been proven to bind
the CRH-FU, with a higher e t y than CRH (Vaughan et al., 1995).
Despite an understanding of some of the phannacological and second
messenger characteristics of CRH receptors, the mechanisms involved in the
differential post-translational processing of CRH receptors remain to be elucidared.
DBerences have been reported in the molecular weight of CRH receptors present in
the rat pituitary (58 H a ) (Grigoriadis and De Souza 1988; 1989b), the rat brain and the
human placenta (75 kDa; Grigoriadis and DeSouza, 1989a; De Souza et al., 1985;
Grigonadis et al., 1993; Clifton et al., 1995) and the human myometriurn (40 and 45
ma; Castro et al., 1996). Grigoriadis et al., (1989a) deglycosylated the carbohydrate
moiety nom the rat brain and pituitary CRH receptor and found that it was
predomimmtiy composed of N-acetyl@ucosamine a d o r terminal sialic acid residues,
as well as some hi& mannose c h a h . The native CRH receptor protein revealed, f i e r
deglycosylation, had molecuiar weight of 40 - 45 kDa This was consistent with the
molecular weight of CRH-Rl (Chen et al., 1 993).
Differences can occur in the CRH receptors at the transcriptional, the post-
transcriptional, the translational and the post-translational level. To understand the
mechanisms functioning at these each of these stages it is important to determine the
site of expression of CRH receptor mRNA and the protein levels. The regionai and
cellular expression of CRH receptor message bas been characterized extensively in the
rat brain and the rat pihiitary (Potter et aï., 1994; Chalmers et al., 1995:). These studies
report that CM-RI mRNA expression is abundant in the cerebrd cortex, the
subcortical limbic structures. the amygdala and in the anterior and intemediate lobe of
the pituitary. CRH-R2 &A expression was found in distinct subcortical regions of
the rat brain including the lateral septal nucleus, the venûomedial hypothalamic
nucleus and the choroid plexus (Chaimers et al., 1995). It was also measured at lower
levels in the olfactory bdb, amygdaioid nuciei, the paraventricular and supraoptic
nuclei of the hypothalamus, as well as in the cerebrai arteries throughout the brain.
Based on the separate pattern of distribution of CRH-R1 mRNA and CRH-R2 mRNA
in the brain it has been proposed that CRH-RI is pRmarily a neuroendocrine receptor
mediating the bioactive effects of CRH in the pituitary and that CRH-R2 is involved in
the hypothalamic nemendocrine, aidonomic and behavioral actions of central CRH
(see rev. Dietench et al., 1997).
The major* of the work reported on CRH receptor expression has been in the
brain. However the pleiotropic effects of CRH in the human (Quartero and Fry, 1989;
Clifton et ai., 1994) suggests the presence of putative CRH recepton at multiple other
sites. Recentty CRH receptors have been identified within the structures of the human
utems including the placenta and the myometnum. This wodd suggest CRH may play
a role in the mechanisms leading to parturition.
i 25 1-oCRH binding sites, have been localized almost exclusively on the
syncytiotrophoblast of human placenta (Hatzoglou et al., 1996) and are thought to
represent a single population of CRH receptos (Clifton et al., 1995; Hatzoglou er al.,
1996). The buiding of L 2 5 ~ - o ~ ~ ~ and CRH analogs to the placenta is dependent on
t h e , temperature, pH and the presence of divalent ions and reversed on addition of
excess oCRH (Clifton er al., 1995; Hatzoglou et al., 1996; Saeed et al., 1997).
Interestingly, the number of ' 2 5 ~ - o ~ ~ binding sites in placenta obtained d e r vaginal
delivery was increased compared to placenta delivered by elective cesarean section.
This suggests an upregulation in CRH receptor numbers in the placenta at the time of
labour in human pregnancy (Petraglia et al., 1990; Clifion et al., 1995). Clifton et
a1.,(1995) also reported that in vaginally delivered placenta, the CRH binding sites had
a much lower aanity than in elective cesarean delivercd placenta, they suggested that
the high concentrations of hCRH-pi in the maternai circulation at term could regulate
the binding properties of the CRH receptor. Despite the apparent presence of CRH
receptors in the human placenta, no midies have, as of yet, examined the expression of
the CRH receptor sub~rpes in this tissue or detemiined if these mbtypes are
differentiaüy regulated with pregnancy andor labour.
Specific '%CRH binding sites are present in human rnyornetrium (Willhouse
et al., 1993). These bhding sites display similar properties to those in the placenta
(Clifton et al., 1995). A majoriv of the studies investigating the binding and second
messenger properties of the CRH receptor in the human myometrium have been
performed by the group of Hiilhouse and Grammatopoulos. These investigaton have
used myometnal samples solely fiom the lower uterine segment, have failed to
differentiate between the existence of C M - R I and/or CRH-R2 and have not studied
receptor concentrations or regdation throughout the time of labour.
Recently CRH-R1 mRNA and CRH-R2 mRNA expression have been
characterized in myometrium fiom nonpregnant and pregnant women (Rodriguez-
Linares et al., 1998). Hillhouse et al., (1993) had initially identified the presence of a
single population of CRH recepton in the human myometrium. Subsequently they
reported the presence of multiple isoforms of the CRH receptor in the rnyometrium
collected fiom nonpregnant and pregnant women (Grammatopoulos et al., 1995). The
CRH receptor is reported to have a higher afnnity in the myometrium of pregnant
women, when compared to the myometriurn of nonpregnant wornen. While this
suggests a role for CRH in myometrial function in pregnancy, it fails to s p e c e
whether there is a differential change in the affinity of al1 the CRH receptor isoforms. It
appears that other binding properties of CRH binding sites are in altered in pregnancy.
In the myomeeiurn of nonpregnant women, the binding of '%OCRH mse with
hcreasing concentrations of myometrial membranes, then stabilized at a plateau, while
in the rnyometriurn of pregnant wornen. the binding of 1 2 5 ~ - o ~ ~ myometrid
membranes increased to a peak, and then decreased (Grammatopouios and Hillhouse,
1994). The authors suggest that the presence of an inhibitor of CRH bindkig exists in
the myometrium in t e m pregnancy. This is in contmst with the previous report fkorn
this group that demonsîrated that the CRH receptor increased in affinity for CRH in
late pregnancy (Hillhow et al., 1993). However this is in agreement with recent work
from this group where they suggest that in human term pregnancy OT is able to
desensitize the CRH receptors in the myometrium by stimulating PKC-mediated
phosphorylation of the receptor (Grammatopouios et al., 1997). A role for CRH in
regulating myometrial tone in pregnancy is evidenced by the ability of CRH to
potentiate contractions of human myomeaium in the presence of OT and PGF2,
(Quartero and Fry, 1989; Benedetto et al., 1994). Despite the demonstrated ability for
CRH to act on human myometrium and the rise in plasma hCRH-pl concentrations
throughout labour (Okamoto er al., 1989) no studies have examined the change in CRH
receptors at the time of labour in the human myometrium.
The ability of CRH recepton to mediate the actions of CRH is dependent
largely on the secondary rnessenger to which the receptor is linked. CRH activates a
dose-dependent increase of both CAMP and PGEz fkom hurnan myometrial membranes
obtahed in term pregnancies. This indicates that CRH recepton in the myornetrium in
term pregnancies are coupled to both the adenylate cyclase and the cyclo-oxygenase
secondary messenger pathways (Grammatopoulos et al., 1994). The ability of the CRH
receptor to stirnulate an increase in iniracellular CAMP, via the adenylate cyclase
pathway, is reduced at the end of pregnancy concurrent with the ability of C M to
stimulate PG synthesis (Grammatopoulos et al., 1994: 1996). This would suggest a shift
tiom the relaxation sbte of the myometrium, which is associated with increased
intracellular CAMP, to the contractile state, associated with cyclooxygenase activity at
term. However, no studies have examined the secondary messenger pathway
mechanisms associated with the CRH receptors at the time of labour or post-parnim.
This review has focused on the roles and mechanisms of CRH and the CRH
receptor subtypes in human pregnancy . The human placenta secretes increasing
concentrations of hCRH-pl into the matemal circulation in the Iast trimester of
pregnancy and throughout labour, suggesting a d e for this hormone in the
mechanisms of parturition. The presence of CRH receptors in the human myometrium,
suggests a local effect of CRH on the muscular component of the utenis. The
demonstrated ability of CRH to stimulate PG synthesis and to enhance the contractile
activity of the human rnyometrium, suggests CRH plays an active role in human
parturition. To potentiate the reported roles of myometrial CRH receptors in the
regdation of myometrial tone in labour, the mRNA expression and peptide
concentrations of the CRH receptor subtypes have to be d e t e d e d both in the absence
and presence of labour. Furthennore, to d e t e d e the putative regdatory mechanisms
involved in myomeaial CRH receptor expression, it is important to establish a suitable
mammalian model. Determining the presence and regdation of CRH recepton in the
human myometrium at the tirne of labour in pre-term and term pregnancies is important
to fùrther our knowiedge in the roie of CRH in the mechanisms of parturition.
Figure 1.1 Schematic di- of the intrauterine environment showing the position of the matemalànd the fetal tissues in pregnancy. A. The membranes (left to right): the myometrium, the decidua w u , the chorion and the amnion. B. The membranes at the site of implantation (righ? to lefi): the perimetrium, the myometrium, the decidua basalis, the placentai villi, the chorion and the amnion (Modiûed fiom Pritchard et ai., 1995).
Figure 1:2 Schematic d i a p m showing the change in the size and shape of the utem in term pregnancy and the development of the upper active and the lower passive segment at the time of Iabour (Modified from Pritchard et al., 1985).
Uterus in thc hnpregnant U'ornan Cterus at thc Time of Labour
Figure 1.3 Schematic diagram depicthg the high intensity and long duration uterine contraction tracings in the upper active segment and the relative absence of contractions in the lower passive segment (Modined fiom Pritchard et al., 1985)
Figure 1.4 Cornparison of the amino acid sequences of human C M , &og sauvagine, sucker urotensin 1 and carp urotensin 1. The one letter amino acid notation is used. The sets of identical residues are enciosed in solid lines (modified fiom Shibatiara ei al., 1983)
M 1 Methionine 1
G H
Glycine Histidine
0- 1 Glutamine 1
N P
Asparagine Proline
I
Y 1 Tyrosine
- - -
R S
Arginine Serine
Figure 1.5 Schematic diagram depicting the seven tranmiembrane structure and the amino acid sequence of CRH-R2a and CRH-R2P. The arrows point to the site in the N terminus where the sequence of CM-R2a and CM-R2P diverge (Modified fkom Chalmers et al., 1 996)
Symbol Identification
Yeilow shaded amino acid amino acid specific to CRH-R2a
Red shaded amino acid
1
1 'P 1 Potential sites for the phosphorylation of PKC 1
amino acid specific to CM-RZP
I
Blue shaded amino acid amino acid that differs between C M - R l and CRH-R2
h Potentiai N glycosylation sites
Table 1: Different Species Homolop of CRH-RI
Perrin et al., 1993
Vita et al., 1993
Ross et ai., 1994
Source of cDNA Humau corticotropic tumor library
Rat brain cDNA iibraiy
Mouse piniitary tumor celf (AtT2O) and human brain cDNA library Cloned cDNA based on identifieci regions amongst ali known rnernbers of 7-TMD Gs coupled receptors
Recep tor Expression Human pituïtary, rat pituitary and braîn
Rat brain
w
r
Mouse pituitary and human brain
Eiuman hippocampus and fetai brain cDNA library
Rat brain
h l m a n brain
Binds CRH to increase CAMP Uitracellular levels
- - - -
Receptor Pro perties 415 aa
Splice variant with 29 amino acid insert in h putative intracellular loop. Receptor with 12 diEerent aa in cornparison to CRH-Rla
Tnincated splice variant containing a
-- --
Second Messenger Pathway Binds rat/hman CRH with high atfinty to increase intracelluiar CAMP levels.
Binds C M to increase CAMP intraceUular levels
eameshift that only encodes the first 185 aa of CRH- R l a
- - -
The receptor has a 40 aa deletion in corn parison to CRH-R 1 a
Binds CRH with hi& affin@ to increase intracelluiar CAMP tevels.
Needs eievated concentrations of CRH to increase intracellular c.hW levels-
Table 2:Different Species Homologs of CRH-R2
Authors
Pemn et ai-, 1995
tovenbe rg et al., 1995
Klshimot O et ai., 1995
Liaw et al., 1996
Source of cDNA
Mouse heart cDNA library
Rat hypothal amus cDNA llibrary
Mouse heat-î cDNA l i brary
Mouse ha r t cDNA library
Hurnan front cerebral CO rt ex cDNA library
Receptor Expression
Mouse heart, GIT, epidydimis and bain
~redominantl y in rat brain, heart and skeletal muscle
Mouse heart and skeletal muscle
Mouse heart, bain and lung
Receptor Properties
431 aa 16 additional aa in the N- terminal in cornparison to CRH-Rla 41 1 aa
Splice variant with a different N-terminal dornain encoding a 431 aa
70 % homology with CRH- R1 a
Second Wessenger Pathway
Human front cerebral cortex
Binds sauvagine with a higher affinity than CRH to stimulate CAMP
41 1 aa
94% identical ta rat CRH- R2a
Sauvagine is > 10 fold more potent in stimulating cAMP levels Binds sauvagine with a higher affÏnity than CRH to stimulate CAMP
Nomenclature
CaAPTER 'IWO: RATIONALE AND HYPOTHESIS
2.1 STUDY 1
CRH-RI mRNA and CRH-R2 mRNA are expressed in the myometrium of
nonpregnant and pregnant women (Rodriguez-Linares et al., 1 998). CRH acts
synergisticaily with OT (Quartero and Fry, 1989) and PGF, (Benedetto et al., 1994) to
potentiate myometriai contractility suggesting it may contribute to the complex
mechanisms involved in the omet and progression of labour in humans. To substantiate
the reported d e s of CRH, in the myornetrium, the mRNA expression and the protein
concentrations of both the CRH-R1 and CRH-EU have to be detennined in the upper
fiuidal region and the lower segment of the utem both in the absence and presence of
labour. Furthemore, the expression of CRH-RI mRNA and CRH-RZ mRNA in human
myometrium have to be compared in pre-tem and term pregnancies to assess the
regulatory effects of gestational age and PTL on CRH receptor expression.
Based on the presence of elevated levels of bioactive hCRH-pl in the matemal
circulation at the time of labour, in both term and PTL pregnancies, the presence of CRH
receptos in the rnyometrium and the ability of CRH to enhance myometnai activity we
hypothesized that the expression of CRH-RI mRNA and/or CRH-R2 mRNA expression
and protein concentrations in the human myometrium would be upregulated at the tirne of
labour.
C h u specific aims were:
1. To estabIish the presence of CRH-RI mRNA and CRH-R2 mRNA and the
locaiization of their proteins in human myometrium h m both nonpregnant patients
and pregnant patients.
2. To detennine whether CRH-RI mRNA andor CRH-R2 mRNA expression in
human myometrium is upregulated at the t h e of labour in term andor in pretem
pregnancies.
3. To determine whether CRH-R1 mRNA expression is differentially expressed in the
myometrium fiom the upper fûndal and the lower segment in nonpregnant,
pregnmt, labouring and postpartum.
4. To determine whether CRH-RI mRNA andor C M - R 2 are expressed in human
decidua, chorion and amnion,
5. To determine ZCRH-RI &A and/or CRH-R2 mRNA expression was increased
at the time of labour in the decidua, chorion and amnion..
CRH receptors are abundantly expressed in rat brain and rat pituitary (Wynn et
al., 1983; Agudera et al., 1987). Extensive studies have been done in the rat brain and
pituitary to determine the factors regulating pituitary and brain CRH receptor
expression (Makino et al., 1995; Pozzoli et al., 1996; Sakai et al., 1996). CurrentIy, no
studies have examined the factors regulating the expression of CRH receptors in the
myometrium. Determinhg the endogenous factors regdating the transcription,
translation and /or postranslationd expression of CRH receptors in the myometrium is
a prerequisite to establish the role of CRH in pregoancy. Because of the obvious
diniculties of performing these studies in vivo it is crucial to identfi a mammalian
mode1 that expresses CRH-RI mRNA a d o r CRH-R2 mRNA in the myometrium.
The goal of our study was to establish a marnmalian mode1 to study the
regdation of CRH receptor expression in the myometrium in pregnancy, in labour and
post-partum. Based on the identical structure of the human and the rat CRH receptors
and the simila. bioactivity of human and rat CRH peptide we hypothesized that CRH-
R1 mRNA and CRH-EU mRNA expression would be increased at the time of labour in
rat myometrium.
Our specific aims were:
1. To detennine if CRH-RI mRNA and CRH-R2 mRNA are expressed in the rat
myometrium fiom day 15 of gestation to 1 day postpartum.
2. To determine if CRH-RI mRNA and CRH-R2 mRNA expression is increased in rat
at the time of labour.
3.1. INTRODUCTION
The human uterus differentiates into an actively contracthg upper segment and a
relatively quiescent lower segment at the time of labour (Pritchard et al., 1985). The
factors involved in this anatomic functional Merence of the uterus remain unclear, CRH
is a 41 amho acid peptide hormone, that has both a priming and potentiating effect on
myometrïal contractility (Quartero and Fry, 1989; Benedetto et al., 1994). Two distinct
subtypes of CRH receptors, CM-RI and CRH-R2 have been isolated and characterïzed
(Chen et al., 1993; Vita et al., 1993; Lovenberg et al., 1995; Liaw et al., 1996). The
cloned cDNA of CRH-RI was isolated fiom human pituitary (Chen et al., 1993) and rat
brain (Perrin et al., 1 993). Recently, the cDNA of CRH-R2 was isolated fkom rat
(Lovenberg et al., 19951; 1995b) and human (Liaw et al-, 1995; Valdenaire et al., 1997)
and encodes two anatomically distinct spliced forms of the receptor (Lovenberg et al.,
1995a; 1995b).
The human placenta secretes increasing concentrations of hCRH-pl into the
materna1 circulation in the third tnmester of pregnancy (Goland et al., 1987 reaching a
peak at the time of labour (Okamoto et al., 1989; McLean et al., 1995). Recently CRH-
RI mRNA and CRH-R2 mRNA have been idenaed in the myometnum of pregnant
women (Rodriguez-Linares et al., 1998). This strongly suggests that CRH may play an
important role in human parturition. We hypothesized that the expression of CRH-RI
mRNA a d o r CRH-R2 mRNA expression and protein concentrations in the
myometrium would be upreguiated at the time of labour in human pregnancies. We
examined the expression of CM-R1 mRNA and CRH-R2 mRNA and their respective
proteins in human rnyometrium in term and preterm pregnancies prior to at the t h e of
labour. Moreover, we disthguished between myometrium collected fiom th: upper
segment and nom the lower segment of the utem. Finally we examined the level of
expression of CRH-RI mRNA and CRH-W mRNA in the decidua and fetal
membranes in term pregnancies prior to and at the time of labour so to establish if the
regulation of CRH receptor expression at the time of labour was specific to the
myometrium.
3.2.1 Biopsy Samples
Myometrial samples were collected fiom 2 groups of women: a) nonpregnant
and b) pregnant. In the nonpregnant, premenopausal women (age 42 f 1.5 years; n = 4)
the myometrid samples were collected fiom the lower region of the uterus at
hysterectomy, performed due to fibroid invasion. Pregnant patients (age 3 1 + 2.4 years),
were divided into the following groups: preterm no labour (mean gestational age
(MGA) = 32 weeks; n = 3, PTL (MGA = 32 weeks; n = 6), terni no labour (MGA = 39
weeks; n = 7) and term in labour (MGA = 39 weeks; n = 7). The myometrial samples
were collected h m the incision Iule withui the lower segment. Regional myometrial
samples were also collected fiom the both the upper and the lower segment of the
utenis collected in the nonpregnant patients (aged 40 + 1.3 years; n = 4) at
hysterectomy performed as a result of fibroids. Upper segment and lower segment
myometrial samples were also collected fiom subjects (aged 32 k 2.1 years; a = 4) at
elective cesarean followed by a hysterectomy performed for progressive cervical cancer
and in active labour (aged 3 5 years; n = 1 ) at a classical cesarean section performed due
to remove conjoint twins. Finaily, a myometrîal sample was couected fkom the upper
and the lower uterine segment at a hysterectomy performed post-parturn [aged 34
years; n=l) for disseminated intravascdar haemorrhage. The pregnant patients were
not admùiistered giucorticoids or oxytocin. All tissue samples were immediately snap
frozen in liquid nitrogen and stored at -80°C.
Decidua (n=8), chorion (n = 4) and amnion (n = 4) were coliected fiom term
patients in the absence (MGA = 39 weeks) and the presence of labour (MGA = 39
weeks). The individual chorion and the decidua were separated by gentle scraping and
immediately individually snap fiozen in liquid nitrogen. Al1 tissue biopsies were
coliected at Mount Sinai Hospital in Toronto, Ontario. informed consent was obtained
&orn al1 the patients and ethical approval for the study was obtahed nom the Bioethic
Cornmittee of Mount Sinai Hospital and the University of Toronto.
To obtain positive and negative control tissues virgin fernale Wistar rats (250-
280 grarns; Charles River Canada, St. Constant, Quebec, Canada) were decapitated and
piNitaries and liver, respectively. Sheep pituitarîes were also coUected fkom fetuses at
term delivery (&y 145 gestation) following euthanasia with Euthanyl (5 mL) via
cardiac puncture to use as positive controls. AU tissue samples were immediately snap
fi-ozen in liquid nitrogen and stored at -80°C.
Al1 animal experimental protocols were approved by the Samuel Lunefeld
Institute Animal Care Cornmittee and Animal Care C o d t t e e of the University of
Toronto according to the Guidelines of the Canadian Council of Animal Care.
3.2.2 Total EWA Extraction
Total RNA was extracted fiom the samples of myometrium, decidua, chorion,
and amnion using the methods desmibed by Chomcynski and Sacchi (1987). Bnefly,
fkozen tissue samples (2- 5 mg) were powdered under liquid nitrogen and homogenized
in 1 mL of a denaturing solution (4M guanidinium thiocyanate, 25 m M sodium citrate,
0.5% sodium lauroylsarcosine, 0.1M LJ-mercaptoethanol (v/v)) using an ULTRA-
TübUUC homogenizer (Janke & Henkel, IKA-Labortechnik, ON, Canada). Sodium
acetate b&er (0.1 mL of 2M ; pH 4) was added to the tissue homogenate, followed by
phenol (water-saturated) ( 1 ml) , and a chloroform-isoarnyl alcohol mixture (0.2 mL ;
49: 1). Each addition was foilowed by thorough rnixing . The samples were incubated on
ice for 15 minutes then cenaifuged (Sorvall RC-SB, Du Pont Instruments, MA, USA)
at 6,500g for 40 minutes at 4OC. The supernatant was tmnsferred to a fresh
polypropylene tube (12 mL; Becton and Dickinson, New Jersey, USA) and mixed with
isopropanol(1 mL) and incubated for 1 hou at -20°C to allow RNA precipitation. The
samples were centrifuged again at 6,500g for 1 hour 4°C. The redting RNA pellet was
dissolved in the same denaturing solution (see above; 1 mL), transferred to an
eppendorf tube (1.5 mL, Diamed, Ont, Canada) and precipitated with an equal volume
of isopropanol ovemight at -20°C. The samples were then centrifbged for 15 minutes at
4°C. The RNA pellet was resuspended in 70% ethanol, (1 mL) vacuum dried and
redissolved in double distilled water (ddH20) with 0.1% diethylpyrocarbonate (DEPC)
water. The total RNA purity and recovery for each sample was determined with a UV
spectrophotometer (Mode1 DU-64, Beckman Instnunents. Inc., CA, USA) at 260 and
280 nanometers.
3.23 Reverse Transcription Poiymerase Chain Reaction (RT-PCR)
Total RNA fkom myomeûium, decidua, chorion, amnion, rat liver and rat
pituitary was converted by reverse transcription into cDNA. The reverse transcription
reaction mix consisted of 1 pg of total RNA, 1 x PCR b s e r (10 mM Tris-HC1,50 m M
KCI, Perkin Eimer, Cetus), 5 mM MgC12 (Perkin Elmer, Cetus), 1 m M each of the
dNTP (dATP, dCTP, dGTP, dTTP, Pharmacia), 5 ng/ pL random (Pharmacia), 1 U/ pL
Mase inhibitor (Boeh~ge r Mannheim) and lOOU Moloney Murine Leukemia V i m
Reverse Transcriptase (MMLV-RT; GibcoBRL, Gaithersburg, MD) in 21 pL DEPC
water. Negative controls were prepared as above but without RNA, to cab there
was no contamination of the PCR reagents. The reaction mixture was incubated at
25°C for 10 minutes, then at 42OC for 30 minutes and finally at 99OC for 5 minutes.
The resuitant cDNA (reverse transcriptase; RT) mixture was stored at -20°C until it
was used.
PCR was performed using the RT mixture. The PCR mixture consisted of 10
pL of the RT mixture, 0.25 mM dNTP, 50 ng of each specific PCR primer (ACGT
Corporation, Toronto), 2.5 U Taq polymerase (Boehringer Mannheim) in 1.25 mM
MgCl,, 50 mM K I , 10 mM Tris-HC1 (pH 8.3) in 24.5 jL DEPC water. Each PCR
reaction undenirent an amplification regime characterized by a pre-incubation stage
(95OC, 5 minutes), a denaturation stage (94°C. 30 seconds), a primer annealing stage
(62OC, 30 seconds), an extension stage (72OC, 30 seconds) and a long extension stage
(72OC, 8 minutes) in a thennal cycler (MJ Research Inc, Mass, USA). PCR reactions
were also performed using RNA that had not been reverse transcribed to establish the
extent of genomic DNA contamination in the RNA. PCR products were
electrophoresed on a 2 % agarose gel, stained with 1.5 % ethidiurn brornide in Tris-
acetatd EDTA b a e r to allow visualization of the PCR product under a
transilliuninator (Lighttools Research, On& Canada).
Specific primers were used to identi& a 333 base pair (bp) product for CRH-RI
in human myometrium, decidua, and fetal membranes (Slorninski et al., 1995). A sense
primer 5' GCC CTG CCC TG€ CTT ï"TT CTA 3' and an antisense primer 5' GCT
CAT GGT TAG CTG GAC CA 3' corresponding to positions 235-255 and 549-568,
respectively were used (Accession number L23 332) (Chen et al., 1993). Similady,
primers were designed to identifjr a 781 bp product for CRH-R3 in human
myometrium, decidua and fetai membranes. A sense primer 5' GGC ATC AAG TAC
AAC ACG AC 3' and an antisense primer 5' CAT CCA GTA CAG GAA GGC AG 3'
corresponding to positions 423-442 and 1 184-1 204, respectively, were used (Accession
number U 1 6253) (Lovenberg et al., 1 995a). p-actin gene expression (intemal control)
was also determined in d sarnples to assess the integrity of the RNA. Prirners were
designed to idenhfy a 2 18 bp product for eactin in ail the samples. A sense primer 5'
AAG AGA GGC ATC CTC ACC CT 3' and an antisense primer 5' TAC ATG GCT
GGG GTG T G AA 3' correspondhg to positions 222- 241 and 420- 439, respectively
were used (Accession number M10278). The designed primers do not flank intron
segments.
3.2.4 Semi-Quantitative PCR
Further anaiysis using semiquantitative PCR (sq-PCR) was performed to compare
myornetrium obtained after different treatments (Kinoshita et al., 1992). To obtain
measurements in the Linear range of C M - R I cDNA we used 28,30, and 32 cycles and
in the decidua and fetal membranes we used 31, 33 and 35 cycles. To obtain
measurements in the linear range of CRH-W cDNA in human myometrium we used
3 1,33, and 35 cycles. The cycles used for p-actin were 16, 18,20. The relative intensity
of cDNA signals was quantified fiom negatives using a computerized image analysis
(Imaging Research Inc., St. Catherine, Ont, Canada).
The identity for the PCR product for CRH-RI was c o b e d using LOU of the
restriction enzymes Alu 1 (GibcoBRL) and BSR 1 (New England Biolabs) in the
appropriate b&er (10 PL) Restriction bufTer A, (GibcoBRL) and B e e r 3 (New
England Biolabs). The PCR product (0.5 - 2 pg cDNA) was incubated at 37'C (Alu 1)
or at 65°C @SR I) for 2-3 hours. The Alu I enyme digest was inactivated at 65°C for
10 minutes then products were nui on a 2% agarose gel to allow visualkation.
33.5 Immunohistochemistry
Myometnal samples from nonpregnant patients (n = 4) and term pregnant
patients in the absence (n = 4) and the presence of labour (n = 4) were removed and
k e d in 4% parafomaldehyde and 0.2% glutaldehyde for 24 hours following tissue
collection. The samples were then washed twice daiIy in phosphate-buEered saline
(PBS, 0.01 M, p H 7.4) for t h e days and stored at in 70% ethanol at 4°C. The samples
were embedded in par& by the Pathology labs at Toronto General Hospital
(Toronto, Ontario, Canada). Sections (5 pm) were cut on a microtome (Leica RM
2035, Nussloch, Gennany) and placed on glass slides coated with 2%
ambopropyltnethoxy-silane (APTEX; Sigma Chernical Co., Missouri, USA) in
acetone and dried for 24 hours at 37°C. The slides were deparafnnized with 3 washes
of xylene substitute (EM Diagnostic Systems, NJ, USA) for 5 minutes each, then
rehydrated in a series of washes 2 minutes each, in 100% ,95%, 70% and 50% ethanol,
followed by washing in 0.01 M PBS (pH 7.4). Endogenous peroxidase activity was
quenched by incubation in 3% hydrogen peroxidase (in PBS) followed by incubating
the sections with 10% normal rabbit s e m to inhibit nonspecific staining.
The slides were incubated with the primary antibodies for CRH-RI and CRH-
R 2 at 4°C ovemight. The primary antibody for CM-RI (Santa Cruz Biotechnology
hc., CA, USA). was a polyclonal antibody raised in a goat against a peptide
corresponding to amino acids 425- 444 of the human and rat CRH-RI (Chen et al.,
1993). The primary antibody for CRH-R2 (Santa Cruz Biotechnology Inc., CA, USA)
is a poiyclonal antibody raised in a goat against a peptide corresponding to amino acids
47-66 of the rat CRH-R;! Covenberg et al., 1995). The CRH-RI antibody cross-reacts
with CRH-R2. Therefore, in some sections we pre-absorbed the CRH-RI d b o d y with
an excess of CRH-R2 peptide (1 pM) (Santa Cruz Biotechnology inc., CA, USA) to
determine CRH-R1 stainuig. The primary antibodies were diluted to 1: 175 in antibody
dilution bmer (lg bovine senun albumin, 0.02 g sodium azide in 100 mL PBS; pH
7.5). M e r 16 hours of incubation with the primary antibody, the slides were washed
twice in PBS for 5 minutes each and incubated with biothylated secondary antibody
(1 : 500; Vectastalli ABC kit; Vector Laboratones, CA, USA) at room temperature for 2
hours. The sections were washed twice in PBS for 5 minutes and incubated with the
avidïn-biotin-peroxidase complex (ABC; Vectastain) in PBS for 2 hours at room
temperature. To visualize antibody staining the sections were incubated with 3',3-
diaminobenzidine tetrahydrochloride dihydrate (DM; Sigma Chernical Co., Miss,
USA) for 10 minutes. nie DAB solution was made by dissolving 50 mg of DAB in
200 mL PBS and adding 2 drops of hydrogen peroxide Mmediately prior to use. The
slides were washed in ddH20 water, counterstained with Carrazi's hematoxylin for 45
seconds, rinsed in ddH20, dehydrated in an alcohoi series; 50%, 70%, 95%, 100% for 2
minutes each and incubated in xylene for 3 washes of 5 minutes each. The slides were
then mounted with Permount (Fisher Scientific, Ontario, Canada) and viewed with a
microscope (Leica, DMRB, Nussloch, Gemany). Sheep pituitaries were used as
positive controls for CRH-RI. For the negative controls, the primary antibody was
preabsorbed with synthetic receptor peptide (1 pM; Santa Cruz Biotechnology Inc.,
CA, USA).
3.2.6 Protein Extraction
Protein was extracted fiom tissue with the use of RIPA lysis buffer. H m a n
myometrial tissue (n=8 term pregnancy and n=7 at the time of labour), rat brain and rat
heart (positive controls) and rat iiver (negative control) tissue (1-2 grams) was ground
in a pestle placed on dry ice. The ground tissue was transferred to a fiesh
polypropylene tube (12 mL; Becton and Dickinson, New Jersey, USA) and pulverized
(Janke & Henkel, MA-Labortechnik, ON, Canada) for approximately 30 seconds in
RIPA lysis buffer (50 mM Tris-HC1; pH 7.5, 150 m M NaCl, 1% Triton X-100; v/v, 1%
sodium deoxycholate; w/v, 0.1% sodium dodecyl sulphate (SDS); w/v, 100 p M Na
orthovanadate, 100 pg/pL leupeptin, 1 rnM phenyhethyl-sulfonyl fluoride while
rnaintained on ice. The tissue homogenates were spun in a microcentrifuge (Sorvall
RC-SB, Du Pont Instruments, MA, USA) at 8,500g for 15 minutes at 4OC. The
supernatant was coliected, aliquoted into eppendorf tubes (1.5 mL, Diamed, Ont,
Canada) and stored at -80°C until used.
Prior to use, a sample (10~1) of the protein was assayed to determine
concentration with a bicinchoninic acid (BCA) protein assay kit (Pierce, Illinois, USA).
A fiesh set of standards ranging fiom 200 pg/ mL to 3 pg/ 2 mL were made by diluting
2 m g l d bovine serum albumin @SA) stock standard in BCA working reagent (50
parts BCA reagent A with 1 part BCA reagent B). The standard or protein sample (10
pl) where added to the working reagent (same as above; 2 mL), in a borosillicate tube
and thoroughly mixed. The tubes where then maintained (45 minutes) in a water bath
(37°C). The protein in the tubes was meanired at absorbance 562 nm versus a water
reference. A standard c w e was prepared by plotting the 562 nm reading for each BSA
standard versus its concentration in pg/mL and used to determine the protein
concentration of the extracted samples.
3.2.7 Western Btots
Western blot gels where made using a miniprotean II ceIl (Bio-Rad. California
USA). A 10% seperating gel mixture was made in an erlmeyer flask with 1. 5 M Tris-
HCl (pH 8.8; 5 mL), 10% SDS, acrylamide/bis (37.5: 1; 3.2 mi.) and ddHzO (4 mL)
was deaerated for 15 minutes. Then* 10% fresh ammonium pesulfate (50 PL) and
N,N,N7&,-temethylenediamine (10 FL; Biorad, Clif, USA) were added. the gel was
poured and lefi to polymerise for 1 hour with a dW20 overlay. A 4% stacking gel was
prepared from 0.5 M Tris-HC1 (pH 6.8; 2.5 mL), acrylamidehis (37.5: 1;l.j mL), and
ddH20 (6.1 mL) and was deaerated for 15 minutes. Immediately afier 10% fresh
ammonium peadfate (50 PL) and N,N.N9,N,-tetramethylenediamine (10 PL) were
added, the gel was poured on top of the stacking gel, a 10 or 15 well comb was
inserted. The mixture was left to polymerise for 1 hour.
An aliquot of the protein samples was thawed and diluted with 0.5 M Tris-HCl
(pH 6.8) to a fmal concentration of 10 pglpL. The protein (100 pg) fiom each sample
was mked with 10 pL of loading b a e r (Tris-HCI; 0.15 g, glycerol; 1 mL, 1 O%SDS; 4
mL, 2-P-me~ptoethanol, 20 mg bromophenol blue and ddHîO; 4 mL) in an eppendod
(1.5 mL, Diarned Ont, Canada). The samples/Ioading b a e r where heated at 9S°C, in a
waterbath, for 4 minutes and cooled at room temperature. The gel apparatus was placed
into the b a e r chamber and the electrode buffer (Tris base; 9 g, glycine; 43.2 g, SDS; 3
g and ddH20; 600 mL) added. The wells were loaded and the gel was run at 100 volts
for 2 hours with a prestakted protein marker (Broad range, New England Biolabs). The
gel was transferred at 15 volts for 30 minutes at room temperature onto nitrocellulose
blotting membrane. The blot was washed 9 X 10 minutes) in ïTBS (1 50 m M NaCl and
50 m M Tris-HCI) with 0.05% Tween-20 and incubated in 4% BSA in TTBS with
0.05% Tween-20 for 30 minutes. The blots where incubated in the primary antibodies
for CRH-RI (same as above) and CRH-R2 (same as above) at 4OC overnight. The
primary antibodies were used at a range of dilutions (1 200 - 12000) to establish the
optimal antibody dilution.
The blot was washed (3 X 10 minutes) in TTBS with 0.05% Tween-70 and
incubated with biotinylated secondary antibody (1 : 1000 in TTBS; same as above) for 2
hours. The blot was washed again and incubated in the avidid biotin complex (1 : 1000
in Ti"E3S; Vectastain) for 2 hours. Finally the biots were developed in 12 mg DAI3
(Sigma Chernical Co., Miss, USA) in ddH20 from 30 seconds to a maximum of 5
minutes to ensure maximal staining of the blot.
3.2.8 Data Analysis
To correct for clifferences in the initial amount of RNA used for RT-PCR we
detemiined O-acM mRNA expression in al1 the samples. The ratio of the optical
densitometry reading measurements for the expression of CRH-RI mRNA or CRH-R2
mRNA (at 3 progressive amplincation cycles) to that of B-actin mRNA expression (at 3
progressive amplification cycles) was detennined for each sample. The mean of the
ratio was determined for each treatment from al1 the samples within that group. A
Mann-Whiîney Rank S u n Test was performed to judge the change in CRH receptor
expression at the rime of labour iri term and then in pretenn prepancies. To detemine
the difference amongst al1 the treatment groups (PTL, no labour, PTL in labour, term no
labour and term in labour) a Kruskal-Wallis One Way Analysis of Variance (ANOVA)
on Ranks was performed followed by an Al1 Painnse Multiple Cornparison Procedure
(Dunn's Method) to isolate the group or groups that differ f?om the othen.
3.3.1 C M Receptor Expression in the Myornetriurn of Nonpregnant Patients
Enzyme digestion of CRH-RI was used to confirm the identity of the receptor
(Figure 3.1) and yielded fragments of the expected size. Digestion with BSR I yielded a
244 bp product and digestion with Alu 1 produced a Both the expression of CRH-RI
rnRNA and CRH-R2 mRNA were present in the myometrium obtained fiom the non-
pregnant hysterectomy patients Figure 3.2). We identified the expected band of 333 bp
representing CRH-R1 (maximally expressed after 32 cycles) and the expected band of
781 bp representing CRH-R;! (maximally expressed after 35 cycles). CRH-RI
mRNAwas present at consistently high levels in di the samples studied whereas CRH-
R2 mRNA expression was variable. When using primers designed to idente CM-W
we observed a second band at 500 bp. We suggest this band represents a spiiced form
of the receptor. Bactin &A (maximally expressed after 20 cycles) expression was
present at similar levels in ai i the patients.
Both the CRH-RI and CRH-R2 protein were localized in the circular and
longitudinal smooth muscle bundles of the human myometrium (Figure H A and
Figure XJA, respectively? and in the smooth muscle of the myometrial vasculature
(Figure 3.5A and Figure MA, respectively). CRH-RI and CRH-R2 staining was
preabsorbed f?om the uterine smooth muscle (Figure M C and Figure 3 . K ) and nom
the smooth muscle of the vasculanire (Figure X5C) with an excess of peptide (1 FM).
CRH-RI was present in the sheep pituitary (Figure 3.7A and Figure 3.7B) and the
staining was pre-absorbed with CRH-RI peptide (Figure 3.7C).CRH-R2 was not
detected in the pituitary (Figure 3.7D). CRH-RI and CRH-R2 were not detected in the
human myometrium, the rat heart, brain and liver by Western blot.
3.3.2 CRH-R1 mRNA and CRH-R2 mRNA Expression in Myometrium Pregnant
Patients
We identified C M - R I mRNA expression in rnyometrium from the lower
segment of women in tenn and preterm pregnancies (Figure 3.8). CRH-R1 mRNA
expression was amplified at 28, 30, and 32 cycles in the sampies from term and preterm
myometrium to obtain linear measurements in the expression of the gene. CRH-RI
rnRNA expression was significantly upregulated @ < 0.03; Mann-Whitney Rank Sum
Test) at the time of labour in term pregnancies (MGA= 39 weeks; Figure 3.9). CRH-Rl
M A expression was also signincantiy upregulated @ c 0.01 ; Mann-Whitney Rank
Sum Test) at the thne of labour in preterm pregnancies (MGA= 32 weeks; Figure 3.10).
CRH-RI mRNA expression showed a trend towards rishg fYom 32 weeks to 39 week's
gestation and rose signincantly at the time of labour (p < 0.05; Al1 Painÿise Multiple
Cornparison Procedures) (Figure 3.1 1). CRH-RI protein was undetectable in human
myometrium at term (Figure 3.3B and Figure 3.5B) but was detected in the uterine
smooth muscle and the smooth muscle of the vasculanire at the time of labour (Figure
3.3D and Figure 3.5D).
We identified CRH-R2 mRNA in the myometnum of some (28%) of the
pregnant patients. We did not fïnd a significant change in CRH-R2 mRNA expression
at the time of labour (Table 3 .l) In the preterm pregnancy, CRH-R2 was present in the
myometrium of 3 of the patients not in labour (n = 5) and in 2 of the patients in labour
(n = 6 ). In term pregnancy CRH-R2 mRNA was not detectable in the myometrium of
the patients not in labour (n = 7) but was present in 2 of the patients in labour (n = 7).
CRH-R2 protein was not detectable in the myometrium of term patients (Figure 3.43
and Figure 3.6B) but was present in the myometrium of pregnant in labour patients
(Figure 3.4D and 3.6C) at the antibody concentrations used.
3.33 Regional Expression of CM-R1 in Myometrium From Noopregnant,
Pregnant, Pregnant in Labour and Post-Partum Patients
Samples were taken from the upper and the lower segments of the uterus to
examine differences in the regionai distribution of C M - R I in the myometriurn. CRH-
RI mRNA expression was present in the upper and lower segment of human
myometrium in nonpregnant (n = 4), pregnant (n = 4), pregnant in labour (n = 1) and in
post-parhm (n = 1) women (Figure 3.12) C M - R I mRNA expression was present at
simiiar levels in myometrium fiom the upper fundal and the lower segment of the
utenis nonpregoant and pregnant women as determined CRH-RI rnRNA/ B-actin
mRNA expression. CRH-R 1 mRNA expression was ap parent1 y higher in my omeûiurn
fiom the lower segment when compared to the upper segment in the at the time of
labour. CRH-RI mRNA expression was apparentfy decreased in the Lower segment
post-pamun and returned to levels sirnilar to that in the nonpregnant patients (Figure
3.12). CRH-RI mRNA expression appears to stay relatively constant in myomeniurn
fiom the fundal segment in the nonpregnant, the pregnant, the labouring and the post-
partum patients.
33.4 CRH-R1 mRNA Expression ui the Fetal Membranes and Decidua
CRH-KI mRNA expression was present in the decidua and the chorion but was
not significantly altered at the time of labour as determîned by anaiysis of sq-PCR
CRH-R 1 mRNA/B-actin rnRN A expression (Figure 3.1 2). C M - R I mRNA expression
was undetectable in the amnion. CRH-R? mRNA expression was undetectable in the
chorion, the amnion and the decidua after 40 amplification cycles.
3.4 DISCUSSION
Using RT-PCR we have shown that CRH-RI mRNA and CRH-R2 mRNA expression
and their proteins are present in the myometnum of both non-pregnant and pregnant
women. CRH-RI mRNA expression is signincantly upregulated at the time of labour
in both pretem and texm pregnancies. The nse in CRH-RI mRNA expression appeared
to be specific to the lower segment of the utem and disappeared immediately
postparhim. CM-R2 mRNA expression was not significantly changed and at the time
of labour in both term and pretem human pregnancies. CRH-RI protein was prevalent
in the myometrium fiom nonpregnant women, was undetectable in term pregnancy and
was iacreased at the t h e of labour in the uterine smooth muscle and the uterine
vascdature. CRH-R2 protein was present in the smooth muscle and the vasculature of
the uterine vessels in the nonpregnant myometrium, was undetectable in term
pregnancy and was modestly increased only in the smooth muscle at the time of labour.
The "classical section", a vertical incision into the body of the utenis fiom the
lower uterine segment to the upper fundal segment of the uterus is rarely used
(Pritchard et al., 1995), therefore making it difficult to obtain samples fiom the active
region of the uterus. Most always the incision is made in the lower uterine segment.
Moreover, because the size and shape of the uterus varies with age and parity it is
impossible to collect rnyometrial samples at the same site in al l the patients. We only
used nonpregnant patients undergohg hysterectomy for fibroidal growth so as to
eliminate the possible effect of this condition on gene expression. However, the tissue
may not be representative of normal tissue. CRH-RI mRNA expression in the
labouring patients was variable, possibly due to cisering stages of labour andor the
duration of labour prior to the surgery however, we no Longer have access to this
information.
We have used PCR to detect the expression of CRK-RI mRNA and CRH-R?
mRNA. There are diffculties associated with the use of this technique for quantitative
analysis of gene expression. To allow us to quantitate gene expression we we have
obtained the linear expression of the CRH-RI and CM-W gene by gradually
increasing the number of amplification cycles. We have used the ratio of the optical
densitometry readings obtained within the linear range of expression of CRH-RI
M A / CRH-EU mRNA to P-actin mRNA. Whereas this method is only semi-
quantitative it is sensitive and effective method for comparing genes expressed at low
levels.
The low levels of CRH-RI mRNA expression and the undetectable levels of
CRH-RI protein in pregnancy suggests there is a decrease in the transcription of the
CRH-RI gene which is associated with a decrease in the translation of CRH-Ri mRNA
in the human myomeûium in pregnancy. It is important to note that the CRH-RI
mRNA may be present in the huma. myometrium at tenn pregnancy but at levels
below the detection limits of the antibody concentration used. The rise in CM-RI
mRNA expression at the t h e of labour could suggest an increase in the transcription
rate of the CRH-RI gene or an increase in the stability of CRH-mRNA at the time of
labour. The significance of the upregulation CRH-RI gene transcription is not known
however it appears to be restncted to the myometrïurn fiom the lower uterine segment
conclusions with regards to the levels of CM-RI protein in the human myomenium.
We suggest that, at the time of labour, CM-RI helps to preserve the quiescence of the
lower utenne segment. In this way the low decreased amplitude and intensity of
myometrial contractions wiU be maintained. TES proposal is consistent with CRH
acting through a CAMP second messenger pathway (Labrie et al., 1982; Aguilera et
al., 1987).
In contrast, reports by Quartero and Fry, (1989) report that CRH acts
synergistically with OT and PGF2= to potentiate myometriai contractility and that this
synergistic effect appears to be mediated via a fdi in CM levels in the lower uterine
segment ( Q ~ e r o et al., 1992). Benedetto et al., (1994) were unable to obtain CRH-
OT potentiated myometrial contractility, however they showed that only when
myometrial strips where pre-incubated in CRH they demonstrated enhanced
myometnal contractility when stirnulated with PGF?,. No contractility was observed
with PGE,. These studies suggest a role for CRH in potentiating myometrial
contractility and are supported by Grammatopoulos et al., (1996). These investigatos
suggested that there was a modification in the coupling between CRH receptors and
adenylate cyclase in term pregnancy. This resulted in a reduction of CRH-stimulated
CAMP production at a time when the CRH receptor was coupled to the cyclooxygenase
pathway (Grammatopoulos et al., 1994). WhiIe this is an atiractive theory to explain
the shift from the relaxed utem at term to the contracthg uterus at the time of labour,
the authors only measured PGEz concentrations in their myometnal membrane media.
PGE2 has the paradoxical ability to mediate relaxation or stimulation dependent on the
receptor nibtype present or if it is converted to PGF?, (Nelen and Breckwoldt, 1994).
The authors fail to explain why CRH receptors in the myomeaium of nonprepant
women pathway are coupled to the cyclooxygenase pathway and not the adenylate
cyclase pathway. This data suggests the cyclooxygenase pathway rnay be constitutve in
the myometrium and possibly not be a major component in the omet of human labour.
In al1 the above mentioned studies the authon have used myometrium from the
lower uterine segment. Our data suggests that the regulation of CRH-RI mRNA
expression in the lower segment at the time of labour does not appear to reflect the
regulation of CRH-RI mRNA at this time in the funda1 region. Differentiaily regulated
CRH-RI rnRNA expression within the human uterus at the time of labour suggests
CRH may mediate different roles in these regions. This brings into question the ability
to assess the role of CRH at the time of labour from samples collected solely in the
lower uterine segment.
Mon of the past snidies in the u tem myometrium have failed to differentiate
between the CRH receptor subtypes, we are the fust group to compare CRH-RI mRNA
and CM-R2 mRNA expression in the hurnan myornetrium at the time of labour. The
presence of heterogeneous CRH receptors in the myorneviurn is not a novel concept,
Grammatopoulos et al., (1995) had previously identified heterogenous populations of
CRH receptors in myometrium. We show that CRH-RI mRNA and CM-R2 mRNA
expression are differentially regulated at the time of labour suggesting that these
receptor subtypes may mediate distinct functions in the hurnan myornetrium at the tirne
of labour. Differential regulation of the CRH receptor subtypes has previously been
reported by Mansi et al., (1 996) and Makino et al., (1 997).
Little is known about CRH receptors in the myometrium of nonpregnant
women. We have detected the expression of CRH-RI mRNA and CM-EU mRNA in
myometrium O btained fkom non-pregnant patients at hy sterectomy . This is in
accordance with Hillhouse et al, (1993) who identified the presence of specific CRH
binding sites on human rnyometrium We have shown that CRH-RI mRNA is the
predominant receptor subtype in the lower region of the utenis, and that it is present at
similar levels in the fundus and the isthmus region. We have Iocalised CM-R1 and
CRH-R2 protein on the u t e ~ e smooth muscle and on the smooth muscle layer of the
uterine vasculature in the myometrium of nonpregnant patients. We are the ftrst to
identiQ both CRH-RI and CRH-W protein in the smooth muscle of the myomeaial
vasculature. Based on the vasoactive effects of CRH (Clifton et al., 1994) we suggest
CRH may be involved in the regulation of myometrial blood flow, however, the
physiological significance of CRH in the myometnum of nonpregnant women remains
to be determined.
In conclusion, CRH-RI mRNA and CRH-EU rnRNA expression was present in
the human myometnum in nonpregnant women and in pregnancy. CRH-RI mRNA
was significantly upregulated in myometrium fiom the lower uterine segment, at the
t h e of labour. Our hding of CRH recepton in myometrium from pregnant women, is
in accordance with reported roles for CRH in the myometrium however the function of
CRH receptors in the myometrium are still unclear and requires further studies.
Figure 3.1 Enzyme digestion to c o d k m the identity of CRH-R1 cDNA: A. original 333 bp prodùct, B. digestion with Ah 1 (244 bp), C. digestion with BSR 1 (277 bp).
Figure 3.2 Photograph of a PCR gel showing the detection of CM-RI mRNA (expected - band size 333 bp; after 32 cycles) and CRH-R2 mRNA (expected band size
781 bp; d e r 35 cycles) in human myometrium (n = 4) obtained at hysterectomy. A 500 bp band was present in association with the 78 1 bp band in the CRH-IU PCR reactions. The detection of pactin mRNA (2 18 bp), the intemal controi is shown.
Figure 3 -3 Photopph showing immunostaining for CRH-R1 protein in the human myornetfium smooth muscle (SM) and blood vessels (BV).Magnified 200x
A. Myometnum in the nonpregnant B. Myometrium in term pregnancy C . Myometnum in nonpregnant (negative conlrol- preabsorbtion with CRH-R 1 antibody) D. Myometrium at the t h e of labour.
Figure 3 -4 Photograph showing immunostaining for CRH-EU protein in the human myomenium smooth muscle (SM). Magaified 200x
A. Myometrium in the nonpregnant B. Myometrium in tenn pregnancy C. Myometrium in nonpregnant (negative control- preabsorbtion with CRH-R2 antibody) D. Myometrium at the time of labour.
Figure 3.5 Photograph showing immunostaining for C M - R I in the smooth muscle layer of the vasculahire (indicated by the arrow). Magnified ZOûx.
A. Myometrium in the nonpregnant B. Myometrium in term pregnancy C. Myomeaium in nonpregnant (negative control- preabsorbtion with CRH-RI antibody) D. Myometnum at the t h e of labour (Magnified 400x).
Figure 3.6 Photograph showing immunostaining for CRH-R2 in the smooth muscle layer of the vascdature (indicated by the arrow). Magnined 200x
A. Myometnum in the nonpregnant B. Myometrium in term pregnancy C. Myometrium at the t h e of labour.
Figure 3.7 Photograph of CRH-RI imrnunosiaining in the sheep pituitary.
A. C M - R I in the region adjacent to the intemediate lobe (IL)(Mapnified 200X) B. CRH-R1 is absent in the IL, (Magnined 200X) C. Preabsorbtion of CRH-RI antibody in the region adjacent to the IL (Magnified 200X) D. CRH-W is absent in the intermediate lobe (Magnined 400X)
Figure 3.8 Photograph of a PCR gel showing the detection of CRH-RI mRNA (333 bp; d e r 32 cycles) in the human myometrïum in pregnancy (NL) and at the time of labour (L). The detection of pactin mRNA (21 8 bp), the intemal control is shown.
A. CM-RI mRNA detection in term pregnancy (n=7) and at the time of labour (n=7). B. CRH-RI mRNA detection in pre-term pregnancy (n = 6) and at the time of labour (~5).
Figure 3.9 Analysis of sq-PCR for CRH-R1 mRNA (333 bp; 28,30, and 32 cycles), in term pregnancy, in myometrium, fiom the lower segment prior to (M,) and after (L) the omet of labour. CRH-RI mRNA expression is significantly increased @ < 0.03) at the time of labour @ < 0.03), in human myomeûium, in term pregnancy.
No labour Labour
No Labour Labour p .= OA13
Figure 3.10 Andysis of sq-PCR for CRH-RI mRNA (333 bp; 28,30, and 32 cycles) in myometriurn, fiom the lower segment, pnor to (NL) and after (L) the onset of labour. CRH-RI mRNA expression is significantiy increased @ < 0.0I)at the time of labour, in the human myometnum, in pre-term pregnancy.
CRH-RI
No labour Labour
1 +- 333 bp
No Labour Labour
Figure 3.1 1 CRH-RI mRNA expression in the human myometrium increases fiom 32 weeks (n = 5) to 39 weeks (n = 7) of gestation and rises M e r to similar levels at the time of labour in pre-term (n = 6) and term (n = 7) pregnancies.
Figure 3.12 Photograph of a PCR gel showing the detection of CRH-RI mRNA (333 bp; after 32 cycles) in human myometrium from the funcial (F) and lower (L) segment of the human utem. CRH-R I mRNA expression is shown in the nonpregnant (n = 4), the pregnant (n = 4), the labouring (n = 1 ) and the postpartum (n = 1 ) patients. Analysis of sq-PCR for CM-RI in human myometrium fiom the F and L segment of the human utenis in the nonpregnant, the pregnant, the Iabouring and the postpamim patients. The detection of P-actin mRNA (218 bp; the interna1 control) is shown.
Figure 3.13 Anaiysis of sq-PCR for CRH-RI mRNA (333 bp; 3 1,33, and 3 5 cycles) in the chorion (n = 4)and the decidua (II = 4) in term pregnancy prior to (NL) and &er (L) the omet of labour.
No labour Labour Na labour Labour Chorion Decidua
NOTE TO USERS
Page(s) missing in num ber only; text follows. Page(s) were microfilmed as received.
UMI
CHAPTER 3: THE RAT- A SUITMU3 MODEL TO STUDY THE
REGULATION OF CRH RECEPTOR EXPRESSION IN
THE MYOMETRIUM?
4.1 INTRODUCTION
We and othen (Rodriguez-Linares et al., 1998) detected CRH-R1 mRNA and
CRH-R2 mRNA expression in the myometrium h m pregnant women. The presence of
CRH receptoa in the myometrium suggests CRH may be of physioiogical importance in
the initiation a d o r progression of human parturition. However, the mechanisms
involved in the regulation of CRH-RI mRNA and CRH-EU mRNA expression in the
myometrium remain unknown. In an effort to further our understanding of CM-mediated
responses, extensive studies have been conducted, in the rat to delineate the factors
involved in the regulation of CRH receptor expression in the pituitary (Wym et al., 1984;
Luo et al., 1995; Sakai et al., 1996) and the brain (Makino et al., 1995; Imaki et al..
1996).
The specific aim of this study was to establish a mamrnalian model in which the
regulation of CRH-RI mRNA and CRK-EU mRNA expression in the myomeû-ium
during pregnancy and at the time of labour could be snidied, in view of the presence and
distribution of remarkably similar CEU receptors in the hurnan and the rat (Perrin et al.,
1995, Liaw et al., 1995; Valdenaire et al., 1997) and the common pharmacological and
secondary messenger pathways of the human and rat CRH receptors, we suggested that
the rat could be a suitable model to snidy the regulation of
CRH receptor expression in the myometrium. We hypothesized that CM-RI mRNA
andor CRH-R2 rnRNA would be upregulated at the time of labour. The objectives of
the study were to determine whether CM-RI mRNA and CRH-R2 mRNA were
expressed in the rat myornetrium and to examine whether the expression of CRH-R1
mRNA andfor CRH-Et2 mRNA was altered at the time of labour. Measurernents were
made relative to Cx 43, mRNA because the increased expression of diis gap junction
protein has been weiI characterized (Lye et al., 1993; Chow and Lye 1994).
4.2 MATERIALS AND METHODS
4.2.1 Animals
Virgin fernale Wistar rats (250- 280 grams; Charles River Canada. S t Constant.
Quebec, Canada) were mated, and the day of the appearance of the vaginal plug was set
as 1 day post-coitum. The animais were housed individdy, under standard conditions
(14 hours lightness and 10 hours darkness; at 22OC) and fed Purina Lab Chow
(Ralston, Purina, , MO, USA) and water ad l i b i ~ m . These animals routhely gave binh
on the moming of day 23. The rats were decapitated and the myometrium removed on
day 15 (n = 4), day 20 (n = 9, day 31 (n = 4), day 22 (n = 4), labour (n = 6) and on 1
day post-partum (n=4 ). Rat pituitary and liver were also collected. Al1 tissue sarnples
were immediately snap fiozen in liquid nitrogen and stored at -80°C.
Al1 experirnents were approved by the Samuel Lunefeld Institute Animal Care
Cornmittee at the University of Toronto according to the Guidelines of the Canadian
Council on Animal Care.
4.22 Total RNA Extraction
Totai RNA was extracted nom the samples of rat myometrium, pituitary and
liver using the methods descnbed by Chomczynski and Sacchi (1987). Briefly, kozen
tissue samples (2- 5 mg) were powdered under liquid nitrogen and hornogenized in 1
mL of a denaturing solution (4M guanidinuim thiocyanate, 25 mM sodium citrate,
0.5% sodium lauroylsarcosine, O.IM f3-rnercaptoethanoi (v/v)) using an ULTRA-
TLlRAXX homogenizer (Janke & Henkei, IKA-Labortechnik, ON, Canada). Sodium
acetate b e e r (0.1 mL of 2M ; pH 4) was added to the tissue homogenate followed by
of phenol (water-saturated) (1 id), and by a chloroform-isoamyl alcohol mixture (0.1
mL ; 49:l). Each addition was followed by thorough rnixing. The samples were
incubated on ice for 15 minutes then centrifùged (Sorvall RCdB, Du Pont Instruments.
MA, USA) at 6,500g for 40 minutes at 4°C. The supernatant was transferred to a fresh
polypropylene tube (12 rnL; Becton and Dickinson. New Jersey, USA) and mixed with
isopropanoI(1 mL) and hcubated for 1 hour at -ZO°C to allow RNA precipitation. The
sarnples were centrifuged again at 6,500g for I hour 4OC. The resulting RNA pellet \vas
dissolved in the denaturing solution (1 mL), transferred to an eppendod tube ( 1.5 mL,
Diarned, Ont, Canada) and precipitated with an equal volume of isopropanol ovemight
at -20°C. The samples were then centrifuged for 15 minutes at 4OC. The RNA pellet
was resuspended in 70% ethanol, (1 d) vacuum dried and redissolved in ddH.20 with
0.1% DEPC water. The total RNA purïty and recovery for each sample was determined
with a W spectrophotometer (Mode1 DU-64, Beckrnan Instruments, Inc., CA, USA)
at 260 and 280 nanometers.
Total RNA fiom rat myometrium, piniitary and liver was reverse transcribed
into cDNA. The reverse transcription reaction mix comisted of 1 pg of total RNA, 1 x
PCR b a e r (10 mM Tris-HCI, 50 mM KCI, Perkin Eher, Cetus), 5 mM MgC12
(Perkin Elmer, Cetus), 1 mM each of the dNTP (dATP, dCTP, dGTP, dTTPy
Pharmacia), 5 ng/@ random hexamers (Pharmacia), 1 U/ pL RNase inhibitor
(Boehringer Mannheim) and 100 U MMLV-RT (GibcoBRL, Gaithersburg, MD) in 21
pL DEPC water. Negative controls were prepared as above but without RNA. The
reaction mixture was incubated at 25°C for 10 minutes, then at 42OC for 30 minutes
and finally at 9g°C for 5 minutes. The resultant cDNA RT mixture was stored at -
20°C until it was used.
PCR was performed using the RT mixture. The PCR mixture consisted of 10
PL of the RT mixture, 0.25 mM dNTP, 50 ng of each specific PCR primer (ACGT
Corporation, Toronto), 2.5 U Taq polymerase (Boehringer Mannheim) in 1.25 mM
MgCl,, 50 mM KCI, 10 mM Tris-HC1 (pH 8.3) in 24.5 pL DEPC water. Each PCR
reaction underwent an amplification regime characterized by a pre-incubation stage
(95OC, 5 minutes), a denaturation stage (94OC, 30 seconds), a primer annealing stage
(62"C, 30 seconds), an extension stage (72OCY 30 seconds) and a long extension stage
(72"C, 8 minutes) in a thermal cycler (MJ Research hc, Mass, USA). PCR reactions
were dso performed using RNA that had not been reverse transcribed to establish the
extent of genomic DNA contamination in the RNA. PCR products were
electrophoresed on a 2 % agarose gel, stained with 1.5 % ethidiurn bromide in Tris-
acetatel EDTA buffer to allow visualkation of the PCR product under a
transilluminator (Lighttools Research, Ont, Canada).
Specific primes were used to ident* CRH-RI, CRH-R2, Cx 43 and p-actin
gene expression in the rat rnyometrium. Primers were designed to detect a 333 bp
product for CRH-R1 in rat myometnum (Slominski et al., 1995) a sense primer 5'
GCC CTG CCC TGC C ï T TTT CTA 3' and an antisense primer 5' GCT CAT GGT
TAG CTG GAC CA 3' corresponding to positions 235-255 and 549-568? respectiveiy
were used (Accession number L23332) (Chen et al., 1993). Similariy, primers were
designed to identie a 509 bp product for CRH-R2 in rat myometrium. A sense primer
5' TAG TGC TGC GGA GTA TCC GC 3' and an antisense primer 5' CAT CCA GTA
CAG G U GGC AG 3' corresponding to positions 63 1-650 and 1 12 1-1 140.
respectively, were used (Accession number U 162%). Cx 43 expression was recognized
by primers were designed to recognize a 433 bp product. A sense primer 5' CCA AGG
AGT TCC ACC AAC TT 3' and an antisense primer 5' AGA CTG ACG GGG TCA
ACG TG 3' corresponding to positions 137- 156 and 551- 570, respectively, were used
(Accession number Ml93 17). p-actin gene expression (intemal control) was ais0
detennined in al1 samples to assess the integrity of the RNA. Primers were designed to
identiQ a 331 bp for B-actin in al1 the samples. A sense primer 5' CGT GGG CCG
CCC TAG GCA CCA 3' and an antisense primer 5' CCC CCC TGA ACC CTA AGG
CCA A 3' corresponding to positions 1343- 1363 and 1653- 1674 , respectively were
used (Accession number JO069 1).
4.2.4 SQ-PCR
Sq-PCR was performed with the CRH-R.2, Cx 43 and i3-actin cDNA to
compare the expression of these genes in rat myomeaium fiom day 15 of gestation
through to labour and 1 day post-parhun. To obtain a linear range in the expression of
CRH-R2 cycles 28, 30, and 32. Cycles 18,20 and 22 were used for Cx 43 and for the
B-actin gene cycles 16,18,20 were used. The relative intensity of cDNA signais was
quantified using computerized image analysis (Imaging Research inc., Ont, Canada).
Enzyme digestion was performed to CO- the identity of the CRH-R2 cDNA
product using 10 U of the restriction enzyme Taq I (Pharmacia) in One-Phor-All buffer
(Pharmacia) The PCR product (0.5- 2pg cDNA) was incubated at 6j°C for 2-3 hours
then the products were nin on a 2% agarose gel (1.5% ethidum bromide) to allow
visualization.
4.2.5 Data Analysis
To correct for differences in the initial amount of RNA used we deterrnined B-
a c h mRNA expression in al1 the samples. The ratio of the optical densitornetry
reading measurernents for the expression of CRH-R2 mRNA or Cx 43 mRNA (at 3
progressive amplification cycles) to that of f3-actin mRNA expression (at 3 progressive
amplification cycles) was detennined for each sample of rat myometrium. The mean of
the ratio was detennined for each treatment fkom d l the samples within that group. To
determine the difference in gene expression amongst the treatment groups (day 15, day
20, day 21, day 32, labour and IPP) Ail Pairwise Multiple Cornparison Procedures
(Student-Newman-Keuls Method; CRH-R2 mRNA and Bonferroni's; Cx 43 mRNA)
were used,
4 3 RESULTS
43.1 CRH-Ri mRNA And CRH-R2 mRNA Expression in Rat Myometrium On
Day 15, Day 20, Day 21, Day 22, At Labour And 1 Day Postpartum
Enzyme digestion (Taq i ) of CRH-R2 was used to confirm the identity of the
product and yielded the product of expected size (409 bp). We have identified CRH-Et2
(expected band of 509 bp) in the rat myornetnum fiom day 1 5-day 22 of gestation, at
the time of delivery and 1 day postpartum (Figure 4.1). The amplification of CRH-RZ
expression increased iinearly in 28, 30 and 32 cycles in al1 the samples studied. We
observed a sidcant nse (p < 0.05; Student-Newman-Keuls Method ) in CM-R2
mRNN 8-actin mRNA expression in rat myometrium at the t h e of labour, with a
significant decrease rise (p < 0.05; Student-Newman-Keuls Method) to pre-labour
levels 1 day post-partum as determineci by anaiysis of sq-PCR CRH-W mRNA / P-
actin mRNA expression (Fig 4.2). CRH-RI mRNA expression was undetectable in the
rat myometrium kom day 15-day 22 of gestation. at the t h e of labour and 1 day
postpartum. The amplification of LI-actin mRNA withui the linear expression of the
gene (cycles 16, 18,20).
432 CI 43 Expression In Rat Myometrium On Day 15, Day 20, Day 21, Day 22,
At Labour And 1 Day Postpartum
Cx 43 mRNA expression (expected band of 433 bp) was present in the rat
myometrium at day 15- day 22 of gestation, at the tirne of labour and 1 day postpartum
(Figure 4.1 ).Cx 43 mRNA expression increased linearly at 1 8, 20 and 22 cycles in al1
the samples shidied. We obsenred a significant rise (p < 0.05; Bonfernoni's method) in
Cx 43 mRNN D-actin mRNA expression in rat rnyo~etrïum at the time of labour and a
s i d c a n t decrease @ < 0.05; Bonferroni's method) to pre-labour leveis 1 day post-
partum as determined by analysis of sq-PCR CRH-R2 mRNA 1 p-actin mRNA
expression (Fig 4.3).
4.1 DISCUSSION
We report the presence of CRH-R2 mRNA expression in the rat rnyornchrn at
day 15- day 22 of gestation, at the time of delivery and 1 day postpartum. CRH-R2
mRNA expression increased significantly and concurrently with Cx 43 mRNA
expression at the time of delivery. We were unable to detect CRH-RI mRNA
expression in the rat myorneûium in pregnancy and post-parnim.
Based on the obvious inappropriate and potentially dangerous reasons for
manipulating the human myometriurn in vivo and the difficulty in obtaining
myometnal samples fiom pregnant women we have used a rat mode1 to study CRH
receptor expression in the myometrium. There is no evidence in the literature
supporting the presence of distinct fiinctional regions within the rat uterus. Hence, no
distinctions were made between regions of the uterine horn, instead we used al1 the
rnyornetnum fiom the hom to determine gene expression. We have used a semi-
quantitative technique to assess CRH-R2 mRNA expression in the rat myometrium.
We have established hear gene expression of the CRH-R2 gene and determined the
ratio of CM-R2 mRNA io the linear gene expression of the Cx 43 gene to evaiuate the
change in receptor expression. However, we have not as yet detexmined the localization
of CRH receptor protein in the rat myometrium in pregnancy and delivery and
examined if the upregulation of CRH-R2 mRNA expression was reflected at the
protein level.
We have previously reported that CRH-RI mRNA expression is significantiy
increased at the Ume of labour in rnyomenium f?om the lower se-ment of the human
uterus. This rise was observed in both terni and pre-texm pregnancies. whereas CRH-
R2 mRNA expression remains unaltered. Our results shows that CRH-RI rnRNA and
CRH-R2 rnRNA are differentially expressed in rat and human myometrium, in
pregnancy. C W R 2 mRNA is upregulated at the tirne of delivery in the rat
myometrium whereas in the human myometriurn CRH-R2 mRNA expression was only
slighrly increased. In contrast, CRH-RI mRNA increased significantly at the time of
labour in the human myornetrium but CRH-RI mRNA expression was undetectable in
the rat myometrium both in pregnancy and at the tirne of delivery. In both the rat and
the human, however the expression of CRH-R.2 m m and CRH-RI &NA,
respectively decreased immediately postpartum suggesting they were regulated by
labour specific mechanisms.
CRH-RI mRNA and CRH-EU mRNA have been reported at simila. sites in the
rat and the human (Chalmers et al., 1996; Perrin et d., 1995; Valdenaire et al.,
1997).However, it is important to keep in mind that the regdatory elements involved in
CRH receptor expression may be species-specific The mechanisms regulating CRH-EC2
mRNA expression in the rat myometrium and the time of labour are unknown. It is
well documented that CRH receptors in the rat pituitary and brain are regulated by
CRH (Wynn et al., 1983; Lu et al., 1994; Makino et al., 1995). Karialis and Mazoub,
(1994) have shown that the rat placenta does iiot produce C M . CRH mRNA
expression and irCRH are present in the rat endometrium in prePancy (Zoumakis et
al., 1996; Makrigiannakis et al., 1997) but the presence of CRH in the rat myometrium
has not been reported. It has been suggested that endornetrial CRH may play a local
role in the regulation of myometrial tone, but the mechanisms of this interaction would
need to be deterrnined. Interestingly, it has recently been suggested that urocortin, a
CRH related peptide, is the preferential ligand for CRH-R2 (Vaughan et al., 1995. In
view of the presence of CRH-R2 in the rat myornetrium it would be of interest to
determine urocortin mRNA expression in the rat myometrium in pregnancy and
delivery .
In conclusion we have detemüned that the rat is a suitable model to study the
factors regulating the expression of CRH-R7 mRNA in the utew. However, the rat is
not an appropriate model to study the regulation of CRH-RI mRNA, which is the
major subtype of the receptor in humau myometrium. A clear understanding of the
mechanisms involved in reguiating CRH receptor expression wiU help decipher some
of the mechanisms regdating CRH-mediateci actions.
Figure 4.1 Enzyme digestion to confïrrn the identity of CM-R2 cDNA: A. Taq 1 digestion (409 bp), B. original size product (509 bp).
Figure 4.2 Photograph of a sq-PCR gel showing the hear expression of CRH-RZ rnRNA, Cx 43 mRNA and P-actin mRNA. The picture shows 3 amplification cycles per sample/ gene: CM-R2 mRNA expression (expected band size 509 bp; cycles 28,30,32). Cx 43 mRNA expression (expected band size 433 bp; cycles 1820,22) and 0-actin mRNA expression (expected band size 33 1 bp: cycles 16, 18,20). The expression of the genes is shown in rat myometrium on day 15 (n = 3), day 20 (n =3), day 21 (n = 3), day 22 (n = 3) of gestation, at the time of labour (day 23; n = 3) and 1 &y postpartum (n = 3).
Figure 4.3 Analysis of sq-PCR for CRHR2 mRNA expression (cycles 28,30,32) in rat myometrium on day 15 (n = 4), &y 20 (n = S), day 21 (n = 4), day 22 (n = 4) of gestation, at the time of labour (&y 23; n = 6) and 1 day postpartum (n = 4). CRH-R2 mRNA expression is signincantiy upregulated at the t h e of labour in rat myometrium @ < 0.05).
dayl5 day2O day2l day22 Labour 1PP
Figure 4.4 Analysis of sq-PCR for Cx 43 mRNA expression (cycles 18,20,22) in rat rnyometrium on day 15 (n= 4), day 20 (n=5), day 21 (II= J), day 22 (n=4) of gestation, at the time of labour (day 23; n= 6) and 1 day postpam (n= 4). Cx 43 mRNA expression is significantly upregulated at the t h e of labour in rat myorneaiurn (p < 0.05).
day15 day20 day21 day22 Labour 1PP
CHAPTER 5: FINAL DISCUSSION
We have shown that CRH-RI mRNA and CRH-R2 mRNA expression and their
proteins are present in the myometrîum of both non-pregnant and pregnant women.
CRH-RI mRNA expression and peptide levels in myometrium are downreguiated with
pregnancy and signincantly upregulated at the time of labour. This rise in CRH-RI
mRNA expression appears to be specific to myometrium nom the lower segment of the
uterus. Moreover, CRH-R1 mRNA expression was increased in both in both pre-term
and term pregnaucies. CRH-RI mRNA expression appears to r e m to pre-labour
levels immediately postpartum. CRH-R2 mRNA expression was not significantly
changed at the t h e of labour in either term or pre-terni human pregnancies. We did not
observe a significant rise in CRH-RI mRNA expression in the chorion and the decidua.
CRH-RI mRNA expression was undetectable in the amnion whereas C M - W mRNA
expression was absent fiom the decidua, the amnion and the chorion.
CRH-RI and CRH-R2 protein was iocalized to the uterine smooth muscle and
to the smooth muscle Iayer of the uterine vessels in the myometrium fiom nonpregnant
women. CM-R1 protein staining was undetectable in the myometnum in term
pregnancy but was apparent once again, but to a lesser degree at the tirne of labour.
CRH-R2 staining was also undetectable in the myometrium in term pregnancy. At the
time of labour, we observed a modest increase of positive CRH-IU protein staining in
the uterine smooth muscle, however, CRH-R2 protein was undetectable in the uterine
vasculanire.
We also report the presence of CRH-R2 mRNA expression in the rat
myornetrium tiom &y 15 to day 22 of gestation, at the t h e of delivery and 1 day
postpartum. C M - R 2 mRNA expression increased signincantly and concomitantly
with Cx 43 M A at the tirne of labour in the rat myometrium. Whereas the nse in
CRH-EU mRNA expression in the myometrium was sudden, the rise in Cx 43 mRNA
expression was gradual and attained a peak at the time of labour. Interestingiy, the
expression of both genes decreased immediately post-parnim. We were unable to detect
CRH-RI mRNA expression in the rat myometnurn in pregnancy, at the time of
delivery and post-partum.
'The observed nse in CRH receptor expression at the time of labour is in
agreement with a physiological mle for CRH in human and rat parturition. The
apparent exclusiveness of this significant rise of CRH-R1 mRNA to the lower segment
of the human uterus rnay suggest hct ional differences for CRH in the fundal and the
lower segment of the utenis. As the lower segment of the uterus is relatively quiescent,
at the time of labour we have suggested that CRH mediates relaxation of this segment
probably via a CAMP-mediated pathway. A CAMP secondary messenger pathway for
CRH is in agreement with studies done by Labne et al., (1982) and Aguilera et al.,
(1987) who have shown CRH receptors are linked to this pathway in the brain and the
pituitary. The increase in CRH-RI mRNA expression, at the tirne of labour in both
pretemi and term pregnancies suggests that the regdation of CRH-RI mRNA
expression is involved in andor mediated by labour related rnechanisms. In addition,
the absence of a nse in CRH-RI mRNA expression in the decidua and fetal membranes
suggests that the upregulation of CRH-R1 mRNA expression is specinc to the human
myometrium.
We observed a nse in CRH-RI protein in the myometrium at the time of labour.
However this rise did not appear to reflect the significant hcrease in CRH-RI mRNA
message that we observed. We are a little cautious in our interpretatîon of this data
because of the antibody used. ui the literature only two CRH-RI antibodies have been
used. The group of E.A. Linton synthesized a CRH-RI antibody which they have used
in a wide array of immmofluorescence and western blot studies (Castro et al., 1996).
Interestingiy, they report different rnolecular weight CRH receptors within and
amongst tissues. The identified a 40 and a 45 kDa CRH-RI in the myometrium (Castro
et al., 1996). In very recent studies they showed a heterogeneous spread of CRH-R1
protein staining in the myometrium of nonpregnant aod pregnant women (Rodriguez-
Linares et al., 1998). We are the first group to our knowledge to use the CRH-RI
antibody in this study. We were unable to detect C M - R I protein in the myometrium
or in positive control tissues with Western blots. Hence we are unable to determine the
size of the product the antibody is detecting. Even though the adbody is pre-absorbed
with the CRH-RI peptide much rem* to be done to characterize the antibody.
Namely the afFinity and specificity of the antibody for the peptide has to be clearly
established. In addition, we need to detennine if the antibody recognizes Merent post-
translational spliced foms of the receptor or furthemore if the antibody cross-reacts
with the CRH-BP.
CRH-R2 mRNA expression was not signifiicantly increased at the time of
labour in human pregnancy. However, CRH-R2 mRNA was present in the
myometrium of more of the preterm patients. We suggest that whiie CRH-R2 mRNA is
not signincantly upreguiated at the time of labour, its expression may be associated to
gestational age andor premature activation of the utenis. The absence of CRH-R2
mRNA fiom the decidua and the fetal membranes suggests that the reported actions of
CRH in these tissues (Jones et al., 1989) may be mediated by CRH-Rl or a possible
unlaiown subtype of the CRH receptor.
We have shown that CRH-R2 mRNA expression is upregulated at the time of
delivery in rat myometrium. The rise in CRH-R2 mRNA was s h o w relative to the
previously reported increase in Cx 43 mRNA (Petrocelli and Lye, 1994) at the t h e of
delivery in the rat myometrium. The presence of CRH-IU mRNA in the rat
myometrium suggests CRH may play a role in delivery. However, CRH is not secreted
by the rat placenta (Robinson et al., 1989). As CRH has not been identified in the rat
myometrium, we suggest that endometrial CRH (Makrigiannakis et al., 1995) may act
via paracrine mechanisms on the rnyornetrial CRH receptor. We aiso suggest that the
CRH-R2 &A may not be associated with relaxation-mediated pathways. This is
because the evidence in the literature suggests the rat uterus is one conûacting entity at
the t h e of labour. In addition, CRH is not thought to be the preferential ligand of
CRH-RZ based on the demonstration that urocortin binds CM-W with higher affinity
than CRH (Lovenberg et al., 1995a). However urocortin mRNA expression has not
been determined in the rat myometrium. The absence of CRH-RI mRNA expression in
the rat myornetriurn is consistent with studies by Chalmers et al., (1995) Lovenberg et
al., (199%) that have shown CRH-RI mRNA is restricted to the brain regions and that
CM-R2 mRNA is expressed in the brain and muscle.
The differential rise of CRH-Rl mRNA and CRH-R2 mRNA expression at the
time of labour in the human and the rat myometriurn, respectively, suggests that
perhaps the receptors are involved in mediahg different actions within this tissue. An
attractive proposal could be that CRHRl mediates relaxation whereas CRH-R2 is
involved in myometrial contractility. The rise in CRH-Rl mRNA levels rise in the
myometriurn nom the lower segment of the human utenis is in agreement with for the
effective expulsion of the fehis fiom the uterus. In the literature, only the genes of
uterotonin receptor mRNA expression have been shown to be tumed on at the time of
labour in the rat myometrium (Fukai et al., 1984). We suggest that CRH is a uterotonic
agent in the rat myometrium and that it mediates its effects via the CRH-R2 receptors.
Future interesthg studies could be to observe the effects of progesterone on CRH-W
mRNA expression. Progesterone is weli-known in its ability to induce refiactoriness of
the myometnum to OT and PGF2, hence leading to a prolongation of pregnancy. We
could examine CRH-W mRNA expression in the rat myometnum, following a)
progesterone infusion, b) progesterone infusion stopped after a period to ailow the rats
to deliver and c) pregnant ovariectomised (progesterone withdrawal) rats.
In this study we predominantly used PCR techniques to assess the level of gene
expression. We are aware of the difnculties in quantitatively assessing the data. We
have shown a linear comlation between the expression of the gene of interest and p-
actin mRNA (an internai controt gene). We have used the ratio of optical densitornetry
w i t b the linear expression of the gene of interest to the linear expression of p-actin
mRNA to assess gene expression. The validity of this technique is well established
(Kinoshita et al., 1 992).
Based on the presence of CRH-W mRNA expression on the rat myometrium
and the upregulation of the CRH-R2 gene expression at delivery, we have determined
that rat is a suitable mammalia. model to study the regulation of CRH-R2 mRNA at
the tirne of labour. However another mammaiim model has to be established to study
the regulation of the expression of CRH-RI mRNA in the myometrium. CRH-RI
mRNA is the predominant CRH receptor subtype in the human myometrium and
elucidating the factors regulating CM-Rl mRNA expression may help decipher the
role of this receptor in human pregnancy. We suggest that the expression of CRH
receptors could also be studied in myometrial cells via culturing or transfection
techniques. However, while these in viîro experiments may M e r our understanding
of the factors regulating CRH receptor expression in the myometrium the inherent
Limitations of in vitro studies prevail.
In conclusion, we have s h o w that CRH-RI mRNA expression is signincantly
upregulated in the human rnyometnum at the t h e of labour, whereas, in the rat CRH-
R2 mRNA expression is upregulated at the tune of delivery. The upregulation of both
CRH-RI mRNA and CRH-R2 mRNA expression in the human and the rat,
respectively, are similarly mediated by labour-related mechanisms, as suggested by the
decrease in the expression of the receptor subtypes immediately postpartum.
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