Citation
Purification, characterization and localization of relaxin in the pregnant guinea pig

Material Information

Title:
Purification, characterization and localization of relaxin in the pregnant guinea pig
Creator:
Pardo, Rube Jose, 1950-
Publication Date:
Language:
English
Physical Description:
ix, 120 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Animals ( jstor )
Antiserum ( jstor )
Bioassay ( jstor )
Guinea pigs ( jstor )
Ligaments ( jstor )
Ovaries ( jstor )
Pregnancy ( jstor )
Rabbits ( jstor )
Rats ( jstor )
Uterus ( jstor )
Anatomical Sciences thesis Ph.D ( mesh )
Dissertations, Academic -- Anatomical Sciences -- UF ( mesh )
Guinea Pigs -- physiology ( mesh )
Relaxin -- physiology ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 63-71.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Rube Jose Pardo.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
028951245 ( ALEPH )
08368430 ( OCLC )
ABX8758 ( NOTIS )

Downloads

This item has the following downloads:

EWHJNYXNO_ZQO987.xml

purificationchar00pard.pdf

purificationchar00pard_0116.txt

purificationchar00pard_0032.txt

purificationchar00pard_0089.txt

purificationchar00pard_0065.txt

purificationchar00pard_0009.txt

purificationchar00pard_0060.txt

purificationchar00pard_0115.txt

purificationchar00pard_0034.txt

purificationchar00pard_0102.txt

purificationchar00pard_0020.txt

purificationchar00pard_0001.txt

purificationchar00pard_0022.txt

purificationchar00pard_0026.txt

purificationchar00pard_0103.txt

purificationchar00pard_0072.txt

purificationchar00pard_0119.txt

purificationchar00pard_0073.txt

purificationchar00pard_0121.txt

purificationchar00pard_0083.txt

purificationchar00pard_0043.txt

purificationchar00pard_0028.txt

purificationchar00pard_0030.txt

purificationchar00pard_0064.txt

purificationchar00pard_0038.txt

purificationchar00pard_0123.txt

purificationchar00pard_0131.txt

purificationchar00pard_0044.txt

purificationchar00pard_0040.txt

purificationchar00pard_0100.txt

purificationchar00pard_0074.txt

purificationchar00pard_0106.txt

purificationchar00pard_0024.txt

purificationchar00pard_0128.txt

purificationchar00pard_0090.txt

purificationchar00pard_0094.txt

purificationchar00pard_0008.txt

purificationchar00pard_0080.txt

purificationchar00pard_0071.txt

purificationchar00pard_0070.txt

purificationchar00pard_0108.txt

purificationchar00pard_0016.txt

purificationchar00pard_0113.txt

purificationchar00pard_0063.txt

purificationchar00pard_0132.txt

purificationchar00pard_0013.txt

purificationchar00pard_0062.txt

purificationchar00pard_0055.txt

purificationchar00pard_0012.txt

purificationchar00pard_0122.txt

purificationchar00pard_0058.txt

purificationchar00pard_0096.txt

purificationchar00pard_0101.txt

EWHJNYXNO_ZQO987_xml.txt

purificationchar00pard_0019.txt

purificationchar00pard_0000.txt

purificationchar00pard_0023.txt

purificationchar00pard_0027.txt

purificationchar00pard_0002.txt

purificationchar00pard_0098.txt

purificationchar00pard_0052.txt

purificationchar00pard_0049.txt

purificationchar00pard_0105.txt

purificationchar00pard_0029.txt

purificationchar00pard_0076.txt

purificationchar00pard_0006.txt

purificationchar00pard_0107.txt

purificationchar00pard_0092.txt

purificationchar00pard_0014.txt

purificationchar00pard_0110.txt

purificationchar00pard_0114.txt

purificationchar00pard_0057.txt

purificationchar00pard_0066.txt

purificationchar00pard_0120.txt

purificationchar00pard_0051.txt

purificationchar00pard_0003.txt

purificationchar00pard_0054.txt

purificationchar00pard_0112.txt

purificationchar00pard_0077.txt

purificationchar00pard_0053.txt

purificationchar00pard_0093.txt

purificationchar00pard_0050.txt

purificationchar00pard_0039.txt

purificationchar00pard_0059.txt

purificationchar00pard_0082.txt

purificationchar00pard_0042.txt

purificationchar00pard_0015.txt

purificationchar00pard_0018.txt

purificationchar00pard_0088.txt

purificationchar00pard_0087.txt

purificationchar00pard_0109.txt

purificationchar00pard_0025.txt

purificationchar00pard_0035.txt

purificationchar00pard_0099.txt

purificationchar00pard_0086.txt

purificationchar00pard_0056.txt

purificationchar00pard_0081.txt

purificationchar00pard_0045.txt

purificationchar00pard_0068.txt

purificationchar00pard_0117.txt

purificationchar00pard_0033.txt

purificationchar00pard_0067.txt

purificationchar00pard_0085.txt

purificationchar00pard_0091.txt

purificationchar00pard_0075.txt

purificationchar00pard_0047.txt

purificationchar00pard_0078.txt

purificationchar00pard_0017.txt

purificationchar00pard_0031.txt

purificationchar00pard_0036.txt

purificationchar00pard_0124.txt

purificationchar00pard_0021.txt

purificationchar00pard_0010.txt

AA00009113_00001.pdf

purificationchar00pard_0005.txt

purificationchar00pard_0127.txt

purificationchar00pard_0118.txt

purificationchar00pard_0095.txt

purificationchar00pard_0046.txt

purificationchar00pard_0069.txt

purificationchar00pard_0004.txt

purificationchar00pard_0079.txt

purificationchar00pard_0041.txt

purificationchar00pard_0037.txt

purificationchar00pard_0048.txt

purificationchar00pard_0130.txt

purificationchar00pard_0011.txt

purificationchar00pard_0104.txt

purificationchar00pard_0061.txt

purificationchar00pard_pdf.txt

purificationchar00pard_0111.txt

purificationchar00pard_0125.txt

purificationchar00pard_0007.txt

purificationchar00pard_0084.txt

purificationchar00pard_0129.txt

purificationchar00pard_0126.txt

purificationchar00pard_0097.txt

AA00009113_00001_pdf.txt


Full Text
















PURIFICATION, CHARACTERIZATION AND LOCALIZATION
OF RELAXIN IN THE PREGNANT GUINEA PIG











BY

RUBE JOSE PARDO


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1982




PURIFICATION, CHARACTERIZATION AND LOCALIZATION
OF RELAXIN IN THE PREGNANT GUINEA PIG
BY
RUBE JOSE PARDO
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1982


I wish to dedicate this dissertation to my parents, Mr. and
Mrs. Rube Pardo, and my grandmother, Gilda de la Torriente. This
dissertation is also dedicated in the memory of my grandfather, Jose
Elias de la Torriente.


ACKNOWLEDGMENTS
I would like to express my appreciation to the members of my
supervisory committee for their help in the work presented in this
dissertation. I especially wish to thank Dr. Lynn Larkin, chairman of
my committee, for his help and financial backing. I also wish to
acknowledge the following individuals who have aided me in my doctoral
studies: Dr. Fuller Bazer, Dr. Don Cameron, Mr. Alberto de LaPaz,
Dr. Asgi Fazleabas, Dr. Michael Fields, Dr. Phillip Fields, Dr. Don
Hay, Dr. Thomas Hollinger, Dr. Satya Kalra, Dr. Ernst Kallenbach, Mr.
Denny Player, Mr. Lane Powell, Dr. Ray Roberts, Dr. Lynn Romrell, Mrs.
Pauletta Sanders, Mr. Will Sanders and Dr. Howard Suzuki. A special
thank you goes to my good friends and fellow graduate students, Phil
Ruiz, Wayne Barbee, and Pat Fitzgerald. Finally and most importantly,
I wish to express my deepest appreciation and love to my parents,
Mr. Rube Pardo and Mrs. Georgina T. Pardo; my grandmother, Gilda de la
Torriente; my sisters, Margarita and Georgina; my brother, Roberto;
and my brother-in-law, Bahram.
in


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF ABBREVIATIONS vi
ABSTRACT viii
INTRODUCTION 1
Relaxin Assays 2
Cellular Localization of Relaxin 8
Isolation and Characterization of Relaxin 13
Relaxin in the Guinea Pig 22
Statement of Problem 25
MATERIALS AND METHODS 26
General Procedures 26
Detection of Relaxin 29
Purification and Characterization of Guinea Pig Relaxin. ... 36
RESULTS 40
Detection of Guinea Pig Relaxin 40
Purification and Characterization of Guinea Pig Uterine
Relaxin 47
DISCUSSION 50
R19 Antiserum: Detection of Guinea Pig Relaxin 50
Detection of Guinea Pig Relaxin with the PAP Technique .... 51
Detection of Guinea Pig Relaxin with Radioimmunoassay 52
Endometrial Glands and Their Role in Relaxin Production. ... 55
Possible Actions of Uterine Relaxin in the Guinea Pig 58
Purification and Characterization of Guinea Pig Relaxin. ... 60
BIBLIOGRAPHY 63
APPENDIX 1 TABLES 72
APPENDIX 2 FIGURES 77
IV


APPENDIX 3 IODINATION OF SUCCINIMIDE RELAXIN Ill
APPENDIX 4 IODINATION OF RELAXIN WITH THE BOLTON AND
HUNTER REAGENT 114
BIOGRAPHICAL SKETCH 120
v


LIST OF ABBREVIATIONS
Bo
CMC
CPM
DAB
EG
EGC
GAR
gww
H 5 E
L
lac
IP
mw
NEPHGE
NSB
NRS
ODS
PAP
PAGE
PBS
R19
RIA
RP
zero count tube
carboxymethyl cellulose
counts per minute
3,3' diaminobenzidine
endometrial gland(s)
endometrial gland cell(s)
goat anti-rabbit IgG
gram wet weight
hematoxylin and eosin
uterine lumen
lactating
late pregnant
molecular weight
non equilibrium polyacrylamide gel electrophoresis
nonspecific binding
normal rabbit serum
octadecylsilica
peroxidase antiperoxidase
polyacrylamide gel electrophoresis
phosphate buffered saline
antiserum made to purified porcine relaxin
radioimmunoassay
peroxidase reaction product
vx


RPM revolutions per minute
SC subcutaneous
SDS sodium dodecyl sulfate
SE surface epithelium of uterine lumen
T total count tube
TCA trichloroacetic acid
U unit(s) of relaxin activity
vi 1


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PURIFICATION, CHARACTERIZATION AND LOCALIZATION
OF RELAXIN IN THE PREGNANT GUINEA PIG
By
Rube Jose Pardo
May 1982
Chairman: Lynn H. Larkin
Major Department: Medical Sciences (Anatomy)
It has been shown using the peroxidase-antiperoxidase immunocyto-
chemical technique that the endometrial glands of the pregnant guinea
pig are the source of the hormone relaxin. The presence of relaxin
has been demonstrated in uteri from day 30, day 45, day 60 pregnant and
late pregnant animals (days 65-67). The density of reaction product
deposition increased as pregnancy proceeded, with high deposition occur
ring in days 45 and 60 of pregnancy and in late pregnant animals. Little
or no immunoperoxidase labeling was observed in tissues from day 15,
nonpregnant and lactating animals (3 days postpartum). Immunoperoxidase
labeling was not seen in nonendometrial gland components of the uterus.
High biological and immunological activities were found in extracts of
uteri taken on days 45 and 60 of pregnancy and in late pregnant animals.
When a crude extract of late pregnant uteri was chromatographed
in Sephadex G-50, a fraction containing relaxin activity eluted in the
6,000 molecular weight range. This fraction was active in the mouse
uterine motility bioassay (1.50 U/mg), and promoted lengthening of the
viii


interpubic ligament in estrogen primed female mice. The bioactive
Sephadex fraction was further purified in a carboxymethylcellulose
(CMC) ion exchange column. A single peak from the CMC column demon
strated relaxin bioactivity (3.87 U/mg) in the mouse uterine motility
bioassay. The CMC purified guinea pig relaxin was compared to CMC
purified porcine relaxin in a two dimensional gel electrophoresis system.
The two purified relaxins were of similar molecular weights, with the
porcine hormone being slightly more basic. The guinea pig relaxin
molecule appears to be similar to porcine relaxin according to the
following criteria: (1) A continuous line of identity was obtained when
a 6,000 molecular weight fraction of relaxin from uteri of day 60 preg
nant guinea pigs was tested with porcine relaxin and antirelaxin serum
in double immunodiffusion plate analyses. (2) Both relaxin molecules
were inactivated when reacted with antirelaxin serum in an antiserum
test employing the mouse uterine motility bioassay. (3) Both relaxin
molecules were inactivated by trypsin and dithiothrietol, but not by
moderate heat.
IX


INTRODUCTION
Many mammals that give birth to large mature young have mechanisms
to compensate for a narrow pelvic width. One of the most dramatic
examples of this is found in the guinea pig, which gives birth to
relatively large young. In the guinea pig, pubic separation is so
extreme that the two halves of the pelvis are freely movable during the
birth process. It was Hisaw's interest in this phenomenon which prompted
him to ask whether certain humoral factors were responsible for the
morphologic changes associated with this process. Hisaw (1926, 1927)
was the first to relate this pelvic separation to the presence of a
blood factor later called relaxin (Fevold, Hisaw and Meyer, 1930). Since
its discovery, relaxin has been recognized as a hormone of pregnancy and
has been detected in many species of animals.
The physiological effects of relaxin are mainly concerned with the
female reproductive tract of mammalian species. Three of these effects
have been extensively reviewed in the literature: (1) relaxation of the
ligaments which stabilize the pelvic bones, (2) inhibition of uterine
contractions, and (3) softening of the cervix at term (Hisaw and Zarrow,
1950; Hall, 1960; Schwabe et al., 1978; Porter, 1979). The relaxation
of pelvic ligaments and inhibition of uterine contractions are the basis
of two important bioassays which are used to detect relaxin.
The following portions of the introduction will concentrate on
four areas of study on relaxin: (1) detection of relaxin, (2) cellular
localization of relaxin, (3) isolation and characterization of relaxin
and (4) description of relaxin research in the guinea pig.
1


2
Relaxin Assays
Relaxation of Pelvic Ligaments
The first qualitative bioassay for relaxin was the guinea pig
pubic symphysis palpation assay developed by Fevold et al. (1930).
An attempt to quantitate this assay was made by Abramowitz et al. (1944).
A guinea pig unit (U) was defined by these investigators as the dose of
relaxin that in 6 hours caused relaxation of the pubic symphysis (deter
mined by palpation) in at least eight of twelve estrogen primed, cas
trated female guinea pigs. Two basic problems were associated with this
assay: (1) the degree of subjectivity was high and (2) repeated use of
the same guinea pigs at first sensitized them to relaxin but then made
the animals refractory to the hormone after several months of use (Noall
and Frieden, 1956). All studies before 1960 exclusively employed the
guinea pig pubic symphysis assay and can, therefore, be questioned for
the reasons explained above.
The mouse interpubic ligament assay was later developed by Steinetz
et al. (1960), and offered a more sensitive and objective method of
assaying relaxin. In this assay, groups of sexually immature female
mice (18-20 g) were primed with a single injection of 5 pg estradiol
and 7 days later received injections of relaxin standards or unknowns
(three dose levels) in 1% benzopurpurine-4B. Eighteen to twenty-four
hours later, the mice were killed and their pubes dissected free of con
nective tissue and fat. The interpubic distance was measured using a
dissecting microscope fitted with an ocular micrometer and a transillum-
inating source. With this assay, dose response curves could be compared
between two relaxin preparations to determine whether the relaxins


3
elicited similar (parallel dose response curves) or dissimilar responses
in the experimental animals.
Inhibition of Uterine Contractions
Krantz et al. (1950) were the first to describe the ability of
relaxin extracts to inhibit spontaneous contractions of rat, guinea pig
and mouse uteri maintained in vivo and in vitro. Kroc et al. (1959)
improved the uterine motility assay further by utilizing uteri from
sexually immature, estrogen primed mice in an in vitro system. This
bioassay is more economical because mice are less costly than the larger
rodents. Also, the mouse uterus requires less relaxin to reduce con
tractions, thereby conserving the hormone. The mouse uterine motility
assay has been recently modified by Larkin et al. (1981). In this
modified assay each uterine horn from sexually immature estrogen primed
mice is divided and suspended in an aerated organ bath of Locke's solu
tion. The uterine segment is attached to a heart lever against 1 g of
tension and contractions are monitored with an ink writing kymograph.
The relaxin standard or unknown is tested for the ability to inhibit
spontaneous uterine contractions. One section of the horn is treated
with the standard, and the other with the unknown. By doubling the con
centrations of standard and unknown in the organ bath at 4 min intervals,
the response of the two uterine segments may be compared and the potency
of the unknown determined.
The guinea pig pubic symphysis assay is the most subjective of the
assays mentioned but was the most widely used until 1960. The mouse
interpubic ligament assay offers the refinement of objectivity, since


4
quantitative comparisons of the slopes of the dose response curves between
unknowns and standards can be made. The mouse uterine motility assay
offers the quickest and most inexpensive method for assaying relaxin
bioactivity, but does not provide the dose response data which are
available with the mouse interpubic ligament assay. Thus a combination
of assays can be used to counteract the shortcomings of one single
assay. All relaxin bioassays are relatively insensitive when compared
with the levels of relaxin detected with radioimmunoassay (RIA).
Radioimmunoassay
In 1972, Bryant developed the first homologous RIA* for porcine
relaxin. In this assay, a relatively impure relaxin preparation (NIH-R-
Pl, 440 U/mg) was iodinated with the chloramine-T-method of Hunter and
Greenwood (1962). This impure preparation was also used for the produc
tion of antiserum and for the relaxin standards. This RIA was used by
Bryant and collaborators for several studies (Bryant, 1972; Bryant and
Stelmasiak, 1974; Bryant et al., 1975; Bryant and Chamley, 1976; Bryant
et al., 1976) before it was discovered by Sherwood and O'Byrne (1974)
that porcine relaxin contained no tyrosine residues and therefore could
not be iodinated by the chloramine-T method. It was likely that Bryant
either iodinated some peptide contaminants within the relaxin preparation
or perhaps labeled a prohormone which contained similar antigenic deter
minants to relaxin. This possibility has been explored by Bryant-
Greenwood and Greenwood (1979) in a recent publication in which they
*A homologous porcine RIA is an RIA where
1. the antirelaxin serum is produced against porcine relaxin
2. the iodinated hormone and the radioinert standards are
porcine relaxin.


5
compared the RIA utilizing NIH-R-P1 relaxin with an RIA using a highly
purified relaxin fraction (CM-a', 3,000 U/mg). The NIH-R-P1 relaxin
was iodinated by the chloramine-T method of Hunter and Greenwood (1962).
The CM-a' relaxin was reacted with a succinimide ester and iodinated by
the method of Bolton and Hunter (1973). It was found that antisera to
CM-a' relaxin crossreacted with NIH-R-P1 relaxin. Also highly purified
CM-a' relaxin crossreacted with antisera made to NIH-R-P1 relaxin.
However, when the two assays were used to detect relaxin in serum of
pregnant ewes, the assay based on the crude relaxin preparation (NIH-
R-Pl) read values of relaxin ten times greater than those read with the
assay utilizing the highly purified hormone (CM-a'). This was interpreted
to mean that the RIA utilizing NIH-R-P1 relaxin was reading a broad
spectrum of immunoactivity and could have been detecting tyrosine contain
ing contaminants or a relaxin prohormone that was not detected by the
RIA utilizing the highly purified hormone.
In 1975, Sherwood and his co-workers developed a homologous RIA
for porcine relaxin which took into account the hormone's total lack of
tyrosine residues (Sherwood et al., 1975). This RIA utilized a highly
purified relaxin preparation containing CM-a', CM-a and CM-B fractions,
which they called native relaxin. Initial efforts to iodinate native
relaxin with the chloramine-T method failed. Therefore, a novel approach
was employed to covalently bind tyrosine to relaxin through an amide
linkage using the agent N-carboxy-L-tyrosine anhydride. The resulting
molecule was named polytyrosyl relaxin, because it contained 1.67 moles
of tyrosine per mole of relaxin, and was used for the development of all
phases of the RIA. Sherwood et al. (1975) reported detecting levels of


6
porcine relaxin as low as 32 pg whereas previously used bioassays were
sensitive in the low microgram range. Utilizing this RIA the presence
of relaxin has been demonstrated in sera of pregnant pigs (Sherwood et
al., 1977a; 1977b). However, the assay did not detect relaxin in sera
from pregnant guinea pigs or pregnant cows (Sherwood et al., 1975).
This observation may have resulted because of a number of reasons, how
ever, two that should be considered are that the antirelaxin serum did
not crossreact with relaxin from these species and that serum levels of
the hormone were below the level of detection of the assay.
A RIA employing polytyrosyl relaxin also has been established in
the laboratory of Dr. B. G. Steinetz. A Sephadex G-50 relaxin fraction
containing 1,000 U/mg was used to develop the antiserum utilized in
Steinetz's RIA. The main difference in the RIA procedures of Steinetz
and of Sherwood was the employment of different antirelaxin sera. The
Steinetz assay system has been used to demonstrate the presence of
relaxin in sera from pregnant rats, mice, hamsters, guinea pigs, dogs,
monkeys and humans (O'Byme and Steintez, 1976; O'Byme et al., 1976;
O'Byrne et al., 1978).
The Bolton and Hunter (1973) method of iodination has been utilized
by several investigators in the development of a RIA for relaxin. In
this method, 3-(4-hydroxyphenyl)-propionic acid N-hydroxy-succinimide
ester is radioiodinated according to the method of Hunter and Greenwood
(1962). The ester is then reacted with relaxin and an iodinated phenyl
group is incorporated into the epsilon amino groups of lysine and N-
terminus of the relaxin molecule. Bryant-Greenwood and her co-workers


7
have used this preparation in the development of a homologous porcine
RIA (Bryant-Greenwood and Greenwood, 1979; Yamamoto et al., 1981). This
homologous porcine RIA has been used by Yamamoto et al. (1981) to
determine relaxin levels in the purification of relaxin from human
placental basal plates. Parallel displacement curves existed between
the porcine and human purified relaxins, although the RIA was less sensi
tive in detecting human relaxin. Parallel displacement curves indicate
similar antigenicity between molecules.
Using the Bolton and Hunter reagent to iodinate relaxin, Loumaye
et al. (1978) also employed a homologous porcine RIA. They were able
to detect relaxin in serum of pregnant women, in extracts of corpora
ltea of pregnancy and in corpora ltea cyst fluid of pregnant and non
pregnant women.
The only homologous nonporcine RIA system has been developed for
the detection of rat relaxin by Sherwood and Cmekovic (1979). Equal
quantities of two ion exchange chromatography fractions (CM-1 and CM-2)
of rat relaxin were pooled and iodinated by the method of Bolton and
Hunter (1973). Antisera were raised in rabbits against the CM-1 and
CM-2 rat relaxin fractions. These relaxin fractions were also employed
as the radioinert standard. The assay could measure in the range of
32-3000 pg of rat relaxin, using an antirelaxin serum dilution of
1:100,000.
The two most commonly used methods of labeling relaxin are the
method of Sherwood et al. (1975), which results in a polytyrosyl relaxin,
and the method of Bolton and Hunter, which employs succinimide relaxin.
Differences in the results of studies utilizing these RIA methods seem


8
to be related to the primary antisera employed in the assays rather than
the iodination procedure used for labeling the hormone.
While RIA and other immunologic techniques are being used increas
ingly to detect relaxin, the bioassay still remains the most widely
used technique for relaxin detection. Although the RIA has the advantage
of increased sensitivity, the bioassay detects the biologically active
hormone.
Cellular Localization of Relaxin
One of the key areas of study concerning relaxin's role in preg
nancy and parturition has been to determine the cellular location of the
hormone during these physiological states. Immunocytochemical tech
niques have been the most commonly employed methods used to detect the
cellular location of relaxin in tissues. These techniques have been
used successfully to detect cells containing relaxin in the pig, cow,
rat and human.
Pig
In the pig, there is good evidence that the corpus luteum of
pregnancy is the principle source of relaxin. Belt et al. (1971) were
the first investigators to correlate levels of relaxin with cytoplasmic
granules in porcine luteal tissue. The accumulation of dense cytoplasmic
granules in granulosa lutein cells of late pregnant pigs, and the
decline in the number of granules after gestation, closely paralleled
the rise and fall of bioassayable corpus luteum relaxin in the same
periods. Kendall et al. (1978) utilized the immunoperoxidase technique
to localize relaxin at the ultrastructural level in cytoplasmic granules
of porcine granulosa lutein cells. Larkin et al. (1977) used


9
immunofluorescent localization methods employing antiporcine relaxin
serum (R8) to localize relaxin in granulosa lutein cells or pregnant
pigs. Furtner studies with the porcine ovary by Arakari et al. (1980)
have shown that the antirelaxin serum employed is of utmost importance
in the localization of relaxin when using immunolabeling techniques. An
antiserum produced against a crude relaxin preparation (NIH-R-P1, 440
U/mg) gave a diffuse pattern of immunofluorescence in the corpus luteum
of pregnancy, with the fluorescence localized mainly in the connective
tissue elements. On the other hand, an antiserum produced against
purified relaxin (CM-a', 3,000 U/mg) gave a sharp and precise localiza
tion within the cytoplasm of the luteal cells. There have been no
reports of localization of relaxin in uterine or placental tissues in
the pig.
Cow
The ovary of the pregnant cow has been shown to be a source of
relaxin with bioassay techniques (Castro-Hemandez, 1976). Fields et al
(1980) detected relaxin with the immunoperoxidase technique in ovaries
taken from cows in the middle and late Stages of pregnancy. Relaxin was
localized in the cytoplasm of the granulosa lutein cells. Measurable
quantities of relaxin were not found with bioassay in bovine uterus or
placenta. The presence of relaxin in the bovine uterus and placenta
was not evaluated using immunocytochemical techniques.
Rat
The ovary of the pregnant rat contains large quantities of extract
able relaxin (Fields and Larkin, 1979; Sherwood and Crnekovic, 1979).
Relaxin has been detected in the rat ovary with bioassay, RIA and


10
inmunocytochemical techniques. Whereas it is well established that the
ovary is a source of relaxin in the pregnant rat, the metrial gland of
the uterus and the placenta have been implicated as tissues which may
also contain relaxin.
Dallenbach-Hellweg et al. (1965) reported immunofluorescent local
ization of relaxin in metrial gland cells of the pregnant rat uterus, but
not in the ovary or placenta. The antiserum utilized was made in rabbits
to porcine relaxin (1,000 U/mg). Results from this study should be viewed
with caution for two reasons. First, controls used in the study were not
stringent, since no attempt was made to absorb the antirelaxin serum
with purified porcine relaxin. Second, work of several laboratories
shows that the metrial gland of the rat does not contain relaxin. Larkin
(1974) tested tissue extracts from day 14 pregnant rats for relaxin bio
activity. Ovarian, but not metrial gland extracts contained bioassay-
able amounts of relaxin. Anderson et al. (1975) could not detect relaxin
in metrial glands of pregnant rats using immunofluorescence, but could
detect labeling in the ovary. The ovarian fluorescence was localized
in the cytoplasm of granulosa lutein cells. The antiserum employed by
Anderson et al. (1975) was raised against an even less pure porcine
relaxin preparation (NIH-R-P1, 440 U/mg), than that employed by Dallenbach-
Hellweg et al. (1965); however, controls were more complete. Other studies
(Anderson and Long, 1978) showed that ovarian extracts contained relaxin
activity, and metrial gland extracts did not. Zarrow and McClintock
(1966) injected I labeled antibody to porcine relaxin into pregnant
rats and discovered substantial accumulations of label in the ovary and


11
placenta. This study may be criticized on two points. First, whole
organs were counted for radioactivity and thus one cannot state with
certainty if cellular relaxin crossreacted with the labeled antibody or
if the antibody crossreacted with receptor bound relaxin present in the
tissue. Also organs with a high blood capacity like the placenta might
have sequestered blood bound labeled antibody. Second, the antibody
utilized in the study was produced against a very crude porcine relaxin
preparation (WL 1164 lot A, 150 U/mg powder).
Rabbit
The ovary, uterus and placenta of the pregnant rabbit have been
reported to contain relaxin. Zarrow and O'Connor (1966) found relaxin in
the rabbit gestational corpus luteum by employing an indirect immuno-
fluorescent labeling technique; however, it was difficult to determine
from the published photographs whether the label was located intra- or
extracellularly. The antibody utilized in the above study was produced
in rabbits to porcine relaxin (WL 1164, lot 8; 622 U/mg powder). No
fluorescence was found in uterine or placental tissue. Zarrow and
Rosenberg (1953) reported bioactive relaxin in the ovary, uterus and
maternal placenta of pregnant rabbits with the highest level appearing
in the maternal placenta. This study also showed that ovariectomy of
pregnant rabbits with subsequent progesterone replacement therapy did
not result in decreased blood levels of relaxin. Fields et al. (1981)
isolated relaxin from extracts of rabbit placentae. However, a cellular
source of the hormone in the placentae was not reported, leaving open
the possibility that the relaxin was blood borne. It appears that the
rabbit is a species which has extra-ovarian sources of relaxin, most
likely the uterus and/or placenta.


12
Human
The ovary and placenta appear to be sources of relaxin in the
pregnant human. Dallenbach and Dallenbach-Hellweg (1964) discovered the
presence of relaxin in basal plate cells of human placentae using an
indirect immunofluorescence technique. The antiserum employed was made
in rabbits to a porcine relaxin preparation (1,000 U/mg). This finding
has been substantiated by several recent studies. Fields and Larkin
(1981) also detected relaxin in basal plate cells of human term placentae
using the immunoperoxidase technique. An antiserum (R19) raised against
purified porcine relaxin was utilized in these studies. Fields and
Larkin (1981) also showed that placentae which gave a positive stain
for relaxin also contained bioassayable relaxin. Yamamoto et al. (1981)
have shown that basal plates of cesarean and vaginally delivered placentae
contain bioactive and immunoreactive relaxin. The decidua of the preg
nant human also has been shown to contain bioactive relaxin by Bigazzi
et al. (1980), but at this time a cellular source of relaxin has not
been found in this tissue.
The ovary has been established as a source of bioactive and immuno
reactive relaxin in the pregnant human (O'Byrne et al., 1978; Szalchter
et al., 1980; Weiss et al., 1976; Weiss et al., 1977). Relaxin also
has been shown to be present in the human gestational corpus luteum
using immunoperoxidase localization (Mathieu et al., 1981). The local
ization of relaxin appeared in the perinuclear area of the luteal cells.
It appears that animals which require the ovary for the maintenance
of pregnancy, i.e., the pig and the rat, also have the ovary as the
principal source of relaxin. On the other hand, animals which do not


13
require the ovary for the maintenance of pregnancy, like the rabbit and
the human, seem to have extraovarian sources of relaxin. The validity
of the above generalization will be tested as future studies encompass
a larger variety of species.
Isolation and Characterization of Relaxin
Relaxin has been isolated and characterized from the ovary of the
pregnant pig (Sherwood and OByme, 1974; Schwabe et al., 1976; 1977),
the ovary of the rat (Fields and Larkin, 1979; Sherwood, 1979; Walsh
and Niall, 1980), the placenta of the rabbit (Fields et al., 1981)
and the placenta and decidua of the human (Bigazzi et al., 1980; Fields
and Larkin, 1981; Yamamoto et al., 1981).
Pig
The ovary of the pig has been shown to be the most abundant source
of relaxin and the majority of the biochemical work has been accomplished
on relaxin extracted from this tissue (Schwabe et al., 1978).
Doczi (1963 U.S. patent 3,096,246) was the first investigator to
extract relaxin from porcine ovaries utilizing an acid-acetone extraction
solution. Griss et al. (1967) utilized a purification technique similar
to that used by Doczi to extract and partially purify relaxin from the
porcine ovary.
An initial extraction in a solution of hydrochloric acid, acetone
and water was conducted and then the relaxin containing extract was
fractionated with gel chromatography and anion exchange chromatography.
These separation techniques yielded a basic polypeptide with a molecular
weight (mw) of 5,000 to 10,000 that had both uterine relaxing activity
and the ability to cause lengthening of the interpubic ligament. Sherwood


14
and O'Byrne (1974) used an extraction procedure similar to that of
Doczi (1963) and Griss et al. (1967) and were the first to fully charact
erize the porcine relaxin molecule. Relaxin obtained by this procedure
could be separated by carboxymethyl cellulose (CMC) ion exchange chroma
tography into three fractions: CM-B, CM-a, and CM-a'. These fractions
had mw and isoelectric points of: 6340 and pH 10.55 (CM-B); 6370 and
pH 10.72 (CM-a) and 6180 and pH 10.77 (CM-a'). None of the fractions
contained amino acid residues of histidine, tyrosine or proline and all
had equal potency (2,000 to 3,000 U/mg), as determined by the mouse
interpubic ligament bioassay. Each fraction consisted of two subunits,
an alpha and a beta chain linked by disulfide bridges. Amino acid
analyses of the CM-a alpha subunit showed it to contain 22 amino acid
residues. The beta subunit contained some microheterogeneity with amino
acids ranging from 28 to 31 in number. Schwabe et al. (1976; 1977),
using the same purification scheme as Sherwood and O'Byme (1974),
sequenced the porcine relaxin molecule. They showed the alpha and beta
chains to contain 22 and 26 amino acid residues, respectively, and also
found that porcine relaxin lacked histidine, tyrosine or proline. James
et al. (1977) also published the primary structure for porcine relaxin.
These investigators used the same purification scheme as Sherwood and
O'Byme (1974), but obtained an amino acid sequence different from that
obtained by Schwabe et al. (1976; 1977). The difference in the alpha
chain was minor (glutamine instead of glutamic acid in the 10 position).
The beta chain was found to contain 29 amino acids, with amino acids
from the twenty-third position to the end terminus being of a different


15
sequence than those found by Schwabe et al. (1976; 1977). Walsh and
Niall (1980) utilized a novel approach in the purification of porcine
relaxin. Tissues were immediately frozen in liquid nitrogen upon
removal from the animals, and homogenized in a cold solution consisting
of trifluoroacetic acid, formic acid, hydrochloric acid and sodium
chloride. After centrifugation of the homogenate, the supernatant was
pumped through an octadecylsilica (ODS) column to which the relaxin and
other peptides adhered. The solution resulting from this procedure was
then chromatographed in gel and CMC ion exchange columns. The resulting
relaxin preparation consisted of one relaxin peak, which contained 31
amino acids in its beta chain, and eluted in the same position as CM-a
porcine relaxin (31 amino acids) in the CMC ion exchange chromatography
column run. The Walsh and Niall technique thus eliminated the molecular
microheterogeneity previously reported by other laboratories, and they
concluded that the microheterogeneity was due to degradation during
extraction.
Further characterization of the porcine relaxin molecule resulted
in the discovery that porcine relaxin and insulin were closely related
molecules. Although the amino acid sequences of the two hormones were
not the same, there was a striking similarity in tertiary structure,
including the presence of disulfide bridges at corresponding positions
in the molecules (Isaacs et al., 1978; Blundell, 1979). Clues that the
three-dimensional configuration of porcine relaxin is important to its
biological activity came from the work of Schwabe and Braddon (1976)
who showed that partial oxidation of the tryptophan at the 18 position


16
of the beta chain led to biological inactivation of the molecule. Reduc
tion of the disulfide bonds of the relaxin molecule with dithiothrietol
also eliminated its bioactivity (Schwabe et al., 1978).
Evidence for a prorelaxin compound has been accumulating from
several sources. James et al. (1977) suggest that relaxin might be
cleaved from a proinsulin like compound by proteolytic enzymes. Since
arginine is present at the N-terminus of the alpha chain as well as the
C terminus of the beta chain, they envision a prorelaxin precursor with
connections between the 30 position in the beta chain and the 1 position
in the alpha chain. The proteolytic cleavage would take place at this
position in the molecule. These investigators have identified forms of
relaxin in pig ovarian extracts which differ in net charge and amino
acid composition from the 6,000 mw relaxin molecule and feel that these
may perhaps be considered intermediates in the conversion of prohormone
to hormone.
Frieden and Yeh (1977) have acquired evidence for a prorelaxin
like compound in porcine ovarian extracts. When these investigators
chromatographed NIH relaxin (440 U/mg), they separated the material
into two protein peaks. Approximately 70% of the relaxin activity was
found in a peak eluting in the 6,000 mw range. However, approximately
10% of relaxin activity was concentrated in a 40,000 mw fraction. When
this higher mw fraction was exposed to trypsin, some of the high mw
material was converted to a 6,000 mw relaxin. This low mw relaxin was
indistinguishable from purified porcine relaxin in gel chromatography,
polyacrylamide gel electrophoresis (PAGE) and biological activity in the
guinea pig interpubic ligament bioassay. It appears from the above


17
studies that relaxin, like insulin, may be cleaved from a larger mw
precursor.
Cow
Bovine relaxin has been purified from ovaries of the late pregnant
cows (Fields et al., 1980). In this study, crude extracts were prepared
from corpora ltea by the technique of Griss et al. (1967). Chromatog
raphy of the crude extract on a Bio-Gel P-10 column demonstrated two
fractions having mw of 1,400 and 6,000. Both fractions were shown to
inhibit mouse uterine contractions in vitro and induce lengthening of
the mouse interpubic ligament. Immunodiffusion analyses showed a con
tinuous precipitin line between the two cow relaxin fractions, the NIH-
R-Pl porcine relaxin and an antiserum (R19), produced against purified
porcine relaxin. The 6,000 mw fraction gave 3 bands when electrofocused:
pH 8.8, pH 10.1 and pH 11.5. The pH 10.1 form of bovine relaxin had the
highest biological activity (250 U/mg) according to the mouse uterine
motility assay. The low mw relaxin lost activity in the presence of
dithiothrietol (Fields et al., 1980).
Rat
In 1979 Sherwood reported the purification and characterization of
rat relaxin. Ovaries were homogenized in a saline solution and two forms
of relaxin were obtained after fractionation of the crude ovarian extract
with Sephadex G-50 gel chromatography and CMC ion exchange chromatography.
The two forms were designated 01-1 and CM-2, and each contained comparable
specific activity when assayed with the mouse interpubic ligament bio
assay. CM-1 and CM-2 had isoelectric points of pH 7.6 and pH 9.4,


18
respectively, and both had raw of approximately 6,000. Unlike pig
relaxin, rat relaxin contained histidine, proline and tyrosine. Also,
although giving a linear log dose-response curve in the mouse interpubic
ligament bioassay, the slope of the line was not parallel to the assay
slope of the purified pig relaxin standard. These results supported
earlier findings by Larkin (1974), who used crude preparations from
rat and pig ovaries. Fields and Larkin (1979) also reported on the
isolation of rat ovarian relaxin. They isolated a fraction from a Bio-
Gel P-10 column which eluted in the mw range of porcine relaxin and
contained a potency of 60 U/mg in the mouse uterus bioassay. Electro-
focusing of the Sephadex fraction yielded 3 peaks with isoelectric
points of pH 9.0, 8.7 and 7.8. These peaks had activities of 325, 425
and 125 U/mg, respectively. Walsh and Niall (1980) isolated ovarian
relaxin from pregnant rats using the ODS technique previously mentioned
and obtained one major relaxin peak after preparation on a CMC ion
exchange chromatography column. No isofocusing data were presented for
the rat relaxin molecule in the Walsh and Niall (1980) study.
John et al. (1981) studied the sequence homologies between rat and
porcine relaxins. They isolated rat relaxin from late pregnant rat
ovaries (days 18-21 of pregnancy), according to the technique of Walsh
and Niall (1980). Amino acid sequencing studies showed the alpha chain
of rat relaxin to be 24 residues long and the beta chain to be 35 resi
dues long. The rat relaxin molecule contained tyrosine and histidine.
Only limited homology existed between rat and porcine relaxin, with
approximately 40% of the amino acid residues in corresponding positions
being identical. Antigenic dissimilarities between the porcine and rat


19
relaxins were also shown by the observation that only slight cross
reactivity existed between antisera produced against porcine relaxin and
rat relaxin (Larkin et al., 1979; Fields and Larkin, 1979; Sherwood and
Cmekovik, 1979) .
Rabbit
The placenta of the rabbit had been reported to contain relaxin by
Zarrow and Rosenberg in 1953. Of the tissues tested (ovary, uterus,
placenta), the maternal portion of the placenta seemed to contain the
highest levels of biologically active relaxin. Lower levels were seen
in the fetal placenta and uterus. Further proof that rabbit placentae
contained relaxin was demonstrated by Larkin et al. (1979) who showed
that antiserum produced against porcine relaxin inhibited the activity
of rabbit placental extracts in the mouse uterine motility assay. Also,
a reaction of identity was obtained when a Bio-Gel P-10 fraction of
rabbit placental extracts was compared with purified porcine relaxin
and an antiserum made against porcine relaxin in an agar double immuno
diffusion assay (Larkin et al., 1979).
This preliminary work led Fields et al. (1981) to purify relaxin
from the rabbit placenta. After extraction using a modified Griss
method (Griss et al., 1967), separation was achieved on a Bio-Gel P-30
column. The Bio-Gel P-30 fraction eluting at 6,000 daltons contained
low bioactivity in the mouse uterine motility bioassay (1.50 U/mg).
When this fraction was chromatographed in a CMC ion exchange column, a
single peak containing 15 U/mg was eluted. The mouse interpubic liga
ment assay was conducted on the CMC fraction. The dose response curve


20
for rabbit placental relaxin was parallel to the dose response curve of
the porcine standard. With this assay, the CMC fraction was calculated
to have a biological activity of 21.6 U/mg. Electrofocusing of the CMC
peak resulted in the separation of four distinct fractions.
Human
Whereas it has been established that the ovary is a source of
relaxin in the pregnant human (Weiss et al., 1976; Weiss et al, 1977;
O'Byrne et al., 1978; Szalchter et al., 1980), characterization of
relaxin from the ovary has not been accomplished. On the other hand,
recent reports of isolation of a placental relaxin have been published
(Fields and Larkin, 1981; Yamamoto et al., 1981). These recent reports
support the earlier work of Dallenbach and Dallenbach-Hellweg (1964) who
found immunoreactive relaxin in the basal plate of the human placenta
using indirect immunofluorescence. Bigazzi et al. (1980) have demon
strated relaxin production by the decidua capsularis in vitro and have
extracted relaxin from decidual tissue collected from term pregnancies.
Fields and Larkin (1981) first isolated and purified human placental
relaxin using the extraction technique of Griss et al. (1967). They
found human relaxin to be a peptide similar to porcine relaxin in molecu
lar weight. The biological activity of an isolated Bio-Gel P-30 fraction
(6,000 mw) as determined by the mouse uterine motility assay, was 11.9
U/mg. The same fraction produced a linear response in the mouse inter-
pubic ligament bioassay, which was parallel to the porcine standard.
Electrofocusing of the active fraction produced one peak having an
isoelectric point of pH 11.4 and a biological activity of 45 U/mg. The
electrofocused fraction exhibited a continuous line of identity with no


21
spurring in double immunodiffusion analyses when tested against purified
porcine relaxin. Incubation of the human relaxin with dithiothrietol
inactivated the hormone, indicating that disulfide bonds were necessary
for its biological activity.
Yamamoto et al. (1981) were also able to detect relaxin in extracts
of basal plates of human placentae. Placentae from cesarean deliveries
were found to contain five times higher relaxin levels than placentae
from normal deliveries. A single immunoreactive peak (as determined by
RIA) eluted in the 6,000 mw range from a Sephadex G-50 gel chromatography
column. The pooled active peak was applied to a CMC ion exchange column
and eluted with a salt gradient. Three relaxin fractions were obtained
from the CMC column and were called CMc-1, CMc-2, and CMc-3. CMc-1 and
CMc-2 eluted prior to the start of the salt gradient, while CMc-3
eluted at the start of the salt gradient (0.1 M NaCl). The three relaxin
peaks had parallel dilution curves when assayed in a homologous porcine
relaxin RIA. The CMc-2 fraction contained the only biological activity
as detected by the mouse uterine motility assay. Electrofocusing and
mouse interpubic ligament data were lacking.
Concurrent with the observations from Larkin's and Bryant-
Greenwood's laboratories were the observations of Bigazzi et al. (1980)
indicating that relaxin could be obtained by scraping the maternal
surface of fetal membranes gathered from normal deliveries. A homogenate
from the decidual tissue was obtained and fractionated. Only one fraction
from a Sephadex G-50 column contained relaxin biological activity as
shown by the rat uterine motility inhibition assay and the mouse inter
pubic ligament assay. This fraction eluted in the mw range of porcine


22
relaxin and had a tissue level of 15.0-33.5 U/mg of fresh tissue.
Further purification of the extract was not reported.
The relaxins studied to date appear similar in that they have
S-S linkages and an approximate mw of 6,000. Some differences, however,
exist among pig relaxin and relaxins purified from other species: (1)
the porcine relaxin has the highest specific activity in the bioassays,
(2) the relaxins from various species differ in isoelectric points, (3)
the two relaxins in which amino acid sequencing has been done appear
to be different, i.e., porcine relaxin contains no tyrosine or histidine
while rat relaxin does, and (4) a low mw form of bovine relaxin has
been reported. While not all relaxins have been studied with RIA, they
have all been characterized utilizing bioassay techniques.
Relaxin in the Guinea Pig
Early literature has suggested that nonovarian sources may be
very important in the production of relaxin in the guinea pig. Hisaw
(1926) was the first to discover that blood serum from pregnant guinea
pigs and rabbits, when injected subcutaneously (SC) into virgin guinea
pigs during early post-estrus, caused pubic symphysis relaxation six
hours later. Hisaw et al. (1944) further demonstrated that the pubic
ligaments of castrated guinea pigs pretreated with estradiol for 4 days
could respond to a single injection of progesterone and exhibit
increased pelvic mobility within 72 hours. Castrated, hysterectomized,
and estrogen-treated guinea pigs, on the other hand, did not respond to
progesterone treatment regardless of the progesterone dose. These same
animals could, however, respond to small quantities of relaxin within


23
six hours. These studies indicated that the estrogen primed uterus
could be induced to produce relaxin with progesterone treatment.
The status of relaxin in the guinea pig was equivocal because
some investigators obtained relaxation of the pelvis of the guinea pig
by estrogen therapy alone (Brouha, 1933) or with combinations of estrogen
and progesterone (Fugo, 1943). It should be pointed out that important
differences existed among Hisaw's observations and those of Brouha and
Fugo. The most obvious difference was that the time required to produce
a reaction in the relaxin treated, castrated animal pretreated with
estrogen was very short (6 hr). On the other hand, estrogen alone
(18-20 days) or combinations of estrogen and progesterone (2-4 days)
took much longer to elicit their effect.
Hisaw's early findings were confirmed by Zarrow (1947; 1948), who
found bioassayable relaxin in the blood of guinea pigs during middle and
late pregnancy, but not after parturition. He also noted relaxin activity
in extracts of the uterus and placentae on days 56 and 63 of pregnancy.
This added credence to Hisaw's theory that the uterus was responsible
for relaxin production in the pregnant guinea pig. Zarrow (1948)
further confirmed this by showing that progesterone could elicit forma
tion of relaxin in a castrated estrogen-primed guinea pig. Progesterone
did not cause production of relaxin in an estrogen-primed castrated and
hysterectomized animal regardless of the dose involved. This work,
although giving strong indication as to the tissue source of relaxin in
the guinea pig, relied exclusively on the cumbersome and subjective
guinea pig pubic symphysis assay. However, recent results have supported
the observations of Hisaw and Zarrow, rather than conclusions obtained by
Brouha and Fugo.


24
Recently, O'Byrne and Steinetz (1976) assayed sera from 4
pregnant guinea pigs at different stages of gestation with RIA. They
used a homologous RIA employing antibodies to porcine relaxin, which
was able to detect as little as 0.1 ng of the guinea pig relaxin. They
found that peripheral blood levels of relaxin gradually increased from
an average of less than 0.2 ng/ml in the 20 day pregnant guinea pigs
to just over 0.4 ng/ml in the 50 day pregnant animals. Postpartum
animals (24 hr after delivery) still contained high relaxin levels
(average 0.5 ng/ml). This study was only concerned with overall serum
levels of immunoreactive relaxin and did not look at individual tissue
levels. Bioassays were not conducted.
Boyd et al. (1981) used a homologous porcine RIA to assay plasma
relaxin immunoactivity in guinea pigs during the estrus cycle, throughout
mid to late pregnancy and parturition, and during lactation. Although
variability among animals was high, several major points could be drawn
from the study: (1) during the estrus cycle, relaxin levels were lowest
during estrus (2 ng/ml) and highest during portions of diestrus and
proestrus (5-6 ng/ml), (2) during the latter stages of pregnancy, relaxin
levels were higher (12-14 ng/ml), decreasing to basal levels after
parturition (2-4 ng/ml), and (3) during lactation, suckling did not
elevate relaxin levels in nursing dams, and in some instances, actually
decreased them.
In summary, work previous to 1950 indicated that relaxin was
present in the blood, uterus and placenta of pregnant guinea pigs. It
also showed that progesterone somehow stimulated production of relaxin


25
by the uterus in estrogen primed, castrated guinea pigs. Only recently
has RIA been employed to detect the presence of relaxin in serum of
pregnant and cycling guinea pigs.
In the past, the guinea pig was used extensively as an experimental
animal in relaxin work. This animal, however, has been neglected in
recent research due possibly to several reasons: (1) interest in guinea
pig relaxin decreased when newer, faster and less expensive bioassay
techniques using other animals became available, (2) corpora ltea of
the pig became established as the main source of relaxin, (3) cost of
keeping guinea pig colonies increased, compared to other laboratory
rodents, and (4) investigators focused on the ovary as being the only
source of relaxin in many mammals. There are, on the other hand, several
compelling reasons to study relaxin in the guinea pig. The guinea pig
is quite similar to the human in placentation, hormonal changes which
occur during gestation and the presence of an extra ovarian source of
relaxin (Zarrow, 1948; Pardo et al., 1980).
Statement of Problem
The primary goal of this research is to study relaxin in the
guinea pig. Studies proposed are designed to answer the following
questions: (1) Is relaxin produced by nonovarian sources in the guinea
pig? If so, what tissue and cell types produce the hormone? (2) What
are the tissue and serum levels of relaxin in the guinea pig throughout
pregnancy and lactation? (3) Can the rise and fall of serum and tissue
levels of relaxin be correlated with immunocytochemical studies? (4)
What are the physical and biochemical characteristics of the guie pig
relaxin molecule?


MATERIALS AND METHODS
General Procedures
Experimental Design, Treatment of Animals and
Collection of Specimens
Guinea pigs obtained from a local vendor were housed in the
University of Florida Health Center Animal Resources Department, and had
access to food and water ad libitum and a photoperiod of 12 hr light
and 12 hr dark. Adult females were housed with a male and pregnancy
was timed from the day on which sperm were found in a vaginal smear.
Animals used in ovariectomy studies were anesthetized with 0.88
ml/Kg Innovar-Vet purchased from Pitman-Moore, Inc., Washington Cross
ing, NJ, and bilaterally ovariectomized through two flank incisions.
Two weeks after the operation, the animals were started on a daily regi
men of hormone injections. Animals were given one of the following:
(1) estrogen alone (10 yg), (2) estrogen (10 yg) and progesterone (1 mg)
together, or (3) no injections. The hormones were mixed in sesame seed
oil and injected SC at the back of the neck. Estradiol dipropionate
was obtained from Ciba Pharmaceutical Products, Inc., Summit, NJ. Pro
gesterone was obtained from Eli Lilly and Co., Indianapolis, IN. Injec
tions were given daily at approximately 11:00 AM for 15 days (time needed
for estrogen-progesterone treated animals to undergo relaxation of the
pelvic ligaments (Zarrow, 1948)). Two animals were used for each of the
three treatments and were monitored daily for pelvic flexibility by
manual palpation.
26


27
All animals were killed at the same time of the day (11:00 am
+_ 1 hr). The animals were anesthetized with pentobarbitol (2.5 mg/100 g
body weight) and exsanguinated via cardiac puncture. The reproductive
tract was removed immediately and portions of the uterus were fixed in
Bouin's solution for histologic study. This tissue was processed for
paraffin embedding. The remainder of the uterus was frozen at -20 C
and later used in the extraction procedure.
Antirelaxin Sera
Antisera against highly purified porcine relaxin was produced in
New Zealand white rabbits as described by Larkin et al. (1977). In this
technique, 2 mg of a pig relaxin preparation (WL 150, 150 U/mg) obtained
from Warner Lambert, Inc., Morris Plains, NJ, were run on PAGE. The
bands were localized by fixing them in trichloroacetic acid (TCA) (15%),
and staining in 0.6% Coomassie blue in 15% TCA. Three bands were present
and were named Cl, C2, and C3; Cl being the closest to the anode. The
C2 bands were then cut out of the gels and homogenized in an equal volume
of Freund's complete adjuvant and injected into New Zealand white rabbits.
Subsequent injections were given with the gels homogenized in Freund's
incomplete adjuvant. The injection schedule was as follows: Rabbit 19
was given one SC injection per week for six weeks. The injections con
taining six-C2 bands were given dorsally between the scapulae. Booster
injections consisting of six-C2 bands were given approximately every
two months.
R19 antiserum has been shown to inhibit the biological activity
of porcine, cow and rabbit relaxins in vitro (Larkin et al., 1979).


28
Also it has been used to detect relaxin immunocytochemically in cells of
the cow ovary (Fields et al., 1980), and human placenta (Fields and
Larkin, 1981).
Tissue Extraction
Preparation of crude uterine extracts was accomplished by utilizing
one of two methods. Initially, tissues were extracted with the acid-
acetone procedure of Griss et al. (1967). This procedure was employed
for the extraction of uteri taken from individual animals and the extract
was used for bioassay and RIA experiments. Recently, a new extraction
procedure for relaxin was reported by Walsh and Niall (1980). This
newer technique was employed to extract relaxin from uteri and the
resulting preparations were used in purification and characterization
studies. A more detailed account of these techniques is given below.
Griss procedure.The extraction procedure of Griss et al. (1967),
was used for extraction of uteri utilized in bioassay and RIA experi
ments. Cold extraction solution (acetone:water:hydrochloric acid, 5.0:
2.83:0.17 ratio) was added to minced frozen tissues at a ratio of 5:1
(ml/g), and homogenized in a Sorvall Omni-mixer at 4 C. The extract
was incubated for 24 hr at 4 C and then centrifuged at 3000 RPM's
(4 C) for 30 min in a Beckman J-21c centrifuge equipped with a JA-14
rotor. Five volumes of acetone were added to the supernatant and the mix
ture was stored at -20 C for 24 hr. The majority of the supernatant
was decanted and the precipitate pelleted by centrifugation at 3000 RPM's
for 10 min in a JA-14 rotor and air dried. The dried powder was weighed
and stored in a sealed container at room temperature.


29
Walsh and Niall procedure.The extraction procedure of Walsh
and Niall (1980) was utilized in the purification and characterization
stages of the research because it had been reported to yield more
relaxin with less proteolysis. Uteri were removed from late pregnant
guinea pigs (65-67 days), immediately frozen in liquid nitrogen and
stored at -80 C in a Reveo freezer until extracted. Twenty gram aliquots
of minced frozen uterus were placed in 200 ml of a cold solution of
15% trifluoroacetic acid, 5% formic acid, 1% NaCl and 1 M HC1. The
tissue was homogenized for 2 min in a Sorvall Omni mixer. The homogen
ate was centrifuged (4 C) for 30 min at 10,000 RPM's in the JA-14
rotor. The resulting supernatant was filtered through Whatman filter
paper (No. 541) and a 0.45 ym pore millipore filter. Octadecylsilica
columns, purchased from Waters Associates, Millford, MA, were preequili
brated by passing 30 ml of an 80% acetonitrile, 0.1% trifluoroacetic
acid solution followed by a 30 ml wash of distilled water. The relaxin
containing supernatant was pumped through three ODS columns twice and
the columns were washed with 30 ml of a 10% acetonitrile, 0.1% tri
fluoroacetic acid solution. The eluate was evaporated to near dryness
at 38 C and resuspended in a known volume of 0.01 M ammonium acetate
buffer pH 5.
Detection of Relaxin
Immunocytochemical Localization of Relaxin
Immunoperoxidase staining was conducted as described by Stemberger
(1979) according to the following protocol. All dilutions of antisera
were carried out with phosphate buffered saline (PBS) pH 7.4, and the
incubations were conducted at room temperature. Paraffin sections


30
(6 pm in thickness) were deparaffinized immediately prior to use. Normal
goat serum (1/30 dilution) was applied to the sections for 30 min. The
slides were drained but not rinsed and 4 drops of either R19 antiserum
or control solutions of varying concentrations were applied to the
sections for 30 min. The slides were rinsed with a stream of PBS and placed
in Copeland jars containing PBS for three, three min rinses. The slides
were drained of excess PBS, and blotted to absorb excess PBS from around
the sections. Four drops of goat antirabbit IgG (GAR) (1/20 dilution),
purchased from Polysciences Inc., Warrington, PA, were applied to the
sections for 30 min. The sections were rinsed, drained and blotted as
described previously. Four drops of peroxidase-antiperoxidase (PAP)
(1/80 dilution with 0.05 M tris saline pH 7.6), purchased from Stem-
berger-Meyer Immunocytochemicals, Jarretsville, MD, were applied to the
sections for 30 min. The sections were rinsed, drained and blotted as
described previously. The PAP was visualized by incubating the slides
in a 5 mg% DAB solution (3,3' diaminobenzidine) type II, purchased from
Sigma, St. Louis, MO, with 0.01% ^2^2 ^or min. The slides were then
washed in distilled water for 5 min, briefly treated with 1% 0^4,
rinsed in distilled water, dehydrated through alcohols and xylene, and
covers lips were applied. The following controls were carried out: (1)
substitution of the R19 antiserum with serum from a male rabbit that had
not been immunized against relaxin (NRS), (2) omission of the R19 anti
serum and replacement with PBS (pH 7.4), (3) absorption of the R19 anti
serum with porcine relaxin standard (NIH-RXN-P1), and (4) successive
dilutions of the R19 antiserum.


31
Bioassay of Relaxin Containing Extracts
All mice used in the bioassays were females of the ICR strain
which were initially obtained from Flow Laboratories (Dublin, VA).
Mouse uterine motility.--The in vitro mouse uterus bioassay as
described by Kroc et al. (1959) and modified by Larkin et al. (1981) was
employed to detect relaxin in tissue extracts. Female mice (16-18 g)
were primed with 0.1 ml of estradiol dipropionate (50 yg/ml). The mice
were killed 7 or 8 days later and their uteri removed. Each horn of the
uterus was divided in to two portions and each portion was suspended in
a test tube containing 20 ml of Locke's solution at 37 C. The uterine
segments were attached to a heart lever against 1 g tension and con
tractions were recorded on an ink writing kymograph. Specific volumes
of either NIH-RXN-P1 standard relaxin preparation or unknown solutions
of known concentrations were added to the tubes so that the bath concen
trations were doubled every 4 min. Specific activities were calculated
using the following equation:
Specific Activity of Unknown = x x ^P^S
VU CU
where VS is the volume (in yl) of standard relaxin preparation needed to
reduce the uterine contractions by half, VU is the volume (in yl) of
unknown relaxin preparation needed to reduce the uterine contractions by
half, CS is the concentration of the standard relaxin preparation (in
yg/ml), CU is the concentration of the unknown relaxin preparation (in
yg/ml), and SpAS is the specific activity of the NIH-RXN-P1 relaxin stand
ard (U/mg protein).
Mouse interpubic ligament.--The in vivo assay for relaxin activity
was employed according to the technique of Steinetz et al. (1960). The


32
length of the interpubic ligament was determined in a three point
parallel line assay employing 20 mice at each dose level of the relaxin
standard and 15 mice at each dose level of the unknown. At day 0,
virgin prepuberal female mice (18-20 g weight) were primed with an SC
injection of 5 yg estradiol cypionate purchased from the Upjohn Co.,
Kalamazoo, MI, in 0.1 ml of sesame seed oil.
On day 7, the relaxin standard (NIH-RXN-P1) and unknowns of com
parable levels of activity (as determined by the mouse uterine motility
bioassay) were injected SC in 0.2 ml of a 1% solution of benzopurpurine-
4B. Control mice received 0.1 ml of estradiol cypionate and 0.2 ml 1%
benzopurpurine-4B. The dose levels for the NIH-RXN-P1 standard were
0.5 yg, 0.25 yg and 0.125 yg per mouse. The dose levels of the guinea
pig Sephadex G-50 fraction were 1 mg, 0.5 mg and 0.25 mg. Eighteen to
twenty-four hours later the mice were killed in a CC^ chamber, the abdom
inal cavities opened and the uteri examined for evidence of estrogen prim
ing. No mice exhibited "threadlike uteri due to lack of priming. The
anal and vulval areas and upper half of the trunk were dissected away
with scissors, thereby removing the skin and all pelvic organs surround
ing the pubic symphysis. The pelvis was positioned under a light source
allowing a beam of light to pass through the pubic ligament. The short
est distance between the edges of the pubic bones was measured with a
dissecting microscope fitted with an occular micrometer. Results were
evaluated by the method of least-squares of variance; the computer
program was PROC CLM of Statistical Analysis System (Barr and Goodnight,
1976). The mathematical model was preparation (NIH versus guinea pig)


33
and dose (3 levels). Dose effects were examined further by polynomial
regression. Differences in dose/trends between preparations (NIH versus
guinea pig) were examined by tests of heterogeneity of regression. A
valid assay is one in which there is a significant (P>0.01) linear
regression of response to log dose, no divergence from parallelism to
the NIH-RXN-P1 standard, no quadratic regression components, and a
lambda value of less than 0.4. The results were expressed as U/mg
relative to the NIH-RXN-P1 porcine relaxin standard.
Radioimmunoassay
Three different iodination methods were attempted in the devel
opment of the homologous porcine RIA for guinea pig relaxin. Since
sufficient amounts of purified guinea pig relaxin were not available
for the RIA experiments, it became necessary to employ porcine relaxin
for both the immunization procedure and for iodination. The first two
procedures involved the use of the Bolton and Hunter reagent for the
iodination of porcine relaxin (NIH-RXN-P1) (Bolton and Hunter, 1973).
These two procedures were not utilized in the research reported and
specific information about these assays will be found in Appendices
3 and 4. In the third procedure, polytyrosyl relaxin was iodinated by
the method of O'Byrne and Steinetz (1976).
Iodination of polytyrosyl relaxin.--The iodination was conducted
according to the technique of O'Byrne and Steinetz (1976) with some
modifications. Polytyrosyl relaxin (5 yg), donated by Dr. B. G. Steinetz
of the Ciba Geigy Corp., Ards ley, NY, was dissolved in 50 yl of 0.1 M
sodium phosphate buffer pH 7.5. The polytyrosyl solution and 1 m Ci
I purchased from the Amersham Corp. were added to a 10 x 75 mm test


34
tube coated with 100 yg dried iodogen. Iodination was achieved util
izing the technique of Markwell and Fox (1978). Iodogen (1,3,4,6-
tetrachloro-3,6-diphenylglycouril) was purchased from the Pierce
Chemical Corp., Rockford, IL. The reaction solution was mixed at room
temperature for 15 min with intermittent shaking and then transferred
to a 1 x 18 cm column of Sephadex G-25 preequilibrated with 0.5 M
sodium phosphate buffer pH 7.0. Fractions (20 drops/tube) from the
gravity fed column were collected in 10 x 75 mm tubes containing 0.5 ml
of PBS 1% ovalbumin pH 7.0. Ten microliters of the pooled assay tubes
of the polytyrosyl relaxin peak contained 160,000 cpm of radioactivity.
Approximately 33% of the -^I-labelled polytyrosyl relaxin was precipi-
1 25
table in antibody excess. The I-labelled polytyrosyl relaxin was
used for four weeks after iodination before a noticeable drop in sensi
tivity was noticed in the RIA.
nr
Development of RIA utilizing I-labelled polytyrosyl relaxin.--
Fractions containing the ^I-labelled relaxin were pooled and employed
in the development of the RIA used to detect guinea pig relaxin. For
the detection of relaxin in crude uterine extracts, 20 mg of the acid-
acetone extracted powder was suspended in 1 ml of PBS-1% ovalbumin,
pH 7.0 and the resulting suspension centrifuged to remove nonsolubilized
material. The supernatant was then diluted 1:1 with PBS-1% ovalbumin,
and tested in the RIA.
Double antibody RIAs were conducted in 10 x 75 mm disposable glass
culture tubes. Quantities of relaxin standard solutions (NIH-RXN-P1)
containing 3.25-2000 pg of relaxin in PBS-1% ovalbumin or volumes of
uterine extract supernatants were added to the culture tubes. Sufficient


35
quantities of PBS-1% ovalbumin were added to each tube to bring the
volume to 500 yl. One hundred microliters of R19 antiserum (1:25,000
final dilution) in 0.05 M ethylene diamine tetraacetic acid-PBS con
taining 6% male rabbit serum were added to each tube. The tubes were
vortexed and then incubated at 4 C for 24 hr. One hundred microliters
1
of I-labelled relaxin (10,000-15,000 CPM) in PBS-1% ovalbumin were
added to each tube, the tubes were vortexed and then incubated for 24 hr
at 4 C. The tubes were then centrifuged at 3000 RPM's for 30 min,
drained of supernatant and the pellets counted in a Searle analytic
gamma counter. A standard curve employing NIH-RXN-P1 relaxin was run
concurrent with every assay. Radioactivity expressed as % bound was
plotted in a % bound versus log dilution curve and unknown guinea pig
values were read off the standard curves and expressed as porcine relaxin
equivalents. The following controls were employed: (1) Total count tube
i or
(T): 100 yl of i'i0I-labelled polytyrosyl relaxin gives the total
amount of isotope added to each tube. (2) Nonspecific binding (NSB) :
primary antiserum (R19) was omitted to determine whether there was
any nonspecific binding of the I-labelled relaxin to other assay
components. (3) "Zero" count tube (Bo): radioinert relaxin was not
added to determine the maximum amount of possible binding of the 1 JI-
labelled relaxin to the antirelaxin serum. Percent binding (% B) was
determined by dividing the radioactivity of the standard or unknown tubes
(bound) by the "zero" count tube (Bo). Nonspecific radioactive binding
was subtracted from all values before calculations were made.
o. n bound-NSB
'O D
Bo-NSB


36
RIA characterization.--The specificity of the homologous porcine
RIA used to detect guinea pig relaxin was tested in two experiments.
First, dilution curves obtained with crude and semi-purified preparations
of guinea pig relaxin were compared to the dilution curves obtained
using purified NIH-RXN-P1 porcine relaxin. Secondly, the levels of
relaxin crossreactivity were determined in preparations of crude
extracts taken from uteri in varying stages of pregnancy. Interassay
reproducibility was determined by measuring the variability in a 125
pg sample of porcine NIH-RXN-P1 relaxin between 4 different standard
dilution curves. Intraassay reproducibility was determined by measur
ing a 125 pg sample of porcine NIH-RXN-P1 relaxin in the same assay 6
different times.
Purification and Characterization of Guinea
Pig Relaxin
Purification
Gel filtration.The Walsh and Niall (1980) procedure was utilized
to extract 118.50 g of uterine tissue from 5 late pregnant guinea pigs
and the crude extract from the ODS extraction (188.8 mg protein) was
suspended in 10 ml ammonium acetate buffer, pH 5.0. Protein was
determined by the method of Lowry et al. (1951). The protein solution
was layered on a Sephadex G-50 (fine) column (2 x 100 cm), purchased
from Pharmacia Fine Chemicals, Uppsala, Sweden, and equilibrated with
the same buffer. The column was developed at room temperature at a rate
of 7.5 ml/hr. Material eluting from the columns was monitored at 280 mm
wavelength with a Beckman Acta III Spectrophotometer. Fractions were
collected every 24 min. Fractions containing the protein peaks were


37
pooled, lyophilized, and assayed using the mouse uterine motility
bioassay. Columns were calibrated by using a series of low mw markers
and by using a porcine relaxin standard. Buffer containing sodium
azide (0.05%) was pumped through the column between experiments to
eliminate bacterial growth.
Bio-Gel P-30, purchased from Bio-Rad Laboratories, Richmond, CA,
was utilized as the gel chromatrography resin to prepare the relaxin
fraction used in the double immunodiffusion experiments. This gel was
equilibrated and run in exactly the same manner as the Sephadex G-50
resin.
Ion exchange chromatography.--The active fraction from the Sephadex
column (35.5 mg) was applied to a 0.8 x 5 cm CMC column (CM-52) purchased
from Whatman Ltd., Springfield, England, and then equilibrated with
0.01 M ammonium acetate buffer, pH 5.0 until all unadsorbed material was
removed. The column was developed at a rate of 9 ml/hr with a linear
NaCl gradient (0.1 M to 0.3 M) in 0.01 M ammonium acetate buffer pH 5.0
to a final conductivity of 20 m Mho. Fractions (1.5 ml) were collected
every 10 min.
Characterization
Double immunodiffusion studies.--Double immunodiffusion plates
were employed as described by Clausen (1969) for the microtechnique.
Petri dishes of 12 x 60 mm (1.5% agar in 0.85% saline with 0.1% sodium
azide) were used. A center well was filled with 5 yl of R19 antiserum
and peripheral wells were filled with 5 pi of a Bio-Gel P-30 6,000 mw
fraction from guinea pig uterus (6 mg/ml), and NIH-RXN-P1 porcine
relaxin. The substances were allowed to diffuse at room temperature for
24 hr.


38
Two dimensional gel electrophoresis.--Two dimensional gel electro
phoresis was conducted using a modification of the Horst et al. (1980)
technique. The first dimension employed a NEPHGE (nonequilibrium pH
gradient electrophoresis) system using tubes 14-15 cm long with an inner
diameter of 2.5 mm. The NEPHGE system was modified to accommodate more
basic polypeptides as described by Sanders et al. (1980), resulting
in an effective pH gradient of 5.4 to 9.8. The lower chamber of the
electrophoresis apparatus was filled with 0.04 M NaOH and the upper
chamber with 0.04 H^SO^. One hundred micrograms of either guinea pig
CMC purified relaxin or NIH-RXN-P1 relaxin were layered on the gels.
The gels were allowed to stack at 75 V for 15 min and then run for 2.5
hr at 400 V. The proteins were separated in the second dimension on
15% sodium dodecyl sulfate (SDS) polyacrylamide slab gels. Electrophor
esis buffer (3 g tris, 14.4 g glycine and 1.0 g per liter SDS) was placed
in the reservoirs and the gels were run toward the anode. Fifteen mamp/
slab were used as the stacking current for 2 hr. The current was turned
up to 30 mamp/slab and the gels were run for 4-5 hr. The gels were fixed
with 7% acetic acid:40% ethyl alcohol, and stained with 0.125% Coomassie
blue R250. The gels were destained in 7% acetic acid:10% ethyl alcohol.
Reduction with dithiothrietol.--Five hundred microliters of an ODS
crude* relaxin fraction (30 mg/ml H2O) from late pregnant guinea pig
*0DS crude relaxin is a partially purified uterine extract taken after the
initial purification step in the ODS procedure. This extract was tested in
the mouse uterine motility assay, without being altered (control), and after
the addition of different agents (experimental). A ratio was determined by
dividing the final volume of the experimental by the final volume of the
control. The assays were run twice and an average value was computed. The
greater the experimental to control ratio, the greater the ability of the
agent to inhibit the action of relaxin.


39
uterus was reduced by the addition of dithiothrietol (final concentra
tion, 0.1 M). The solution was incubated for 1 hr at room temperature.
A nontreated sample from the same fraction was tested as a control, and
potencies were compared using the mouse uterine motility bioassay.
Heating.--One hundred microliters of an ODS crude relaxin fraction
(30 mg/ml H?0) from late pregnant guinea pig uterus was heated at 70 C
for 1 hr. A nontreated sample from the same fraction was tested as a
control, and potencies were compared using the mouse uterine motility
bioassay.
Trypsin digestion.--Seven hundred microliters of an ODS crude
relaxin fraction (30 mg/ml H^O) from late pregnant guinea pig uterus
was incubated with the trypsin at a final concentration of 0.1 mg
trypsin/mg protein. The solution was incubated for 1.5 hr at room
temperature (pH 7.0). A nontreated sample from the same fraction was
tested as a control, and potencies were compared using the mouse uterine
motility bioassay.
In vitro assay of antisera (Larkin et al., 1979).--Fifty micro
liters of R19 antiserum were added to one of the tubes in the mouse
uterine motility bioassay, while the other tube received 50 pi of NRS
as a control. An ODS relaxin preparation of late pregnant guinea pig
uterus (30 mg/ml H^0) was then added to each tube in equal concentrations,
and the volumes required to inhibit the uterine contractions were com
pared.


RESULTS
Detection of Guinea Pig Relaxin
Immunocytochemical Localization of Uterine Relaxin
The PAP immunocytochemical technique was used to examine the
ovary, placenta, uterus, spleen and liver of guinea pigs. The endo
metrial gland cells (EGC) of the uterus was the only cell type that
showed heavy deposition of peroxidase reaction product (RP), indicating
the presence of relaxin. The ovary demonstrated weak staining, while
the liver, spleen and placentae did not stain. Therefore, subsequent
studies employed the uterus. Tissue samples were taken from guinea
pigs at different stages of pregnancy, during lactation and in ovari-
ectomized animals undergoing estrogen-progesterone treatments to deter
mine periods of relaxin production.
Relaxin was not detected in sections of uteri taken from nonpreg
nant ovariectomized control (no hormone treatment or estrogen treated
animals) or day 15 pregnant animals. Day 30 was the earliest stage of
pregnancy studied that showed accumulation of relaxin in the EGC. At
this stage, only a few glands contained relaxin (Figure 1). Control
(NRS treated) sections did not exhibit RP (Figure 2).
Hematoxylin and eosin (H 5 E) stained sections viewed at higher
magnification revealed that the EGC were low columnar cells with basally
located nuclei, and could be easily distinguished from uterine surface
epithelium (SE) (Figure 3). A section adjacent to that shown in
Figure 3 stained with R19 antiserum and the PAP technique demonstrated
40



41
even deposition of RP in the cytoplasm of EGC with no nuclear staining
(Figure 4). Not all EGC within a single gland exhibited RP (Figure 4).
Sections of uteri taken on day 45 of pregnancy showed that a higher
percentage of endometrial glands (EG) contained relaxin than did day 30
tissue (Figure 5). Control (NRS treated) sections did not exhibit RP
(Figure 6). No remarkable features were noted at a higher magnification
in H § E stained sections beyond those mentioned for the day 30 EGC
(Figure 7). A section adjacent to that shown in Figure 7 treated with
R19 antiserum and the PAP technique showed RP in most, but not all of
the EGC (Figure 8). The EGC that were stained had RP evenly distributed
throughout the cytoplasm (Figure 8).
All EG in sections of the day 60 uteri demonstrated dense accumu
lations of RP (Figure 9). Control (NRS treated) sections did not have
RP (Figure 10). At higher magnifications, H 5 E stained EGC appeared to
contain granular accumulations of acidophilic material in the luminal
portions of the cytoplasm (Figure 11). Individual granules could not
be clearly resolved in these cells although under higher magnification
structures resembling granules could be detected in the luminal portion
of the cells. Electron micrographs of EGC from day 60 pregnant animals
showed dense accumulations of granules located in the apical areas of
the cells (data not shown). A section adjacent to that shown in Figure 11
treated with R19 antiserum and the PAP technique, showed that every EGC
in every gland exhibited RP (Figure 12). While the pattern of staining
varied somewhat between animals, the most characteristic feature was a
dense accumulation of RP in the apical portion of the cells, with little
or no stain observed in the basal cytoplasmic regions.


42
As in day 60 tissue, sections of uteri taken from the late preg
nant group of animals revealed that all glands gave a positive reaction
for relaxin (Figure 13). Control (NRS treated) sections did not exhibit
RP localization (Figure 14). High magnification of H § E stained tissue
showed little differences between individual EGC (Figure 15). A section
adjacent to that shown in Figure 15 stained with R19 antiserum and the
PAP technique showed a striking pattern of RP deposition in some EGC
which differed markedly from the day 60 tissue (Figure 16). Some cells
demonstrated RP throughout the cytoplasm. Other cells had a dense
accumulation of labeling localized in a juxtanuclear region in the apical
portion of the EGC, with a conspicuous absence of stain from the other
areas of the cell cytoplasm (Figure 16).
Sections of uteri from the lactating group of guinea pigs demon
strated a low percentage of glands that gave a positive reaction for
relaxin (Figure 17). Control (NRS treated) sections did not exhibit
deposition of RP (Figure 18). High magnification of H § E treated
sections showed the EGC to be tall columnar type cells with a large
number of mitotic figures (Figure 19). Few endometrial glands showed
deposition of RP, and those that did had RP evenly distributed throughout
the cytoplasm of the cells (Figure 20).
The group of animals ovariectomized and treated with estradiol
and progesterone produced preparations that most resembled tissue taken
from animals on day 45 of pregnancy. When a section of uterus from this
group of animals was treated with R19 antiserum and the PAP technique,
the majority of the EG gave a positive reaction for relaxin (Figure 21).


43
Control (NRS treated) tissue did not show deposition of RP (Figure 22).
High magnification of H § E treated sections revealed the EGC to be
cuboidal cells with few distinguishing features (Figure 23). A section
adjacent to that shown in Figure 23, treated with R19 serum and the PAP
technique, showed that the deposition of RP in this uterus was less
dense than in glands of uteri taken from animals in the latter stages
of pregnancy (Figure 24). In all stages studied, RP was found only in
EGC, that is, no RP was noted in the uterine stroma, luminal epithelium
or uterine cervical glands. The following represent results of control
studies utilized in the PAP procedure: (1) immunoperoxidase labeling
was abolished when antiserum R19 was absorbed with purified porcine
(NIH-RXN-P1) relaxin prior to incubation with tissue sections, (2) suc
cessive dilutions (1/10-1/100,000) of antiserum R19 eventually abolished
tissue immunolabeling, and (3) staining was eliminated when NRS was sub
stituted for R19, GAR or PAP in the immunolabeling procedure.
Overall, these results support evidence obtained by others that
the uterus is a source of relaxin in the guinea pig.
Biologically Active Uterine Relaxin During
Pregnancy and Lactation
The preceding cytological evidence is extended by bioassays which
show that biologically active relaxin is only detected in the uterus, and
that the activity peaks in the later stages of pregnancy (Table 1; Fig
ures 25 and 26). Uteri from day 30 pregnant animals contained low bio
logical activity (0.15 +^0.09 units per gram wet weight (U/gww); 0.63 +_
0.40 U total). At day 45 of pregnancy, a significant increase in uterine
relaxin biological activity was noted (2.19 +_0.51 U/gww; 13.71 ^2.52
U total). Uterine relaxin levels further increased at day 60 of pregnancy


44
(2.62 j^0.20 U/gww; 46.80 +_ 2.85 U total). Uteri from late pregnant
animals showed the highest biological activity levels (3.76 + 0.76
U/gww; 57.75 _+ 7.35 U total). Relaxin levels dropped in lactating
animals (0.23 +_0.14 U/gww; 1.50 +_ 1.17 U total).
Statistical Analyses of Bioassay Data
Total activity.A regression analysis of the bioassay data
(Table 1; Figure 25) showed that total biological activity of the
uterine extracts was different over time of pregnancy with a high level
of significance (p<0.0001), and a quadratic component with a high cor-
2
relation coefficient (r = 0.907). A Duncan's multiple range test
for total biological activity values showed the following results (alpha
level = 0.05).
Ip 60_ 45 lac 30
Stages interconnected by bars are statistically indistinguishable from
each other according to the Duncan's multiple range test.
Specific activity.A regression analyses of bioassay data (Table 1;
Figure 26) showed that specific activity (U/gww) of the uterine extracts
was different over time of pregnancy with a high level of significance
(p<0.0001) and a quadratic component (r = 0.739). A Duncan's multiple
range test for specific biological activity values showed the following
results (alpha level = 0.05)
lp 6£ 45 lac 30
Crude extracts of liver and placenta of late pregnant guinea pigs
showed no relaxin activity in the mouse uterine motility bioassay. In
an attempt to find some relaxin activity, the placental extract was
further purified by passing it through a Bio-Gel P-30 column. The


45
fraction eluting in the 6,000 mw range contained very low activity
(0.27 U/mg) (data not shown). The liver extract was not purified or
tested further. It should be emphasized that neither of these two
tissues exhibited any demonstrable immunolabeling when tested with R19
antiserum and the PAP immunocytochemical technique. Therefore, the
relaxin activity in the placental extract may have been the result of
blood borne relaxin.
Radioimmunoassay of Uterine Relaxin During
Pregnancy and Lactation
RIA characterization.--The iodination curve (Figure 27), antiserum
titration curve (Figure 28), and dilution curves (Figure 29) obtained
with NIH-RXN-P1 relaxin support the validity of the RIA employed in the
present study. The dilution curves of the NIH-RXN-P1 relaxin and the two
guinea pig relaxin preparations were of similar slopes (Figure 29).
Therefore, it was deemed feasible to utilize NIH-RXN-P1 for the develop
ment of standard curves. The RIA did not detect relaxin in serum of
pregnant guinea pigs. The interassay coefficient of variation for the
RIA (4 assays) was 15.4% at 125 pg and the intra-assay coefficient of
variation (6 samples) at 125 pg was 4.2%. The RIA was capable of detect
ing levels of relaxin that ranged from 32 pg to 1,000 pg.
RIA of uterine extracts.
(1) Total amount of immunoreactive relaxin per uterus.--The RIA
of crude extracts showed that uteri from day 15 pregnant guinea pigs
(Table 2; Figure 30) contained the least amount of relaxin (0.40 +_0.14
ng). Data are expressed as nanogram (ng) porcine relaxin equivalents.
Amounts of relaxin increased in uterine extracts from day 30 pregnant
animals (11.44 +_ 4.51 ng). At day 45 of pregnancy, uterine relaxin


46
levels increased to 148.47 +_ 21.52 ng and were highest by day 60 of
pregnancy (172.67 +_ 19.17 ng). By late pregnancy, total uterine relaxin
levels had decreased to 101.90 +_ 25.78 ng and a wide variability existed
between animals. In the lactating animals the uterine relaxin levels had
decreased to 4.78 +^0.61 ng. A regression analysis of the RIA data
(Table 2; Figure 30) showed that total amounts of immunoreactive relaxin
were different (p<0.0001) over time of pregnancy, with a quadratic com-
ponent (r^ = 0.831). A Duncan's multiple range test for total relaxin
immunoactivity showed the following results (alpha level = 0.05).
60 45 lp 30 lac 15
(2) Concentration of immunoreactive relaxin.--Uterine extracts
from day 15 pregnant animals (Figure 31) contained low concentrations of
relaxin (0.25 0.05 ng/gww) which increased at day 30 of pregnancy
(2.06 ^0.44 ng/gww). The highest specific activity was found in the
uteri of day 45 pregnant animals (24.72 +_ 6.31 ng/gww). Uteri of day
60 and late pregnant animals had lower relaxin concentrations (9.83 +_
0.48 ng/gww and 6.45 +_ 1.74 ng/gww, respectively). While uteri from
lactating animals again contained very low concentrations of relaxin
(1.01 +_ 0.29 ng/gww).
A regression analysis of the RIA data (Table 2 and Figure 31)
showed that specific relaxin immunoactivity (ng/gww) of the crude uterine
extracts was different (p<0.0001) over time of pregnancy, with a quad-
ratic component (r = 0.754). A Duncan's multiple range test for specific
immunoactivity values showed the following results (alpha level = 0.05).
45_ 60 l£ 30 lac 15


47
Purification and Characterization of Guinea Pig
Uterine Relaxin
Purification: OPS Procedure
Uteri of the late pregnant guinea pigs were purified with the
extraction procedure of Walsh and Niall (1980). Five guinea pigs in the
late stages of pregnancy (days 65-67) were killed and 118.5 g wet weight
of uteri were utilized (Table 3). The ODS extracted material (188.8 mg)
contained low but detectable activity in the mouse uterine motility
assay (0.32 U/mg). The ODS material was fractionated in a Sephadex
column (Figure 32) and the 6,000 mw fraction had an activity of 1.50
U/mg. This active fraction from the Sephadex G-50 column (35.5 mg) was
further chromatographed in a CMC ion exchange column. The most active
fractions from the CMC column (tubes 55-90) contained 1.7 mg protein,
had an activity of 3.87 U/mg, and the peak protein fraction (tube 70)
eluted in the 7 m Mho conductance range (Figure 33). Similarly run
NIH-RXN-P1 porcine relaxin eluted in the 10 m Mho conductance range (data
not shown). RIA of every tenth tube of the CMC column run showed that
immunoreactive relaxin was present in the eluate. The regions containing
the highest immunoreactive relaxin (^300 ng/ml/OD; tubes 80-90) also
exhibited bioactivity (Figure 33).
Characterization
Double immunodiffusion studies.--Analyses of a Bio-Gel P-30
relaxin fraction (6 mg/ml) from the guinea pig uterus (day 60 of
pregnancy) tested against R19 antiserum to porcine relaxin and NIH-
RXN-P1 porcine relaxin showed a single precipitin line with no spurring
(Figure 34).


48
Two dimensional gel electrophoresis.--Carboxymethyl cellulose
purified guinea pig relaxin tested in a two dimensional gel electro
phoresis system showed that guinea pig relaxin migrated in the same mw
range as did NIH-RXN-P1 relaxin, which is known to have a mw of 6,000
(Figure 35). An equilibrium isoelectric point of a molecule cannot be
resolved with NEPHGE. Nevertheless, it was apparent that the guinea
pig relaxin molecule did not migrate as far in the first dimension
(pH 6.9) as did NIH-RXN-P1 porcine relaxin (pH 7.2), indicating that the
guinea pig relaxin molecule had a lower pH than the porcine molecule
(Figure 35). This observation supported data from the CMC column run,
which showed guinea pig relaxin eluting earlier (7 m Mho range) than
NIH-RXN-P1 porcine relaxin (10 m Mho range (Figure 33)). Figure 35 is
a representation of a slab gel, made up of a composite of two separately
run gels.
Mouse interpubic ligament assay.--A Sephadex G-50 6,000 mw fraction
from guinea pig uterine extracts was tested in the mouse interpubic
ligament assay (Figure 36). A positive response was obtained as noted
by linear response to log-dose of the Sephadex fraction. The data
indicate a valid assay according to the following criteria: (1) parallel
ism existed between the guinea pig relaxin fraction and the NIH-RXN-P1
porcine relaxin standard, and (2) a lambda value (standard error/slope)
of less than 0.4 was obtained. The specific activity of the Sephadex
fraction was calculated to be 0.8 U/mg protein. The mouse uterine motil
ity assay of the same fraction gave a biological activity of 1.5 U/mg.
The best fit curve for the NIH-RXN-P1 porcine relaxin was y = 1.2 (log x)
+ 0.83, with a standard error (SE) = 0.086 and a lambda value of 0.08.


49
The guinea pig CMC purified relaxin had a best fit curve of y = 0.87
(log x) + 0.64 with an SE = 0.097 and a lambda value of 0.12. Control
(estrogen treated) animals had a mean interpubic ligament length of
0.89 + 0.75 (X + SEM) .
Physiochemical characteristics.Treatment of the ODS crude
extract with dithiothrietol, trypsin and R19 reduced its ability to
inhibit uterine contractions (Table 4). These results indicate that
the guinea pig relaxin molecule depends on intact disulfide linkages
and structural integrity for its biological activity. Also, blocking
the immunologically active sites of the relaxin molecule with anti-
relaxin antibodies inhibits the hormone's biological activity. Heat
(70 C) did not adversely affect the guinea pig relaxin molecule.
Dithiothrietol and trypsin tested by themselves did not alter the ampli
tude or frequency of the contractions.


DISCUSSION
R19 Antiserum: Detection of Guinea Pig Relaxin
A major portion of this dissertation utilized techniques which
employed antirelaxin serum (R19). R19 was produced in rabbits against
highly purified porcine relaxin (Larkin et al., 1977). This antiserum
has been shown to crossreact with relaxin from different species; i.e.,
cow (Fields et al., 1980), human (Fields and Larkin, 1981), and rabbit
(Fields et al., 1981). R19 also has the ability to detect guinea pig
relaxin, as demonstrated by the following observations from the current
study: (1) Double immunodiffusion agar plate assays showed a reaction
of identity when partially purified uterine relaxin from day 60 preg
nant guinea pigs was tested against highly purified pig relaxin and
R19. (2) R19 was shown to be effective in inhibiting the action
of guinea pig relaxin using the in vitro mouse uterine motility anti
serum assay. (3) The immunoperoxidase labeling data from different
stages of gestation correlated well with bioassay and radioimmunoassay
data of crude uterine extracts from these stages; that is, the stages
demonstrating the greatest degree of labeling were also the stages
with the highest relaxin levels (days 45, 60 of pregnancy and late
pregnant animals). (4) All the controls in the immunolocalization
experiments were negative, including the observation that immunoperoxi
dase labeling was eliminated when the R19 antiserum was absorbed with
purified porcine relaxin standard (NIH-RXN-P1) prior to tissue incubation.
50


51
Detection of Guinea Pig Relaxin with the
PAP Technique
The R19 antiserum discussed in the previous section was employed
in immunocytochemical studies on sections of uteri from pregnant and
lactating guinea pigs, as well as in uteri of guinea pigs induced to
undergo pelvic relaxation with estrogen and progesterone treatments.
The immunocytochemical studies show an increase during pregnancy in
RP localized in the EG. This suggests that there is an increase in the
accumulation of relaxin in these cells, supporting the contention that
the EG are the sites for relaxin production. Of the intervals evalu
ated, day 30 was the first stage of pregnancy where a small amount of
immunoperoxidase labeling (relaxin) was noted in some EG. At this
stage, very few glands were labeled and in those glands that did label,
the reaction production was detected in only a few of the gland cells.
In uterine tissue from day 48 animals almost all of the EG appeared to
contain relaxin, but even at this stage, not all of the EGC of a given
gland were labeled. Thus it appears that there exists a gradual build up
of relaxin by the EGC. This is most clearly seen in day 60 and late
pregnant stages where dense deposits of RP were localized in the apical
cytoplasm of the EGC. In uteri from lactating animals (3 days post
partum), relaxin was absent from most of the EG, and a high incidence
of mitotic figures were noticed in the glandular epithelium.
Results from experiments involving nonpregnant, ovariectomized-
hormone treated animals provide strong evidence for a non-ovarian source
of relaxin. When ovariectomized animals were treated with estrogen,
no RP was seen localized over the EGC. When ovariectomized animals
were treated with estrogen and progesterone, RP was seen localized over


52
the EGC. These results agree with and extend the results of Zarrow
(1948), who noted that hysterectomized and ovariectomized guinea pigs
did not undergo pelvic relaxation or contain serum relaxin when treated
with a similar course of steroid injections. It is not known how
estrogen and progesterone induce the accumulation or synthesis of
relaxin in the EGC.
Detection of Guinea Pig Relaxin with
Radioimmunoassay
There have been no publications to date that report on the produc
tion of antisera to purified guinea pig relaxin. All studies dealing
with radioimmunological detection of relaxin in the guinea pig have
employed antiporcine relaxin sera (Sherwood et al., 1975; O'Byrne and
Steinetz, 1976; Bryant-Greenwood and Greenwood, 1979; Boyd et al., 1981;
Nagao and Bryant-Greenwood, 1981). It seems logical that differences
in the specificity and sensitivity of the various antisera could be
responsible for the wide variations in relaxin levels reported between
different laboratories which have studied relaxin in the guinea pig.
For example, the present RIA (employing R19 antiserum) detected porcine
relaxin as well as relaxin from guinea pig uterine extracts, but not
guinea pig serum relaxin. This antiserum was produced in rabbits to
highly purified porcine realxin. Sherwood et al. (1975) also reported
that an antiserum produced in rabbits to highly purified porcine relaxin
detected only porcine relaxin. These investigators were not able to
detect relaxin in sera of pregnant guinea pigs. This is in agreement
with the present study. However, Sherwood et al. (1975) did not report
attempts to detect relaxin in extracts of relaxin containing tissues.


53
O'Byrne and Steinetz (1976) on the other hand demonstrated that ani-
serum R6 crossreacted with relaxin in serum from a variety of pregnant
animals: humans, baboons, rhesus monkeys, dogs, cats, guinea pigs, rats
and mice. The R6 antiserum was produced in rabbits to a relaxin
fraction (Sephadex G-50: 1,000 U/mg) O'Byme and Steinetz (1976) did
not publish bioassay results. The RIA employed in the present studies,
as well as in the experiments of Sherwood et al. (1975) and O'Byme and
Steinetz (1976), used polytyrosyl relaxin. It seems that the difference
in immunological specificity encountered by the three laboratories
stemmed not from the polytyrosyl relaxin, but from the use of different
antisera or possibly from the technique employed. Nagao and Bryant-
Greenwood (1981) found that an antiserum produced to a relatively impure
porcine relaxin fraction (Sephadex G-50 column cut) appeared capable of
detecting a greater range of "relaxin" immunoactivity than an antiserum
produced against a purified relaxin fraction (porcine CM-a'). Perhaps
this extended range is due to the ability of the more "impure" antisera
to crossreact with metabolites of relaxin or with nonrelaxin components
of serum. This is not unreasonable to propose since it has been shown
by investigators working in Dr. Bryant-Greenwood's laboratory (Arakari
et al., 1980) that the antiserum to impure relaxin recognizes connective
tissue elements in immunofluorescence studies involving the pregnant
sow ovary. Nagao and Bryant-Greenwood (1981) utilized the antiserum
raised against CM-a' to assay uterine extracts taken from guinea pigs in
different stages of gestation. The highest levels encountered by these
investigators were of an order of magnitude 10 to 100 times greater than


54
the levels encountered in the present study. If the bioassay levels of
the present study are converted to ng porcine relaxin equivalents,*
they fall in the range of relaxin levels found by Nagao and Bryant-
Greenwood (1981) utilizing RIA. It appears that the antiserum used by
Bryant-Greenwood detected greater amounts of relaxin than the R19
antiserum used in the present study. It seems obvious from these com
parisons, that a direct correlation cannot be made between RIA data
obtained from different laboratories if different labeling techniques
and antisera were used. It is reasonable, however, to compare relative
data from the same system if the appropriate controls are carried out.
An important aspect of the present investigation was the correla
tion of RIA studies with bioassay and immunological localization studies.
Stages of pregnancy which demonstrated high levels of biologically and
immunologically active relaxin in uterine extracts, were also the stages
which displayed increased immunoperoxidase labeling in the uterine tissue
sections. A comparison of relaxin levels detected with bioassay and
radioimmunoassay at the different stages of pregnancy raises an interest
ing point. Bioassay of crude uterine extracts detected the highest con
centration of relaxin in late pregnant animals. On the other hand,
concentrations of immunoreactive relaxin were highest in the day 45
pregnant animals, but decreased by day 60 of pregnancy and continued to
decline during late pregnancy. It seems that the bioassay detected levels
*This comparison is made by using the following conversion.
ng equivalent relaxin =
biological activity (Units) x 1,000 ng of porcine relaxin
3 Units


55
of relaxir! that were not detected by the RIA. One may speculate on
the possibility that accumulations of relaxin (prorelaxin) that are
biologically active, but not immunoreactive, are present in those latter
stages of pregnancy. The present study did not report on serum relaxin
levels. Serum studies undertaken in other laboratories, however, showed
that biologically active (Zarrow, 1948), and immunoreactive (O'Byrne
and Steinetz, 1976; Boyd et al., 1981) serum relaxin levels increased
as pregnancy proceeded in guinea pigs, dropping just prior to parturition.
This trend seen in serum relaxin levels approximates activity detected
in the tissue extracts and tissue sections with techniques employed in
the present investigation, supporting the hypothesis that uterine relaxin
may play an influential role in pregnancy and parturition in the guinea
pig-
Endometrial Glands and Their Role in Relaxin Production
EG classically have been assumed to play a role during pregnancy
where they serve a nutritive and/or supportive role to the preimplanta
tion embryo (Finn, 1977). In the mouse, the end result of glandular
differentiation is the secretion of periodic acid Schiff positive
material from the EG into the uterine lumen, and it has been shown that
estrogen and progesterone together can induce uterine glandular secre
tions (Finn, 1971; Finn and Martin, 1971). In the pig, Bazer (1975)
has shown that a uterine specific purple protein is secreted by the
glandular endometrial epithelium throughout pregnancy. Most animals
studied have been shown to produce uterine specific proteins, especially
during the early stages of pregnancy (Aitken, 1979). Animals with an


56
epitheliochorial or syndesmochorial type of placentation may provide a
source of nutrition to the developing embryo through the production of
uterine specific proteins which diffuse through the placenta. Animals
with a hemochorial type of placentation, like the human and the guinea
pig, derive most of their embryonic nutrition from the maternal blood
stream. It has, nevertheless, been shown that amniotic fluid from
humans in the second trimester of pregnancy contains uterine specific
proteins (Sutcliffe et al., 1978). The EG and/or the SE are most
likely active during pregnancy and produce proteins which possibly come
into contact with the embryo and fetus.
Direct and indirect evidence from other laboratories has been
accumulated which implicates the uterus as an important source of
relaxin in the guinea pig: (1) Frieden and Adams (1977) have shown
that softening of the pelvic ligaments can be detected by palpation as
early as mid-pregnancy in the guinea pig, which is the approximate time
(day 30) when accumulation of relaxin was initially detected in the EGC
with immunoperoxidase labeling, RIA, and bioassay. (2) Porter (1972)
has shown that a uterine quieting substance, most likely relaxin, is
present in the blood of pregnant guinea pigs. (3) Zarrow (1948) and
Nagao and Bryant-Greenwood (1981) have detected the presence of relaxin
in sera of ovariectomized estrogen-progesterone treated guinea pigs.
(4) Catchpole (1969) has shown that the guinea pig is able to proceed
through a normal pregnancy and parturition after ovariectomy as early
as day 38 of pregnancy, thus strongly indicating a nonovarian source of
relaxin for this species.


57
The uterus of the guinea pig has been known to be a source of
relaxin for some time (Zarrow, 1948). Day 30 of pregnancy seems to be
the approximate time when measurable levels of relaxin first appear
in the uterus and blood of guinea pigs (O'Byme and Steinetz, 1976;
Boyd et al., 1981; Nagao and Bryant-Greenwood, 1981). This is also
approximately 10-15 days after the first detectable rise in serum
estrogen and progesterone (Challis et al., 1971). As shown by Zarrow
(1948) 10-15 days is the time required for injections of estrogen and
progesterone to evoke the synthesis of relaxin by the uterus of non
pregnant ovariectomized guinea pigs.
This latency period appears to be shorter than 15 days, since by
this time, treatment with estrogen and progesterone had evoked a change
in the interpubic ligament length of the guinea pigs. Nagao and Bryant-
Greenwood (1981) detected a rise in uterine relaxin 11 days after ovari
ectomized guinea pigs were primed with estrogen and progesterone. However,
no one has reported a systematic study to determine when relaxin is first
produced by the uterus following estrogen and progesterone stimulation.
Autoradiographic evidence indicates that estrogen primed, ovari
ectomized guinea pigs contain progesterone receptors in the endometrial
7
glands, as evidenced by accumulation of H progesterone in the cytoplasm
and nuclei of the EGC (Stumpf, 1968; Sar and Stumpf, 1974; Warembourg,
1974). This is consistent with the contention that the EG are a target
tissue for the steroid hormones,and all evidence indicates that estrogen
and progesterone are necessary for relaxin synthesis by the EG.


58
Possible Actions of Uterine Relaxin in the Guinea Pig
As evidence accumulates that the corpus luteum of the pregnant
female is not the only source of relaxin, it becomes apparent that
relaxin may be produced by other tissues and act locally as well as
systemically. Perhaps this is best illustrated by the guinea pig,
which may have an ovarian source of relaxin (Nagao and Bryant-Greenwood,
1981), but also contains a uterine source as well. Relaxin produced by
the uterus of this animal may have systemic as well as local effects.
Relaxin has been shown to be present in the systemic blood of pregnant
guinea pigs with bioassay (Zarrow, 1948) and RIA (O'Byrne and Steinetz,
1976; Boyd et al., 1981). Also Zarrow (1948) has shown that relaxin is
present in systemic blood of ovariectomized-estrogen and progesterone
treated guinea pigs. Effects such as changes which occur in the inter-
pubic ligament prior to parturition, in mammary gland development, as
well as in the maintenance of uterine quiescence could be proposed as
possible systemic effects of relaxin.
An increasing body of evidence on the other hand, indicates that
relaxin may act as a local hormone in the guinea pig. Immunolocalization
studies from this laboratory illustrate a distinct staining pattern in
some of the EGC in uterine tissue taken from guinea pigs in the latter
stages of pregnancy. In day 60 and late pregnant animals, RP was
localized apically in some EGC, i.e. immunolabeling was not seen in the
basal areas of the cytoplasm in these cells. Also electron micrographs
of EG taken from the same stages showed a large number of apical gran
ules. These observations may suggest that relaxin is being released
into the uterine lumen of the endometrial glands. Relaxin synthesized


59
and released from the EG into the uterine lumen could: (1) Have free
access to fetal-maternal tissues during pregnancy. Harkness and Hark-
ness (1956; 1957) have shown that the tensile strength of rat fetal
membranes is greatly reduced during the birth process. Relaxin may
be responsible for this phenomenom. (2) Maintain uterine quiescence
during pregnancy. Although there is no direct evidence for this assump
tion, the proximity of the uterine endometrium to the myometrium could
possibly allow for local diffusion of relaxin to occur. Porter (1972)
has shown that relaxin is most likely the hormone responsible for keeping
the uterus quiescent in the guinea pig during pregnancy. (3) Effect
cervical softening at term. MacLennan et al. (1980), have found that
topical application of porcine relaxin to the posterior fornix of the
vagina resulted in softening of the cervix in a significant number of
women.
One of the most interesting problems remaining to be investigated
is how relaxin enters the systemic circulation from the EG and what
course it follows to reach the target tissues. Studies to identify
target organs for relaxin become very important when one considers the
possibilities of local effects of relaxin.
Relaxin may be released into the uterine lumen, and make its way
back through the endometrial stroma into the bloodstream. While the
findings in the present study did not indicate direct secretion of
relaxin into the uterine stromal compartment, this cannot be ruled out.
Work in other systems by Bazer and Thatcher (1977) suggested an
endocrine-exocrine mechanism for the release of prostaglandin F 2-alpha


60
(PGF 2-alpha) by the uterine endometrial glands of the pig. It was
postulated by these investigators that in the nonpregnant state, PGF
2-alpha is produced in an endocrine fashion by the EG under the control
of progesterone. On the other hand, in a pregnant animal, the estrogen
released by the conceptus changes the pattern of release to an exocrine
fashion, and the hormone is released into the uterine lumen.
Purification and Characterization of Guinea Pig Relaxin
The guinea pig relaxin molecule demonstrated characteristics similar
to relaxins isolated from other species (Sherwood and O'Byrne, 1974;
Fields and Larkin, 1979; Sherwood, 1979; Fields and Larkin, 1981; Fields
et al., 1980; 1981; Reinig et al., 1981) according to the following
criteria: (1) mw of approximately 6,000, (2) basic isoelectric point,
(3) ability to inhibit uterine contractions (mouse uterine motility
bioassay), (4) ability to induce interpubic ligament formation in estrogen
primed mice (mouse interpubic ligament assay), (5) susceptibility to
enzyme digestion with trypsin and to the strong reducing agent dithio-
thrietol, indicating its proteinaceous nature, and its reliance on
disulfide bonds for its biological activity, and (6) resistance to
moderate heat. Guinea pig relaxin was antigenically similar to porcine
relaxin in that: (1) a reaction of identity was obtained when an extract
of guinea pig uterus from a day 60 pregnant animal was reacted against
antiserum to purified porcine relaxin and porcine relaxin (NIH-RXN-P1),
(2) an antiserum produced against porcine relaxin inhibited the ability
of guinea pig extracts to retard spontaneous uterine contractions,
(3) parallel dilution curves were obtained between relaxin containing
uterine extracts of late pregnant guinea pigs and porcine relaxin


61
(NIH-RXN-P1) in a homologous porcine relaxin RIA, and (4) the biologic
ally active guinea pig relaxin CMC peak (Figure 33) was also immuno
logical ly active in the homologous porcine RIA.
Guinea pig relaxin displayed a very low specific biological
activity when compared to porcine relaxin. The present studies confirmed
preliminary work done by Pardo et al., 1980, utilizing a different extrac
tion procedure, who showed that crude uterine extracts of day 60 preg
nant guinea pigs contained low biological activity (0.14 U/mg) when
tested with the mouse uterine motility bioassay. Higher activity levels
were reported for relaxin separated on a column of Bio-Gel (36 U/mg)
(Pardo et al., 1980). This is in contrast to levels reported in the
present study. The significance of this discrepancy is unclear and may
relate to different separation techniques, as well as variability of the
bioassays. The low biological activity of guinea pig relaxin is not
unreasonable to expect since relaxins isolated from all species except
the pig had low biological activity. This discrepancy in activity levels
between the pig and other species perhaps reflects differences in
specificity in the different bioassays. A good example of this phenome-
nom was found in relaxin isolated from the shark (Reinig et al., 1981).
Shark relaxin has been found to be ineffective in the mouse bioassays
(uterine motility and interpubic ligament formation). However, shark
relaxin is active when guinea pigs were employed for the uterine motility
and interpubic ligament assays. It is clear, however, more than one
bioassay should be employed when considering whether a preparation
contains relaxin. Guinea pig relaxin was effective in both the mouse


62
uterine motility bioassay and in the mouse interpubic ligament bioassay,
and thus at least in these aspects it remains similar to relaxin from
other mammalian species.
Future studies can be proposed to answer many of the questions as
yet unexplained. First, what, if any, is the contribution of the ovary
to the synthesis of relaxin? Second, how do the steroid hormones
initiate the synthesis of relaxin by the EGC, and why a synchronization
of secretion is not apparent under a uniform hormonal milieu? Third,
what is the possible mechanism of relaxin release by the EGC? Fourth,
how does uterine relaxin reach its target organs with respect to the
events of pregnancy and parturition; e.g., maintaining the uterus
quiescent during pregnancy and preparing the cervix and pelvic ligaments
for parturition?
It is hoped that the research presented in this dissertation will
amplify the knowledge of relaxin physiology in the guinea pig, and
advance an understanding of the parturition process in this animal as
well as in other species.


BIBLIOGRAPHY
Abramowitz, A. A., W. L. Money, M. X. Zarrow, R. V. N. Talmage, L. H.
Kleinholz, and F. L. Hisaw. (1944) Preparation, biological assay
and properties of relaxin. Endocrinology, 34: 103-114.
Abramson, D., E. Hurwitt, and G. Lesnik. (1937) Relaxin in human
serum as a test of pregnancy. Surg. Gynecol. Obstet., 65: 335-339.
Aitken, R. J. (1979) Uterine proteins. In: Oxford Reviews of
Reproductive Biology. (A. A. Finn, ed.) Clarendon Press, Oxford,
England.
Anderson, M. L., and J. A. Long. (1978) Localization of relaxin in the
pregnant rat. Bioassay of tissue extracts and cell fractionation
studies. Biol. Reprod., 18: 110-117.
Anderson, M. L., J. A. Long, and T. Hayashida. (1975) Immunofluorescence
studies on the localization of relaxin in the corpus luteum of
the pregnant rat. Biol. Reprod., 13: 499-504.
Arakari, R. F., R. G. Kleinfeld, and G. D. Bryant-Greenwood. (1980)
Immunofluorescence studies using antisera to crude and to purified
porcine relaxin. Biol. Reprod., 23: 153-159.
Barr, A. J., and J. H. A. Goodnight. (1976) Users Guide to the Statisti
cal Analysis System. North Carolina State University, Raleigh,
N. C.
Bazer, F. W. (1975) Uterine protein secretions: Relationship to
development of the conceptus. J. Anim. Sci., 41: 1376-1382.
Bazer, F. W., and W. W. Thatcher. (1977) Theory of maternal recognition
of pregnancy in swine based on estrogen controlled endocrine
versus exocrine secretion of Prostaglandin F2-alpha by the uterine
endometrium. Prostaglandins, 14: 397-401.
Belt, W. D., L. L. Anderson, L. F. Cavazos, and R. M. Melampy. (1971)
Cytoplasmic granules and relaxin levels in porcine corpora ltea.
Endocrinology, 89: 1-10.
Bigazzi, M., E. Nardi, P. Bruni, and F. Petrucci. (1980) Relaxin in
human decidua. J. Clin. Endocrinol. Metab., 51: 939-941.
Blundell, T. (1979) Conformation and molecular biology of polypeptide
hormones. I. Insulin, insulin-like growth factor and relaxin.
TIBS. March, 1979: 51-54.
63


64
Bolton, A. E., and W. M. Hunter. (1973) The labelling of proteins to
high specific radioactivities by conjugation to a 12Si_contaj.nj.ng
acylating agent. Biochem. J., 133: 529-539.
Boyd, S., J. Z. Kendall, N. Ment, and G. D. Bryant-Greenwood. (1981)
Relaxin immunoactivity in plasma during the reproductive cycle of
the female guinea pig. Biol. Reprod., 24: 405-414.
Brouha, L. (1933) Recherches sur la mobilisation de la symphyse
pubienne chez le cobaye impubere. Compte Rendu Soc. Biol., 113:
406-408.
Bryant, G. D. (1972) The detection of relaxin in porcine, ovine and
human plasma by radioimmunoassay. Endocrinology, 91: 1113-1117.
Bryant, G. D., and W. A. Chamley. (1976) Changes in relaxin and pro
lactin immunoactivities in ovine plasma following suckling. J.
Reprod. Frtil., 46: 457-459.
Bryant, G. D., M. E. A. Panter, and T. Stelmasiak. (1975) Immunore-
active relaxin in human serum during the menstrual cycle. J.
Clin. Endocrinol. Metab., 41: 1065-1069.
Bryant, G. D., J. F. Sassin, E. D. Weitzman, S. Kapen, and A. Frantz.
(1976) Relaxin immunoactivity in human plasma during a 24-hour
period. J. Reprod. Frtil., 48: 389-392.
Bryant, G. D., and T. Stelmasiak. (1974) The specificity of radio
immunoassay for relaxin. Endo. Res. Commun., 1: 415-433.
Bryant-Greenwood, G. D., and F. C. Greenwood. (1979) Specificity of
radioimmunoassays for relaxin. J. Endocrinol., 81: 239-247.
Castro-Hernandez, A. (1976) Isolation and purification of bovine
luteal polypeptides with relaxin hormone activity (Doctoral
Dissertation, University of Florida).
Catchpole, H. R. (1969) Hormonal mechanisms during pregnancy and
parturition. In: Reproduction in Domestic Animals. (H. H. Cole
and P. T. Cupps, eds.) Academic Press, New York.
Challis, J. R. G., R. B. Heap, and D. V. Illingsworth. (1971) Concentra
tions of oestrogen and progesterone in the plasma of non-pregnant,
pregnant and lactating guinea pigs. J. Endocrinol., 51: 333-345.
Clausen, J. (1969) Immunochemical Techniques for the Identification and
Estimation of Macromolecules. (T. S. Work, E. Work, eds.)
American Elsevier, New York, p. 521.


65
Dallenbach, F. D., and G. Dallenbach-Hellweg. (1964) Immunohistologische
untersuchungen zur lokalisierung des relaxins in menschlicher
plazenta and decidua. Virch. Arch. Path. Anat., 337: 301-316.
Dallenbach-Hellweg, G., J. V. Battista, and F. D. Dallenbach. (1965)
Immunohistological and histochemical localization of relaxin in
the metrial gland of the pregnant rat. Am. J. Anat., 117:
435-450.
Fevold, H., F. L. Hisaw, and R. K. Meyer. (1930) The relaxative hor
mone of the corpus luteum, its purification and concentration.
J. Am. Chem. Soc., 52: 3340-3348.
Fields, M. J., P. A. Fields, A. Castro-Hernandez, and L. H. Larkin.
(1980) Evidence for relaxin in corpora ltea of late pregnant
cows. Endocrinology, 107: 869-875.
Fields, P. A., and L. H. Larkin. (1979) Isolation of rat relaxin.
Anat. Rec., 193: 537.
Fields, P. A., and L. H. Larkin. (1981) Purification and immunohisto-
chemical localization of relaxin in the human term placenta. J.
Clin. Endocrinol. Metab., 52: 79-85.
Fields, P. A., L. H. Larkin, and R. J. Pardo. (1981) Purification of
relaxin from the placenta of the rabbit. Ann. N. Y. Acad. Sci.
380: 76-86.
Finn, C. A. (1977) The implantation reaction. In: Biology of the
Uterus (R. M. Wynn, ed.) Plenum Press, New York.
Finn, C. A., and L. Martin. (1971) Endocrine control of the prolifer
ation and secretion of uterine glands in the mouse. Acta Endocrinol.
Suppl., 155: 139.
Frieden, E. H., and W. C. Adams. (1977) The response to endogenous
relaxin of guinea pigs refractory to porcine relaxin. Proc. Soc.
Exp. Biol. Med., 155: 558-561.
Frieden, E. H., and F. L. Hisaw. (1953) The biochemistry of relaxin.
Rec. Prog. Horm. Res., 8: 333-372.
Frieden, E. H., and L. Yeh. (1977) Evidence for a "pro-relaxin" in
porcine relaxin concentrates. Proc. Soc. Exp. Biol. Med., 154:
407-411.
Fugo, N. W. (1943) Relaxation of the pelvic ligaments of castrate
hysterectomized guinea pigs induced by progesterone. Proc. Soc.
Exp. Biol. Med., 54: 200-201.


66
Griss, G., J. Keck, R. Engelhom, and H. Tuppy. (1967) The isolation
and purification of an ovarian polypeptide of uterine-relaxing
activity. Biochim. Biophys. Acta, 140: 45-54.
Hall, K. (1960) Relaxin. J. Reprod. Frtil., 1: 368-384.
Harkness, M. L. R., and R. D. Harkness. (1956) Changes in the foetal
membrane during pregnancy in the rat. J. Physiol., 129: 788.
Harkness, M. L. R., and R. D. Harkness. (1957) Changes in the physical
properties of the uterine cervix of the rat during pregnancy.
J. Physiol., 148: 524-547.
Hisaw, F. L. (1926) Experimental relaxation of the pubic ligament of
the guinea pig. Proc. Soc. Exp. Biol. Med., 23: 661-663.
Hisaw, F. L. (1927) Experimental relaxation of the symphysis pubis of
the guinea pig. Anat. Rec., 37: 126.
Hisaw, F. L., and M. X. Zarrow. (1950) The physiology of relaxin.
Vit. and Horm., 8: 151-178.
Hisaw, F. L., M. X. Zarrow, W. L. Money, R. V. N. Talmage, and A. A.
Abramowitz. (1944) Importance of the female reproductive tract
in the formation of relaxin. Endocrinology, 34: 122-134.
Horst, M. N., S. M. M. Basha, G. A. Baumbach, E. H. Mansfield, and R. M.
Roberts. (1980) Alkaline urea solubilization, two-dimensional
electrophoresis and lectin staining of mammalian cell plasma
membrane and plant seed proteins. Anal. Biochem., 102: 399-408.
Hunter, W. M., and F. C. Greenwood. (1962) Preparation of iodine-131
labelled human growth hormone of high specific activity. Nature,
194: 495-496.
Isaacs, N., R. James, H. Niall, G. Bryant-Greenwood, G. Dodson, A. Evans,
and A. C. T. North. (1978) Relaxin and its structural relation
ship to insulin. Nature, 271: 278-281.
James, R., H. Niall, S. Kwok, and G. Bryant-Greenwood. (1977) Primary
structure of porcine relaxin: Homology with insulin and related
growth factors. Nature, 267: 544-546.
John, M. J., B. W. Borjesson, J. R. Walsh, and H. Niall. (1981)
Limited sequence homology between porcine and rat relaxins: Impli
cations for physiological studies. Endocrinology, 108: 726-729.
Kendall, J. Z., C. G. Plopper, and G. D. Bryant-Greenwood. (1978)
Ultrastructural immunoperoxidase demonstration of relaxin in
corpora ltea from a pregnant sow. Biol. Reprod., 18: 94-98.


67
Krantz, J. C., H. H. Bryant, and C. J. Carr. (1950) The action of
aqueous corpus luteum extract upon uterine activity. Surg.
Gynecol. Obstet., 90: 372-375.
Kroc, R. L., B. G. Steinetz, and V. L. Beach. (1959) The effects of
estrogens, progestagens, and relaxin in pregnant and non-pregnant
laboratory rodents. Ann. N. Y. Acad. Sci., 75: 942-980.
Larkin, L. H. (1974) Bioassay of rat metrial gland extracts for
relaxin using the mouse interpubic ligament technique. Endocrin
ology, 94: 567-570.
Larkin, L. H., P. A. Fields, and R. M. Oliver. (1977) Production of
antisera against electrophoretically separated relaxin and immuno-
fluorescent localization of relaxin in the porcine corpus luteum.
Endocrinology, 101: 679-683.
Larkin, L. H., P. A. Fields, and R. J. Pardo. (1981) Mouse uterus bio
assay for relaxin. In: Relaxin. (G. D. Bryant-Greenwood, H. D.
Niall and F. C. Greenwood, eds.) Elsevier, North Holland.
Larkin, L. H., C. A. Suarez-Quian, and P. A. Fields. (1979) In vitro
analyses of antisera to relaxin. Acta Endocrinol., 92: 568-576.
Loumaye, E., B. Teuwissen, and K. Thomas. (1978) Characterization of
relaxin radioimmunoassay using Bolton-Hunter reagent. Gynecol.
Obstet. Invest., 9: 262-267.
Lowry, 0. H., N. J. Roseborough, A. L. Farr, and R. J. Randall. (1951)
Protein measurement with the folin phenol reagent. J. Biol. Chem.,
193: 265-275.
MacLennan, A. H., R. C. Green, G. D. Bryant-Greenwood, F. C. Greenwood,
and R. F. Seamark. (1980) Ripening of the human cervix and
induction of labor with purified porcine relaxin. Lancet, Feb. 2,
1980, pp. 220-223.
Marcus, G. J. (1974) Mitosis in the rat uterus during the estrus cycle,
early pregnancy, and early pseudopregnancy. Biol. Reprod. 10:
447-452.
Markwell, M. A. K., and C. F. Fox. (1978) Surface-specific iodination
of membrane proteins of viruses and eukaryotic cells using 1, 3,
4, 6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. Biochem.,
17: 4807-4817.
Mathieu, P. H., J. Rathier, and K. Thomas. (1981) Localization of
relaxin in human gestational corpus luteum. Cell Tiss. Res., 219:
213-216.


68
Nagao, R., and G. D. Bryant-Greenwood. (1981) Evidence for a uterine
relaxin in the guinea pig. In: Relaxin. (G. D. Bryant-Greenwood,
H. D. Niall and F. C. Greenwood, eds.) Elsevier, North
Holland.
Noall, M. W., and E. H. Frieden. (1956) Variations of sensitivity of
ovariectomized guinea pigs to relaxin. Endocrinology, 5: 659-664.
O'Byrne, E. M., F. F. Flitcraft, W. K. Sawyer, J. Hochman, G. Weiss, and
B. G. Steinetz. (1978) Relaxin bioactivity and immunoactivity
in human corpora ltea. Endocrinology, 102: 1641-1644.
O'Byrne, E. M., W. K. Sawyer, M. C. Butler, and B. G. Steinetz. (1976)
Serum immunoreactive relaxin and softening of the uterine cervix
in pregnant hamsters. Endocrinology, 99: 1333-1335.
O'Byrne, E. M., and B. G. Steinetz. (1976) Radioimmunoassay (RIA) of
relaxin in sera of various species using an antiserum to porcine
relaxin. Proc. Soc. Exp. Biol. Med., 152: 272-276.
Pardo, R., L. H. Larkin, and P. A. Fields. (1980) Immunocytochemical
localization of relaxin in endometrial glands of the pregnant guinea
pig. Endocrinology, 107: 2110-2112.
Porter, D. G. (1972) Myometrium of the pregnant guinea pig: The
probable importance of relaxin. Biol. Reprod., 7: 458-464.
Porter, D. G. (1979) Relaxin: Old Hormone, new prospect. In: Oxford
Reviews of Reproductive Biology, Vol. 1 (C. A. Finn, ed.) Clarendon
Press, Oxford, England.
Reinig, J. W., D. N. Lambert, C. Schwabe, L. K. Gowan, B. G. Steinetz,
and E. M. O'Byrne. (1981) Isolation and characterization of
relaxin from the sand tiger shark (odontaspis taurus). Endocrin
ology, 109: 537-543.
Sanders, M. M., V. E. Groppi, Jr., and E. T. Browning. (1980) Resolu
tion of basic cellular proteins including histone variants by
two-dimensional gel electrophoresis: Evaluation of lysine to
arginine ratios and phosporylation. Anal. Biochem., 103: 157-
165.
Sar, M., and W. E. Stumpf. (1974) Cellular and subcellular localization
of ^H-progesterone or its metabolites in the oviduct, uterus,
vagina and liver of the guinea pig. Endocrinology, 94: 1116-1125.
Schwabe, C., and S. A. Braddon. (1976) Evidence for one essential
tryptophan residue at the active site of relaxin. Biochem.
Biophys. Res. Commun., 68: 1126-1132.


69
Schwabe, C., J. K. McDonald, and B. G. Steinetz. (1976) Primary
structure of the A-chain of porcine relaxin. Biochem. Biophys.
Res. Commun., 70: 397-405.
Schwabe, C., J. K. McDonald, and B. G. Steinetz. (1977) Primary
structure of the B-chain of porcine relaxin. Biochem. Biophys.
Res. Commun., 75: 503-510.
Schwabe, C., B. G. Steinetz, G. Weiss, A. Segaloff, J. K. McDonald,
E. O'Byrne, J. Hochman, B. Carriere, and L. Goldsmith. (1978)
Relaxin. Rec. Prog. Horm. Res., 34: 123-211.
Sherwood, 0. D. (1979) Purification and characterization of rat
relaxin. Endocrinology, 104: 886-892.
Sherwood, 0. D., and V. E. Crnekovic. (1979) Development of a homo
logous radioimmunoassay for rat relaxin. Endocrinology, 104:
893-897.
Sherwood, 0. D., P. A. Martin, C. C. Chang, and P. J. Dziuk. (1977a)
Plasma relaxin levels in pigs with corpora ltea induced during
late pregnancy. Biol. Reprod., 17: 97-100.
Sherwood, 0. D., P. A. Martin, C. C. Chang, and P. J. Dziuk. (1977b)
Plasma relaxin levels during late pregnancy and at parturition in
pigs with altered utero-ovarian connections. Biol. Reprod.,
17: 101-103.
Sherwood, 0. D., and E. M. O'Byrne. (1974) Purification and character
ization of porcine relaxin. Arch. Biochm. Biophys., 160: 185-196.
Sherwood, 0. D., K. R. Rosentreter, and M. L. Birkhimer. (1975) Develop
ment of a radioimmunoassay for porcine relaxin using bI labelled
polytyrosyl-relaxin. Endocrinology, 96: 1106-1112.
Steinetz, B. G., V. L. Beach, R. L. Kroc, N. R. Stasilli, R. E. Nussbaum,
P. J. Nemith, and R. K. Dun. (1960) Bioassay of relaxin using
a reference standard: A simple and reliable method utilizing
direct measurement of interpubic ligament formation in mice.
Endocrinology, 67: 102-115.
Sternberger, L. A. (1979) Immunocytochemistry. John Wiley and Sons,
New York.
3
Stumpf, W. E. (1968) Subcellular distribution of H-estradiol m rat
uterus by quantitative autoradiography--a comparison between ^H-
estradiol and %-norethynodrel. Endocrinology, 83: 777-782.


70
Sutcliffe, R. G., D. J. H. Brock, L. B. V. Nicholson, and E. Dunn.
(1978) Fetal- and uterine-specific antigens in human amniotic
fluid. J. Reprod. Frtil., 54: 85-90.
Szalchter, N., E. O'Byrne, L. Goldsmith, B. G. Steinetz, and G. Weiss.
(1980) Myometrial inhibiting activity of relaxin-containing
extracts of human corpora ltea. Am. J. Obstet. Cynecol., 136:
584-586.
Walsh, J. R., and H. D. Niall. (1980) Use of an octadecylsilica
purification method minimizes proteolysis during isolation of
porcine and rat relaxins. Endocrinology, 107: 1258-1260.
Warembourg, M. (1974) Radiographic study of the guinea pig uterus
after injection and incubation with "^H-progesterone. Endocrinology,
94: 665-670.
Weiss, G., E. M. O'Byrne, J. A. Hochman, L. T. Goldsmith, I. Rifkin, and
B. G. Steinetz. (1977) Secretion of progesterone and relaxin by
the human corpus luteum at midpregnancy and at term. Obstet.
Gynecol., 50: 679-681.
Weiss, G., E. M. O'Byrne, J. A. Hochman, B. G. Steinetz, L. Goldsmith,
and J. G. Flitcraft. (1978) Distribution of relaxin in women
during pregnancy. Obstet. Gynecol., 52: 568-570.
Weiss, G., E. M. O'Byme, and B. G. Steinetz. (1976) Relaxin: A
product of the corpus luteum of pregnancy. Science, 194: 948-949.
Yamamoto, S., S. C. M. Kwok, F. C. Greenwood, and G. D. Bryant-Greenwood.
(1981) Relaxin purification from human placental basal plates.
J. Clin. Endocrinol. Metab., 52: 601-607.
Zarrow, M. X. (1947) Relaxin content of blood, urine and other tissues
of pregnant and postpartum guinea pigs. Proc. Soc. Exp. Biol.
Med., 66: 488-491.
Zarrow, M. X. (1948) The role of the steroid hormones in the relaxation
of the symphysis pubis of the guinea pig. Endocrinology, 42: 129-
140.
Zarrow, M. X., E. G. Holmstrom, and H. A. Salhanick. (1955) The con
centration of relaxin in the blood serum and other tissues of
women during pregnancy. J. Clin. Endocrinol. Metab., 15: 22-27.
Zarrow, M. X., and J. A. McClintock. (1966) Localization of I
labelled antibody to relaxin. J. Endocrinol., 36: 377-387.


71
Zarrow, M. X., and W. B. O'Connor. (1966) Localization of relaxin in
the corpus luteum of the rabbit. Proc. Soc. Exp. Biol. Med.,
121: 612-614.
Zarrow, M. X., and B. Rosenberg. (1953) Sources of relaxin in the
rabbit. Endocrinology, 53: 593-598.


APPENDIX 1
TABLES


Table 1. Bioactlve relaxin content of uteri from guinea pigs in different stages of pregnancy and lactation
STAGE OF ANIMAL
GESTATION NO.
(g) (mg)
WET WT. DRY WT.
TOTAL UNITS OF
RELAXIN PER UTERUS
UNITS OF RELAXIN TER
GRAM WET WEIGHT
DAY 30
4
6.1
61.0
(2)
1.65
0.25
5
2.6
23.0
(2)
0.87
(0.63 + 0.40)**
0.33
(0.15 + 0.09)
6
7.2
106.0
(1)
0
0
7
4.0
38.0
(1)
0
0
DAY 45
8
8.0
443.5
(2)
20.84
2.61
9
3.2
195.0
(2)
9.17
(13.71 + 2.52)
2.86
(2.19 + 0.51)
10
5.0
280.0
(1)
13.16
2.63
11
17.5
248.0
(2)
11.66
0.66
DAY 60
12
17.0
273.4
(3)
42.83
2.19
13
18.0
346.5
(3)
43.43
(46.80 + 2.85)
2.41
(2.62 + 0.20)
14
20.0
391.0
(2)
55.13
2.76
15
14.7
243.7
(2)
45.82
3.12
Late
16
13.0
321.0
(2)
50.08
3.85
Pregnant
17
16.0
268.2
(2)
41.84
2.62
18
26.0
495.6
(2)
77.31
(57.75 + 7.35)
2.97
(3.76 + 0.76)
19
11.0
234.9
(2)
73.52
6.68
20
17.2
194.3
(4)
46.00
2.68
Lactating
21
6.3
53.9
(1)
0
0
22
4.0
50.0
(1)
0
(1.50 + 1.17)
0
(0.23 + 0.14)
23
3.0
35.6
(1)
1.07
0.36
24
9.2
105.0
(1)
4.94
0.54
* Twenty milligrams
(20 mg) of
Acid Acetone
ext racted
powder was homogenized in
1 ml of distilled water.
** Units of
activity
are expressed as porcine NIU-RXN-
PI standard relaxin equivalents (X + SEM) determined
by the mouse uterine motility bioassay.
( )
denotes
the number of bioassays
conducted.


Table 2
Immunoreactive relaxin content of uteri taken from guinea pigs in different stages of pregnancy and lactation.
(ng)
( ng)
STAGE OF
ANIMAL
(*>
fag)
TOTAL G.P. RELAXIN
AMT.
RELAXIN
PREGNANCY
NO.
WET WT.
DRY WT.
PER UTERUS PER GRAM
WET WEIGHT
DAY 15
1
2.0
13.0
0.65
0.33
2
1.5
10.0
0.40 (0.40 + 0.14)**
0.27
(0.25 + O.i
3
1.0
9.0
0.16
0.16
DAY 30
4
6.1
61.0
12.20
2.00
5
2.6
23.0
1.46
6
7.2
106.0
23 85 (H'44 1 4.51)
3.31
(2.06 + 0.<
7
4.0
38.0
5.89
1.47
DAY 45
8
8.0
443.5
199.58
24.95
9
3.2
195.0
33.52
10
5.0
280.0
168.00 (148.47 + 21.52)
33.60
(24.72 + 6
11
17.5
248.0
119.04
6.80
DAY 60
12
17.0
273.4
158.57
9.33
13
18.0
346.5
173 25
9.63
14
20.0
391.0
22^83 d72-67 19.17)
11.24
(9.83 + 0.'
15
14.7
243.7
134.04
9.12
LATE
16
13.0
321.0
72.23
5.56
PREGNANT
17
16.0
268.2
64.37
4.02
18
26.0
495.6
183.37 (101.90 + 25.78)
7.05
(6.45 + l.;
19
11.0
234.9
140.94
12.81
20
17.2
194.3
48.58
2.82
1.ACTATING
21
6.3
53.9
5.66
0.90
22
A .0
50.0
3.00 ,, _
0.75
23
3.0
35.6
5 52 (4.78 + 0.61)
1.84
(1.01 + o.:
24
9.2
105.0
4.94
0.54
Twenty milligrams
of Acid
Acetone extracted
powder was
homogenized In 1 ml of distilled
water.
**Activlty expressed as ng porcine NIH-RXN-P1 standard relaxin equivalents (X + SFH) as determined by a
homologous porcine radioimmunoassay. All assays were conducted In duplicate
'-J
4-*-


Table 3. Protein yield and potencies of late pregnant guinea pig uterus throughout the ODS purification procedure.
Fraction
Recovery (mg)
Protein Yielda
(nig/g Fresh Tissue)
Total Units
Spec 1fic Activity
(U/mr.)
ODS extracted guinea pig
uterus
188.80
1.59
60.00
(2) 0.32
Bioactlve Sepliadex C-50
f raction^
35.50
0.30
53.19
(2) 1.50
Carboxymethylcellulose ion
exchange chromatography (CHC)c
1.70
0.01
6.39
(2) 3.87
Yields represent the values calculated from 5 uteri. The mouse uterine molility bloassay was utilized to determine
the potency of the preparations. ( ) denotes the number of bioassays conducted.
a Total wet weight of the ute-rl was 118.5 grams.
Bioassays were conducted on the pooled relaxln containing fractions
C Bioassays were conducted on the pooled relaxln containing fractions
from the chromatography run.
from the CMC run.


76
Table 4. Physiochemical characteristics of guinea pig
uterine relaxin. The source of relaxin was an
ODS* purified uterine preparation.
Reduced Activity
No Change
Dithiothrietol
24x
Heating at 70 C
2x
Trypsin
24x
R19 antiserum
1 lx
*ODS crude relaxin is a partially purified uterine extract
taken after the initial purification step in the ODS pro
cedure. This extract was tested in the mouse uterine motility
assay, without being altered (control), and after experimental
treatments. A ratio was determined by dividing the final
volume of the experimental by the final volume of the control.
The assays were run twice and an average value was computed.
The greater the experimental to control ratio, the greater
the ability of the agent to inhibit the action of relaxin.


APPENDIX 2
FIGURES


Figures 1-4 represent sections of uteri taken from guinea pigs
on day 30 of pregnancy.
1.Transverse section of uterus stained using the PAP tech
nique with R19 antiserum (1:500 dilution). Arrow endometrial
glands exhibiting RP; arrowhead endometrial glands lacking RP.
X 40.
2. Section adjacent to that shown in Figure 1 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo
metrial glands (arrows). X 40.
3. Hematoxylin and eosin stained section. X 500.
4. Section stained using the PAP technique with R19 anti
serum (1:500 dilution). Note that not all endometrial gland cells
demonstrate RP. Clear areas in basal regions of the endometrial
gland cells represent unstained nuclear profiles. X 500.


79


Figures 5-8 represent sections of uteri taken from guinea pigs
on day 45 of pregnancy.
5. Transverse section of the guinea pig uterus taken on
day 45 of pregnancy and stained using the PAP technique with R19
antiserum (1:500 dilution). Arrow endometrial glands exhibiting
RP; arrowhead endometrial glands lacking RP. A higher percentage
of glands are labeled than in day 30 tissue, however, not all glands
have RP at this stage. X 40.
6. Section adjacent to that shown in Figure 5 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over
endometrial glands (arrows). X 40.
7. Hematoxylin and eosin stained section. X 500.
8. Section stained using the PAP technique with R19 anti
serum (1:500 dilution). While not all cells in each gland show
the presence of RP, note that the majority of cells in each gland
show a heavy accumulation of RP. X 500.


81


Figures 9-12 represent sections of uteri taken from guinea
pigs on day 60 of pregnancy.
9.Transverse section of the guinea pig uterus taken on
day 60 of pregnancy stained using the PAP technique with R19 anti
serum (1:500 dilution). Note that all of the endometrial glands
exhibit RP. X 40.
10. Section adjacent to that shown in Figure 9 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo
metrial glands. X 40.
11. Hematoxylin and eosin stained section. Note what appear
to be dense aggregates of material near the luminal surface of the
endometrial gland cells. X 500.
12. Section stained using the PAP technique with R19 anti
serum (1:500 dilution). Dense aggregates of RP similar to that
demonstrated in Figure 9 are shown in the luminal surfaces of the
EGC. Clear areas in base of EGC represent unstained nuclear
profiles. X 500.


83


Figures 13-16 represent sections of uteri taken from late
pregnant guinea pigs.
13. Transverse section of the guinea pig uterus from late
pregnant animals (day 65 or 66 of pregnancy) and stained using the
PAP technique with R19 antiserum (1:500 dilution). Note all of the
endometrial glands exhibit RP. X 40.
14. Section adjacent to that shown in Figure 13 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo
metrial glands. X 40.
15. Hematoxylin and eosin stained section. Continuity
between the lumen of an endometrial gland and the lumen of the
uterus can be seen in the lowest of the three gland profiles. Note
the difference in cytoplasmic and nuclear staining densities between
the EGC and cells of the uterine lumen epithelium. X 500.
16. Section adjacent to that shown in Figure 15 stained
using the PAP technique with R19 antiserum (1:500 dilution). Note
that some of the endometrial gland cells have RP distributed through
out the cytoplasm, some have no RP and in some cells (lower gland)
the RP is localized in a specific supranuclear region. Note also
that the extent of the gland can be determined by the region where
deposition of RP ceases in cells that are continuous with the
uterine lumen epithelium. This pattern of deposition of RP
corresponds with differences in staining noted in H § E stained
tissue (Figure 15). X 500.


85
16


Figures 17-20 represent sections of uteri taken from lactating
guinea pigs.
17.Transverse section of guinea pig uterus taken from lac
tating animals (3 days postpartum) and stained using the PAP
technique with R19 antiserum (1:500 dilution). Arrow endometrial
glands exhibiting RP; arrowhead endometrial glands lacking RP.
X 40.
18. Section adjacent to that shown in Figure 17 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo
metrial glands (arrows). X 40.
19. Hematoxylin and eosin stained section. Note the large
number of mitoses in the glands (arrows). X 500.
20. Section stained using the PAP technique with R19 anti
serum (1:500 dilution). Note that RP is located in only a few
cells of the gland and that the pattern of deposition of RP is
variable from cell to cell. X 500.


87


Figures 21-24 represent sections of uteri taken from ovari-
ectomized hormone treated animals.
21. Transverse section of guinea pig uterus taken from
ovariectomized animals treated with estrogen (10 yg) and progester
one (2 mg) daily for 15 days stained using the PAP technique with
R19 antiserum (1:500 dilution). Arrow endometrial glands exhibit
ing RP. X 40.
22. Section adjacent to that shown in Figure 21 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo
metrial glands (arrows). X 40.
23. Hematoxylin and eosin stained section. Glandular cells
are cuboidal with densely staining nuclei. X 500.
24. Section stained using the PAP technique with R19 anti
serum (1:500 dilution). Only a few endometrial gland cells do not
show the presence of relaxin. Location of RP varies from cell to
cell, however, the majority of cells appear to have RP distributed
throughout the cytoplasm. X 500.


89


Figure 25. Biologically active relaxin content o_f uteri
taken from guinea pigs during pregnancy and lactation (X +_SEM) .
Data expressed as total units per uterus as determined by the mouse
uterine motility bioassay of uterine extracts.


Full Text
UNIVERSITY OF FLORIDA
3 1262 08554 3493


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EWHJNYXNO_ZQO987 INGEST_TIME 2012-03-02T22:14:13Z PACKAGE AA00009113_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

385,),&$7,21 &+$5$&7(5,=$7,21 $1' /2&$/,=$7,21 2) 5(/$;,1 ,1 7+( 35(*1$17 *8,1($ 3,* %< 58%( -26( 3$5'2 $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( &281&,/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

, ZLVK WR GHGLFDWH WKLV GLVVHUWDWLRQ WR P\ SDUHQWV 0U DQG 0UV 5XEH 3DUGR DQG P\ JUDQGPRWKHU *LOGD GH OD 7RUULHQWH 7KLV GLVVHUWDWLRQ LV DOVR GHGLFDWHG LQ WKH PHPRU\ RI P\ JUDQGIDWKHU -RVH (OLDV GH OD 7RUULHQWH

PAGE 3

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

PAGE 4

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

PAGE 5

$33(1',; ,2',1$7,21 2) 68&&,1,0,'( 5(/$;,1 ,OO $33(1',; ,2',1$7,21 2) 5(/$;,1 :,7+ 7+( %2/721 $1' +817(5 5($*(17 %,2*5$3+,&$/ 6.(7&+ Y

PAGE 6

/,67 2) $%%5(9,$7,216 %R &0& &30 '$% (* (*& *$5 JZZ + ( / ODF ,3 PZ 1(3+*( 16% 156 2'6 3$3 3$*( 3%6 5 5,$ 53 ]HUR FRXQW WXEH FDUER[\PHWK\O FHOOXORVH FRXQWV SHU PLQXWH n GLDPLQREHQ]LGLQH HQGRPHWULDO JODQGVf HQGRPHWULDO JODQG FHOOVf JRDW DQWLUDEELW ,J* JUDP ZHW ZHLJKW KHPDWR[\OLQ DQG HRVLQ XWHULQH OXPHQ ODFWDWLQJ ODWH SUHJQDQW PROHFXODU ZHLJKW QRQ HTXLOLEULXP SRO\DFU\ODPLGH JHO HOHFWURSKRUHVLV QRQVSHFLILF ELQGLQJ QRUPDO UDEELW VHUXP RFWDGHF\OVLOLFD SHUR[LGDVH DQWLSHUR[LGDVH SRO\DFU\ODPLGH JHO HOHFWURSKRUHVLV SKRVSKDWH EXIIHUHG VDOLQH DQWLVHUXP PDGH WR SXULILHG SRUFLQH UHOD[LQ UDGLRLPPXQRDVVD\ SHUR[LGDVH UHDFWLRQ SURGXFW Y[

PAGE 7

530 UHYROXWLRQV SHU PLQXWH 6& VXEFXWDQHRXV 6'6 VRGLXP GRGHF\O VXOIDWH 6( VXUIDFH HSLWKHOLXP RI XWHULQH OXPHQ 7 WRWDO FRXQW WXEH 7&$ WULFKORURDFHWLF DFLG 8 XQLWVf RI UHOD[LQ DFWLYLW\ YL

PAGE 8

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH &RXQFLO RI WKH 8QLYHUVLW\ RI )ORULGD LQ 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ 385,),&$7,21 &+$5$&7(5,=$7,21 $1' /2&$/,=$7,21 2) 5(/$;,1 ,1 7+( 35(*1$17 *8,1($ 3,* %\ 5XEH -RVH 3DUGR 0D\ &KDLUPDQ /\QQ + /DUNLQ 0DMRU 'HSDUWPHQW 0HGLFDO 6FLHQFHV $QDWRP\f ,W KDV EHHQ VKRZQ XVLQJ WKH SHUR[LGDVHDQWLSHUR[LGDVH LPPXQRF\WR FKHPLFDO WHFKQLTXH WKDW WKH HQGRPHWULDO JODQGV RI WKH SUHJQDQW JXLQHD SLJ DUH WKH VRXUFH RI WKH KRUPRQH UHOD[LQ 7KH SUHVHQFH RI UHOD[LQ KDV EHHQ GHPRQVWUDWHG LQ XWHUL IURP GD\ GD\ GD\ SUHJQDQW DQG ODWH SUHJQDQW DQLPDOV GD\V f 7KH GHQVLW\ RI UHDFWLRQ SURGXFW GHSRVLWLRQ LQFUHDVHG DV SUHJQDQF\ SURFHHGHG ZLWK KLJK GHSRVLWLRQ RFFXUn ULQJ LQ GD\V DQG RI SUHJQDQF\ DQG LQ ODWH SUHJQDQW DQLPDOV /LWWOH RU QR LPPXQRSHUR[LGDVH ODEHOLQJ ZDV REVHUYHG LQ WLVVXHV IURP GD\ QRQSUHJQDQW DQG ODFWDWLQJ DQLPDOV GD\V SRVWSDUWXPf ,PPXQRSHUR[LGDVH ODEHOLQJ ZDV QRW VHHQ LQ QRQHQGRPHWULDO JODQG FRPSRQHQWV RI WKH XWHUXV +LJK ELRORJLFDO DQG LPPXQRORJLFDO DFWLYLWLHV ZHUH IRXQG LQ H[WUDFWV RI XWHUL WDNHQ RQ GD\V DQG RI SUHJQDQF\ DQG LQ ODWH SUHJQDQW DQLPDOV :KHQ D FUXGH H[WUDFW RI ODWH SUHJQDQW XWHUL ZDV FKURPDWRJUDSKHG LQ 6HSKDGH[ D IUDFWLRQ FRQWDLQLQJ UHOD[LQ DFWLYLW\ HOXWHG LQ WKH PROHFXODU ZHLJKW UDQJH 7KLV IUDFWLRQ ZDV DFWLYH LQ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ 8PJf DQG SURPRWHG OHQJWKHQLQJ RI WKH YLLL

PAGE 9

LQWHUSXELF OLJDPHQW LQ HVWURJHQ SULPHG IHPDOH PLFH 7KH ELRDFWLYH 6HSKDGH[ IUDFWLRQ ZDV IXUWKHU SXULILHG LQ D FDUER[\PHWK\OFHOOXORVH &0&f LRQ H[FKDQJH FROXPQ $ VLQJOH SHDN IURP WKH &0& FROXPQ GHPRQn VWUDWHG UHOD[LQ ELRDFWLYLW\ 8PJf LQ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ 7KH &0& SXULILHG JXLQHD SLJ UHOD[LQ ZDV FRPSDUHG WR &0& SXULILHG SRUFLQH UHOD[LQ LQ D WZR GLPHQVLRQDO JHO HOHFWURSKRUHVLV V\VWHP 7KH WZR SXULILHG UHOD[LQV ZHUH RI VLPLODU PROHFXODU ZHLJKWV ZLWK WKH SRUFLQH KRUPRQH EHLQJ VOLJKWO\ PRUH EDVLF 7KH JXLQHD SLJ UHOD[LQ PROHFXOH DSSHDUV WR EH VLPLODU WR SRUFLQH UHOD[LQ DFFRUGLQJ WR WKH IROORZLQJ FULWHULD f $ FRQWLQXRXV OLQH RI LGHQWLW\ ZDV REWDLQHG ZKHQ D PROHFXODU ZHLJKW IUDFWLRQ RI UHOD[LQ IURP XWHUL RI GD\ SUHJn QDQW JXLQHD SLJV ZDV WHVWHG ZLWK SRUFLQH UHOD[LQ DQG DQWLUHOD[LQ VHUXP LQ GRXEOH LPPXQRGLIIXVLRQ SODWH DQDO\VHV f %RWK UHOD[LQ PROHFXOHV ZHUH LQDFWLYDWHG ZKHQ UHDFWHG ZLWK DQWLUHOD[LQ VHUXP LQ DQ DQWLVHUXP WHVW HPSOR\LQJ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ f %RWK UHOD[LQ PROHFXOHV ZHUH LQDFWLYDWHG E\ WU\SVLQ DQG GLWKLRWKULHWRO EXW QRW E\ PRGHUDWH KHDW ,;

PAGE 10

,1752'8&7,21 0DQ\ PDPPDOV WKDW JLYH ELUWK WR ODUJH PDWXUH \RXQJ KDYH PHFKDQLVPV WR FRPSHQVDWH IRU D QDUURZ SHOYLF ZLGWK 2QH RI WKH PRVW GUDPDWLF H[DPSOHV RI WKLV LV IRXQG LQ WKH JXLQHD SLJ ZKLFK JLYHV ELUWK WR UHODWLYHO\ ODUJH \RXQJ ,Q WKH JXLQHD SLJ SXELF VHSDUDWLRQ LV VR H[WUHPH WKDW WKH WZR KDOYHV RI WKH SHOYLV DUH IUHHO\ PRYDEOH GXULQJ WKH ELUWK SURFHVV ,W ZDV +LVDZnV LQWHUHVW LQ WKLV SKHQRPHQRQ ZKLFK SURPSWHG KLP WR DVN ZKHWKHU FHUWDLQ KXPRUDO IDFWRUV ZHUH UHVSRQVLEOH IRU WKH PRUSKRORJLF FKDQJHV DVVRFLDWHG ZLWK WKLV SURFHVV +LVDZ f ZDV WKH ILUVW WR UHODWH WKLV SHOYLF VHSDUDWLRQ WR WKH SUHVHQFH RI D EORRG IDFWRU ODWHU FDOOHG UHOD[LQ )HYROG +LVDZ DQG 0H\HU f 6LQFH LWV GLVFRYHU\ UHOD[LQ KDV EHHQ UHFRJQL]HG DV D KRUPRQH RI SUHJQDQF\ DQG KDV EHHQ GHWHFWHG LQ PDQ\ VSHFLHV RI DQLPDOV 7KH SK\VLRORJLFDO HIIHFWV RI UHOD[LQ DUH PDLQO\ FRQFHUQHG ZLWK WKH IHPDOH UHSURGXFWLYH WUDFW RI PDPPDOLDQ VSHFLHV 7KUHH RI WKHVH HIIHFWV KDYH EHHQ H[WHQVLYHO\ UHYLHZHG LQ WKH OLWHUDWXUH f UHOD[DWLRQ RI WKH OLJDPHQWV ZKLFK VWDELOL]H WKH SHOYLF ERQHV f LQKLELWLRQ RI XWHULQH FRQWUDFWLRQV DQG f VRIWHQLQJ RI WKH FHUYL[ DW WHUP +LVDZ DQG =DUURZ +DOO 6FKZDEH HW DO 3RUWHU f 7KH UHOD[DWLRQ RI SHOYLF OLJDPHQWV DQG LQKLELWLRQ RI XWHULQH FRQWUDFWLRQV DUH WKH EDVLV RI WZR LPSRUWDQW ELRDVVD\V ZKLFK DUH XVHG WR GHWHFW UHOD[LQ 7KH IROORZLQJ SRUWLRQV RI WKH LQWURGXFWLRQ ZLOO FRQFHQWUDWH RQ IRXU DUHDV RI VWXG\ RQ UHOD[LQ f GHWHFWLRQ RI UHOD[LQ f FHOOXODU ORFDOL]DWLRQ RI UHOD[LQ f LVRODWLRQ DQG FKDUDFWHUL]DWLRQ RI UHOD[LQ DQG f GHVFULSWLRQ RI UHOD[LQ UHVHDUFK LQ WKH JXLQHD SLJ

PAGE 11

5HOD[LQ $VVD\V 5HOD[DWLRQ RI 3HOYLF /LJDPHQWV 7KH ILUVW TXDOLWDWLYH ELRDVVD\ IRU UHOD[LQ ZDV WKH JXLQHD SLJ SXELF V\PSK\VLV SDOSDWLRQ DVVD\ GHYHORSHG E\ )HYROG HW DO f $Q DWWHPSW WR TXDQWLWDWH WKLV DVVD\ ZDV PDGH E\ $EUDPRZLW] HW DO f $ JXLQHD SLJ XQLW 8f ZDV GHILQHG E\ WKHVH LQYHVWLJDWRUV DV WKH GRVH RI UHOD[LQ WKDW LQ KRXUV FDXVHG UHOD[DWLRQ RI WKH SXELF V\PSK\VLV GHWHUn PLQHG E\ SDOSDWLRQf LQ DW OHDVW HLJKW RI WZHOYH HVWURJHQ SULPHG FDVn WUDWHG IHPDOH JXLQHD SLJV 7ZR EDVLF SUREOHPV ZHUH DVVRFLDWHG ZLWK WKLV DVVD\ f WKH GHJUHH RI VXEMHFWLYLW\ ZDV KLJK DQG f UHSHDWHG XVH RI WKH VDPH JXLQHD SLJV DW ILUVW VHQVLWL]HG WKHP WR UHOD[LQ EXW WKHQ PDGH WKH DQLPDOV UHIUDFWRU\ WR WKH KRUPRQH DIWHU VHYHUDO PRQWKV RI XVH 1RDOO DQG )ULHGHQ f $OO VWXGLHV EHIRUH H[FOXVLYHO\ HPSOR\HG WKH JXLQHD SLJ SXELF V\PSK\VLV DVVD\ DQG FDQ WKHUHIRUH EH TXHVWLRQHG IRU WKH UHDVRQV H[SODLQHG DERYH 7KH PRXVH LQWHUSXELF OLJDPHQW DVVD\ ZDV ODWHU GHYHORSHG E\ 6WHLQHW] HW DO f DQG RIIHUHG D PRUH VHQVLWLYH DQG REMHFWLYH PHWKRG RI DVVD\LQJ UHOD[LQ ,Q WKLV DVVD\ JURXSV RI VH[XDOO\ LPPDWXUH IHPDOH PLFH Jf ZHUH SULPHG ZLWK D VLQJOH LQMHFWLRQ RI SJ HVWUDGLRO DQG GD\V ODWHU UHFHLYHG LQMHFWLRQV RI UHOD[LQ VWDQGDUGV RU XQNQRZQV WKUHH GRVH OHYHOVf LQ b EHQ]RSXUSXULQH% (LJKWHHQ WR WZHQW\IRXU KRXUV ODWHU WKH PLFH ZHUH NLOOHG DQG WKHLU SXEHV GLVVHFWHG IUHH RI FRQn QHFWLYH WLVVXH DQG IDW 7KH LQWHUSXELF GLVWDQFH ZDV PHDVXUHG XVLQJ D GLVVHFWLQJ PLFURVFRSH ILWWHG ZLWK DQ RFXODU PLFURPHWHU DQG D WUDQVLOOXP LQDWLQJ VRXUFH :LWK WKLV DVVD\ GRVH UHVSRQVH FXUYHV FRXOG EH FRPSDUHG EHWZHHQ WZR UHOD[LQ SUHSDUDWLRQV WR GHWHUPLQH ZKHWKHU WKH UHOD[LQV

PAGE 12

HOLFLWHG VLPLODU SDUDOOHO GRVH UHVSRQVH FXUYHVf RU GLVVLPLODU UHVSRQVHV LQ WKH H[SHULPHQWDO DQLPDOV ,QKLELWLRQ RI 8WHULQH &RQWUDFWLRQV .UDQW] HW DO f ZHUH WKH ILUVW WR GHVFULEH WKH DELOLW\ RI UHOD[LQ H[WUDFWV WR LQKLELW VSRQWDQHRXV FRQWUDFWLRQV RI UDW JXLQHD SLJ DQG PRXVH XWHUL PDLQWDLQHG LQ YLYR DQG LQ YLWUR .URF HW DO f LPSURYHG WKH XWHULQH PRWLOLW\ DVVD\ IXUWKHU E\ XWLOL]LQJ XWHUL IURP VH[XDOO\ LPPDWXUH HVWURJHQ SULPHG PLFH LQ DQ LQ YLWUR V\VWHP 7KLV ELRDVVD\ LV PRUH HFRQRPLFDO EHFDXVH PLFH DUH OHVV FRVWO\ WKDQ WKH ODUJHU URGHQWV $OVR WKH PRXVH XWHUXV UHTXLUHV OHVV UHOD[LQ WR UHGXFH FRQn WUDFWLRQV WKHUHE\ FRQVHUYLQJ WKH KRUPRQH 7KH PRXVH XWHULQH PRWLOLW\ DVVD\ KDV EHHQ UHFHQWO\ PRGLILHG E\ /DUNLQ HW DO f ,Q WKLV PRGLILHG DVVD\ HDFK XWHULQH KRUQ IURP VH[XDOO\ LPPDWXUH HVWURJHQ SULPHG PLFH LV GLYLGHG DQG VXVSHQGHG LQ DQ DHUDWHG RUJDQ EDWK RI /RFNHnV VROXn WLRQ 7KH XWHULQH VHJPHQW LV DWWDFKHG WR D KHDUW OHYHU DJDLQVW J RI WHQVLRQ DQG FRQWUDFWLRQV DUH PRQLWRUHG ZLWK DQ LQN ZULWLQJ N\PRJUDSK 7KH UHOD[LQ VWDQGDUG RU XQNQRZQ LV WHVWHG IRU WKH DELOLW\ WR LQKLELW VSRQWDQHRXV XWHULQH FRQWUDFWLRQV 2QH VHFWLRQ RI WKH KRUQ LV WUHDWHG ZLWK WKH VWDQGDUG DQG WKH RWKHU ZLWK WKH XQNQRZQ %\ GRXEOLQJ WKH FRQn FHQWUDWLRQV RI VWDQGDUG DQG XQNQRZQ LQ WKH RUJDQ EDWK DW PLQ LQWHUYDOV WKH UHVSRQVH RI WKH WZR XWHULQH VHJPHQWV PD\ EH FRPSDUHG DQG WKH SRWHQF\ RI WKH XQNQRZQ GHWHUPLQHG 7KH JXLQHD SLJ SXELF V\PSK\VLV DVVD\ LV WKH PRVW VXEMHFWLYH RI WKH DVVD\V PHQWLRQHG EXW ZDV WKH PRVW ZLGHO\ XVHG XQWLO 7KH PRXVH LQWHUSXELF OLJDPHQW DVVD\ RIIHUV WKH UHILQHPHQW RI REMHFWLYLW\ VLQFH

PAGE 13

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f 5DGLRLPPXQRDVVD\ ,Q %U\DQW GHYHORSHG WKH ILUVW KRPRORJRXV 5,$r IRU SRUFLQH UHOD[LQ ,Q WKLV DVVD\ D UHODWLYHO\ LPSXUH UHOD[LQ SUHSDUDWLRQ 1,+5 3O 8PJf ZDV LRGLQDWHG ZLWK WKH FKORUDPLQH7PHWKRG RI +XQWHU DQG *UHHQZRRG f 7KLV LPSXUH SUHSDUDWLRQ ZDV DOVR XVHG IRU WKH SURGXFn WLRQ RI DQWLVHUXP DQG IRU WKH UHOD[LQ VWDQGDUGV 7KLV 5,$ ZDV XVHG E\ %U\DQW DQG FROODERUDWRUV IRU VHYHUDO VWXGLHV %U\DQW %U\DQW DQG 6WHOPDVLDN %U\DQW HW DO %U\DQW DQG &KDPOH\ %U\DQW HW DO f EHIRUH LW ZDV GLVFRYHUHG E\ 6KHUZRRG DQG 2n%\UQH f WKDW SRUFLQH UHOD[LQ FRQWDLQHG QR W\URVLQH UHVLGXHV DQG WKHUHIRUH FRXOG QRW EH LRGLQDWHG E\ WKH FKORUDPLQH7 PHWKRG ,W ZDV OLNHO\ WKDW %U\DQW HLWKHU LRGLQDWHG VRPH SHSWLGH FRQWDPLQDQWV ZLWKLQ WKH UHOD[LQ SUHSDUDWLRQ RU SHUKDSV ODEHOHG D SURKRUPRQH ZKLFK FRQWDLQHG VLPLODU DQWLJHQLF GHWHUn PLQDQWV WR UHOD[LQ 7KLV SRVVLELOLW\ KDV EHHQ H[SORUHG E\ %U\DQW *UHHQZRRG DQG *UHHQZRRG f LQ D UHFHQW SXEOLFDWLRQ LQ ZKLFK WKH\ r$ KRPRORJRXV SRUFLQH 5,$ LV DQ 5,$ ZKHUH WKH DQWLUHOD[LQ VHUXP LV SURGXFHG DJDLQVW SRUFLQH UHOD[LQ WKH LRGLQDWHG KRUPRQH DQG WKH UDGLRLQHUW VWDQGDUGV DUH SRUFLQH UHOD[LQ

PAGE 14

FRPSDUHG WKH 5,$ XWLOL]LQJ 1,+53 UHOD[LQ ZLWK DQ 5,$ XVLQJ D KLJKO\ SXULILHG UHOD[LQ IUDFWLRQ &0Dn 8PJf 7KH 1,+53 UHOD[LQ ZDV LRGLQDWHG E\ WKH FKORUDPLQH7 PHWKRG RI +XQWHU DQG *UHHQZRRG f 7KH &0Dn UHOD[LQ ZDV UHDFWHG ZLWK D VXFFLQLPLGH HVWHU DQG LRGLQDWHG E\ WKH PHWKRG RI %ROWRQ DQG +XQWHU f ,W ZDV IRXQG WKDW DQWLVHUD WR &0Dn UHOD[LQ FURVVUHDFWHG ZLWK 1,+53 UHOD[LQ $OVR KLJKO\ SXULILHG &0Dn UHOD[LQ FURVVUHDFWHG ZLWK DQWLVHUD PDGH WR 1,+53 UHOD[LQ +RZHYHU ZKHQ WKH WZR DVVD\V ZHUH XVHG WR GHWHFW UHOD[LQ LQ VHUXP RI SUHJQDQW HZHV WKH DVVD\ EDVHG RQ WKH FUXGH UHOD[LQ SUHSDUDWLRQ 1,+ 53Of UHDG YDOXHV RI UHOD[LQ WHQ WLPHV JUHDWHU WKDQ WKRVH UHDG ZLWK WKH DVVD\ XWLOL]LQJ WKH KLJKO\ SXULILHG KRUPRQH &0Dnf 7KLV ZDV LQWHUSUHWHG WR PHDQ WKDW WKH 5,$ XWLOL]LQJ 1,+53 UHOD[LQ ZDV UHDGLQJ D EURDG VSHFWUXP RI LPPXQRDFWLYLW\ DQG FRXOG KDYH EHHQ GHWHFWLQJ W\URVLQH FRQWDLQn LQJ FRQWDPLQDQWV RU D UHOD[LQ SURKRUPRQH WKDW ZDV QRW GHWHFWHG E\ WKH 5,$ XWLOL]LQJ WKH KLJKO\ SXULILHG KRUPRQH ,Q 6KHUZRRG DQG KLV FRZRUNHUV GHYHORSHG D KRPRORJRXV 5,$ IRU SRUFLQH UHOD[LQ ZKLFK WRRN LQWR DFFRXQW WKH KRUPRQHnV WRWDO ODFN RI W\URVLQH UHVLGXHV 6KHUZRRG HW DO f 7KLV 5,$ XWLOL]HG D KLJKO\ SXULILHG UHOD[LQ SUHSDUDWLRQ FRQWDLQLQJ &0Dn &0D DQG &0% IUDFWLRQV ZKLFK WKH\ FDOOHG QDWLYH UHOD[LQ ,QLWLDO HIIRUWV WR LRGLQDWH QDWLYH UHOD[LQ ZLWK WKH FKORUDPLQH7 PHWKRG IDLOHG 7KHUHIRUH D QRYHO DSSURDFK ZDV HPSOR\HG WR FRYDOHQWO\ ELQG W\URVLQH WR UHOD[LQ WKURXJK DQ DPLGH OLQNDJH XVLQJ WKH DJHQW 1FDUER[\/W\URVLQH DQK\GULGH 7KH UHVXOWLQJ PROHFXOH ZDV QDPHG SRO\W\URV\O UHOD[LQ EHFDXVH LW FRQWDLQHG PROHV RI W\URVLQH SHU PROH RI UHOD[LQ DQG ZDV XVHG IRU WKH GHYHORSPHQW RI DOO SKDVHV RI WKH 5,$ 6KHUZRRG HW DO f UHSRUWHG GHWHFWLQJ OHYHOV RI

PAGE 15

SRUFLQH UHOD[LQ DV ORZ DV SJ ZKHUHDV SUHYLRXVO\ XVHG ELRDVVD\V ZHUH VHQVLWLYH LQ WKH ORZ PLFURJUDP UDQJH 8WLOL]LQJ WKLV 5,$ WKH SUHVHQFH RI UHOD[LQ KDV EHHQ GHPRQVWUDWHG LQ VHUD RI SUHJQDQW SLJV 6KHUZRRG HW DO D Ef +RZHYHU WKH DVVD\ GLG QRW GHWHFW UHOD[LQ LQ VHUD IURP SUHJQDQW JXLQHD SLJV RU SUHJQDQW FRZV 6KHUZRRG HW DO f 7KLV REVHUYDWLRQ PD\ KDYH UHVXOWHG EHFDXVH RI D QXPEHU RI UHDVRQV KRZn HYHU WZR WKDW VKRXOG EH FRQVLGHUHG DUH WKDW WKH DQWLUHOD[LQ VHUXP GLG QRW FURVVUHDFW ZLWK UHOD[LQ IURP WKHVH VSHFLHV DQG WKDW VHUXP OHYHOV RI WKH KRUPRQH ZHUH EHORZ WKH OHYHO RI GHWHFWLRQ RI WKH DVVD\ $ 5,$ HPSOR\LQJ SRO\W\URV\O UHOD[LQ DOVR KDV EHHQ HVWDEOLVKHG LQ WKH ODERUDWRU\ RI 'U % 6WHLQHW] $ 6HSKDGH[ UHOD[LQ IUDFWLRQ FRQWDLQLQJ 8PJ ZDV XVHG WR GHYHORS WKH DQWLVHUXP XWLOL]HG LQ 6WHLQHW]nV 5,$ 7KH PDLQ GLIIHUHQFH LQ WKH 5,$ SURFHGXUHV RI 6WHLQHW] DQG RI 6KHUZRRG ZDV WKH HPSOR\PHQW RI GLIIHUHQW DQWLUHOD[LQ VHUD 7KH 6WHLQHW] DVVD\ V\VWHP KDV EHHQ XVHG WR GHPRQVWUDWH WKH SUHVHQFH RI UHOD[LQ LQ VHUD IURP SUHJQDQW UDWV PLFH KDPVWHUV JXLQHD SLJV GRJV PRQNH\V DQG KXPDQV 2n%\PH DQG 6WHLQWH] 2n%\PH HW DO 2n%\UQH HW DO f 7KH %ROWRQ DQG +XQWHU f PHWKRG RI LRGLQDWLRQ KDV EHHQ XWLOL]HG E\ VHYHUDO LQYHVWLJDWRUV LQ WKH GHYHORSPHQW RI D 5,$ IRU UHOD[LQ ,Q WKLV PHWKRG K\GUR[\SKHQ\OfSURSLRQLF DFLG 1K\GUR[\VXFFLQLPLGH HVWHU LV UDGLRLRGLQDWHG DFFRUGLQJ WR WKH PHWKRG RI +XQWHU DQG *UHHQZRRG f 7KH HVWHU LV WKHQ UHDFWHG ZLWK UHOD[LQ DQG DQ LRGLQDWHG SKHQ\O JURXS LV LQFRUSRUDWHG LQWR WKH HSVLORQ DPLQR JURXSV RI O\VLQH DQG 1 WHUPLQXV RI WKH UHOD[LQ PROHFXOH %U\DQW*UHHQZRRG DQG KHU FRZRUNHUV

PAGE 16

KDYH XVHG WKLV SUHSDUDWLRQ LQ WKH GHYHORSPHQW RI D KRPRORJRXV SRUFLQH 5,$ %U\DQW*UHHQZRRG DQG *UHHQZRRG
PAGE 17

WR EH UHODWHG WR WKH SULPDU\ DQWLVHUD HPSOR\HG LQ WKH DVVD\V UDWKHU WKDQ WKH LRGLQDWLRQ SURFHGXUH XVHG IRU ODEHOLQJ WKH KRUPRQH :KLOH 5,$ DQG RWKHU LPPXQRORJLF WHFKQLTXHV DUH EHLQJ XVHG LQFUHDVn LQJO\ WR GHWHFW UHOD[LQ WKH ELRDVVD\ VWLOO UHPDLQV WKH PRVW ZLGHO\ XVHG WHFKQLTXH IRU UHOD[LQ GHWHFWLRQ $OWKRXJK WKH 5,$ KDV WKH DGYDQWDJH RI LQFUHDVHG VHQVLWLYLW\ WKH ELRDVVD\ GHWHFWV WKH ELRORJLFDOO\ DFWLYH KRUPRQH &HOOXODU /RFDOL]DWLRQ RI 5HOD[LQ 2QH RI WKH NH\ DUHDV RI VWXG\ FRQFHUQLQJ UHOD[LQnV UROH LQ SUHJn QDQF\ DQG SDUWXULWLRQ KDV EHHQ WR GHWHUPLQH WKH FHOOXODU ORFDWLRQ RI WKH KRUPRQH GXULQJ WKHVH SK\VLRORJLFDO VWDWHV ,PPXQRF\WRFKHPLFDO WHFKn QLTXHV KDYH EHHQ WKH PRVW FRPPRQO\ HPSOR\HG PHWKRGV XVHG WR GHWHFW WKH FHOOXODU ORFDWLRQ RI UHOD[LQ LQ WLVVXHV 7KHVH WHFKQLTXHV KDYH EHHQ XVHG VXFFHVVIXOO\ WR GHWHFW FHOOV FRQWDLQLQJ UHOD[LQ LQ WKH SLJ FRZ UDW DQG KXPDQ 3LJ ,Q WKH SLJ WKHUH LV JRRG HYLGHQFH WKDW WKH FRUSXV OXWHXP RI SUHJQDQF\ LV WKH SULQFLSOH VRXUFH RI UHOD[LQ %HOW HW DO f ZHUH WKH ILUVW LQYHVWLJDWRUV WR FRUUHODWH OHYHOV RI UHOD[LQ ZLWK F\WRSODVPLF JUDQXOHV LQ SRUFLQH OXWHDO WLVVXH 7KH DFFXPXODWLRQ RI GHQVH F\WRSODVPLF JUDQXOHV LQ JUDQXORVD OXWHLQ FHOOV RI ODWH SUHJQDQW SLJV DQG WKH GHFOLQH LQ WKH QXPEHU RI JUDQXOHV DIWHU JHVWDWLRQ FORVHO\ SDUDOOHOHG WKH ULVH DQG IDOO RI ELRDVVD\DEOH FRUSXV OXWHXP UHOD[LQ LQ WKH VDPH SHULRGV .HQGDOO HW DO f XWLOL]HG WKH LPPXQRSHUR[LGDVH WHFKQLTXH WR ORFDOL]H UHOD[LQ DW WKH XOWUDVWUXFWXUDO OHYHO LQ F\WRSODVPLF JUDQXOHV RI SRUFLQH JUDQXORVD OXWHLQ FHOOV /DUNLQ HW DO f XVHG

PAGE 18

LPPXQRIOXRUHVFHQW ORFDOL]DWLRQ PHWKRGV HPSOR\LQJ DQWLSRUFLQH UHOD[LQ VHUXP 5f WR ORFDOL]H UHOD[LQ LQ JUDQXORVD OXWHLQ FHOOV RU SUHJQDQW SLJV )XUWQHU VWXGLHV ZLWK WKH SRUFLQH RYDU\ E\ $UDNDUL HW DO f KDYH VKRZQ WKDW WKH DQWLUHOD[LQ VHUXP HPSOR\HG LV RI XWPRVW LPSRUWDQFH LQ WKH ORFDOL]DWLRQ RI UHOD[LQ ZKHQ XVLQJ LPPXQRODEHOLQJ WHFKQLTXHV $Q DQWLVHUXP SURGXFHG DJDLQVW D FUXGH UHOD[LQ SUHSDUDWLRQ 1,+53 8PJf JDYH D GLIIXVH SDWWHUQ RI LPPXQRIOXRUHVFHQFH LQ WKH FRUSXV OXWHXP RI SUHJQDQF\ ZLWK WKH IOXRUHVFHQFH ORFDOL]HG PDLQO\ LQ WKH FRQQHFWLYH WLVVXH HOHPHQWV 2Q WKH RWKHU KDQG DQ DQWLVHUXP SURGXFHG DJDLQVW SXULILHG UHOD[LQ &0Dn 8PJf JDYH D VKDUS DQG SUHFLVH ORFDOL]Dn WLRQ ZLWKLQ WKH F\WRSODVP RI WKH OXWHDO FHOOV 7KHUH KDYH EHHQ QR UHSRUWV RI ORFDOL]DWLRQ RI UHOD[LQ LQ XWHULQH RU SODFHQWDO WLVVXHV LQ WKH SLJ &RZ 7KH RYDU\ RI WKH SUHJQDQW FRZ KDV EHHQ VKRZQ WR EH D VRXUFH RI UHOD[LQ ZLWK ELRDVVD\ WHFKQLTXHV &DVWUR+HPDQGH] f )LHOGV HW DO f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f 5HOD[LQ KDV EHHQ GHWHFWHG LQ WKH UDW RYDU\ ZLWK ELRDVVD\ 5,$ DQG

PAGE 19

LQPXQRF\WRFKHPLFDO WHFKQLTXHV :KHUHDV LW LV ZHOO HVWDEOLVKHG WKDW WKH RYDU\ LV D VRXUFH RI UHOD[LQ LQ WKH SUHJQDQW UDW WKH PHWULDO JODQG RI WKH XWHUXV DQG WKH SODFHQWD KDYH EHHQ LPSOLFDWHG DV WLVVXHV ZKLFK PD\ DOVR FRQWDLQ UHOD[LQ 'DOOHQEDFK+HOOZHJ HW DO f UHSRUWHG LPPXQRIOXRUHVFHQW ORFDOn L]DWLRQ RI UHOD[LQ LQ PHWULDO JODQG FHOOV RI WKH SUHJQDQW UDW XWHUXV EXW QRW LQ WKH RYDU\ RU SODFHQWD 7KH DQWLVHUXP XWLOL]HG ZDV PDGH LQ UDEELWV WR SRUFLQH UHOD[LQ 8PJf 5HVXOWV IURP WKLV VWXG\ VKRXOG EH YLHZHG ZLWK FDXWLRQ IRU WZR UHDVRQV )LUVW FRQWUROV XVHG LQ WKH VWXG\ ZHUH QRW VWULQJHQW VLQFH QR DWWHPSW ZDV PDGH WR DEVRUE WKH DQWLUHOD[LQ VHUXP ZLWK SXULILHG SRUFLQH UHOD[LQ 6HFRQG ZRUN RI VHYHUDO ODERUDWRULHV VKRZV WKDW WKH PHWULDO JODQG RI WKH UDW GRHV QRW FRQWDLQ UHOD[LQ /DUNLQ f WHVWHG WLVVXH H[WUDFWV IURP GD\ SUHJQDQW UDWV IRU UHOD[LQ ELRn DFWLYLW\ 2YDULDQ EXW QRW PHWULDO JODQG H[WUDFWV FRQWDLQHG ELRDVVD\ DEOH DPRXQWV RI UHOD[LQ $QGHUVRQ HW DO f FRXOG QRW GHWHFW UHOD[LQ LQ PHWULDO JODQGV RI SUHJQDQW UDWV XVLQJ LPPXQRIOXRUHVFHQFH EXW FRXOG GHWHFW ODEHOLQJ LQ WKH RYDU\ 7KH RYDULDQ IOXRUHVFHQFH ZDV ORFDOL]HG LQ WKH F\WRSODVP RI JUDQXORVD OXWHLQ FHOOV 7KH DQWLVHUXP HPSOR\HG E\ $QGHUVRQ HW DO f ZDV UDLVHG DJDLQVW DQ HYHQ OHVV SXUH SRUFLQH UHOD[LQ SUHSDUDWLRQ 1,+53 8PJf WKDQ WKDW HPSOR\HG E\ 'DOOHQEDFK +HOOZHJ HW DO f KRZHYHU FRQWUROV ZHUH PRUH FRPSOHWH 2WKHU VWXGLHV $QGHUVRQ DQG /RQJ f VKRZHG WKDW RYDULDQ H[WUDFWV FRQWDLQHG UHOD[LQ DFWLYLW\ DQG PHWULDO JODQG H[WUDFWV GLG QRW =DUURZ DQG 0F&OLQWRFN f LQMHFWHG ODEHOHG DQWLERG\ WR SRUFLQH UHOD[LQ LQWR SUHJQDQW UDWV DQG GLVFRYHUHG VXEVWDQWLDO DFFXPXODWLRQV RI ODEHO LQ WKH RYDU\ DQG

PAGE 20

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f 5DEELW 7KH RYDU\ XWHUXV DQG SODFHQWD RI WKH SUHJQDQW UDEELW KDYH EHHQ UHSRUWHG WR FRQWDLQ UHOD[LQ =DUURZ DQG 2n&RQQRU f IRXQG UHOD[LQ LQ WKH UDEELW JHVWDWLRQDO FRUSXV OXWHXP E\ HPSOR\LQJ DQ LQGLUHFW LPPXQR IOXRUHVFHQW ODEHOLQJ WHFKQLTXH KRZHYHU LW ZDV GLIILFXOW WR GHWHUPLQH IURP WKH SXEOLVKHG SKRWRJUDSKV ZKHWKHU WKH ODEHO ZDV ORFDWHG LQWUD RU H[WUDFHOOXODUO\ 7KH DQWLERG\ XWLOL]HG LQ WKH DERYH VWXG\ ZDV SURGXFHG LQ UDEELWV WR SRUFLQH UHOD[LQ :/ ORW 8PJ SRZGHUf 1R IOXRUHVFHQFH ZDV IRXQG LQ XWHULQH RU SODFHQWDO WLVVXH =DUURZ DQG 5RVHQEHUJ f UHSRUWHG ELRDFWLYH UHOD[LQ LQ WKH RYDU\ XWHUXV DQG PDWHUQDO SODFHQWD RI SUHJQDQW UDEELWV ZLWK WKH KLJKHVW OHYHO DSSHDULQJ LQ WKH PDWHUQDO SODFHQWD 7KLV VWXG\ DOVR VKRZHG WKDW RYDULHFWRP\ RI SUHJQDQW UDEELWV ZLWK VXEVHTXHQW SURJHVWHURQH UHSODFHPHQW WKHUDS\ GLG QRW UHVXOW LQ GHFUHDVHG EORRG OHYHOV RI UHOD[LQ )LHOGV HW DO f LVRODWHG UHOD[LQ IURP H[WUDFWV RI UDEELW SODFHQWDH +RZHYHU D FHOOXODU VRXUFH RI WKH KRUPRQH LQ WKH SODFHQWDH ZDV QRW UHSRUWHG OHDYLQJ RSHQ WKH SRVVLELOLW\ WKDW WKH UHOD[LQ ZDV EORRG ERUQH ,W DSSHDUV WKDW WKH UDEELW LV D VSHFLHV ZKLFK KDV H[WUDRYDULDQ VRXUFHV RI UHOD[LQ PRVW OLNHO\ WKH XWHUXV DQGRU SODFHQWD

PAGE 21

+XPDQ 7KH RYDU\ DQG SODFHQWD DSSHDU WR EH VRXUFHV RI UHOD[LQ LQ WKH SUHJQDQW KXPDQ 'DOOHQEDFK DQG 'DOOHQEDFK+HOOZHJ f GLVFRYHUHG WKH SUHVHQFH RI UHOD[LQ LQ EDVDO SODWH FHOOV RI KXPDQ SODFHQWDH XVLQJ DQ LQGLUHFW LPPXQRIOXRUHVFHQFH WHFKQLTXH 7KH DQWLVHUXP HPSOR\HG ZDV PDGH LQ UDEELWV WR D SRUFLQH UHOD[LQ SUHSDUDWLRQ 8PJf 7KLV ILQGLQJ KDV EHHQ VXEVWDQWLDWHG E\ VHYHUDO UHFHQW VWXGLHV )LHOGV DQG /DUNLQ f DOVR GHWHFWHG UHOD[LQ LQ EDVDO SODWH FHOOV RI KXPDQ WHUP SODFHQWDH XVLQJ WKH LPPXQRSHUR[LGDVH WHFKQLTXH $Q DQWLVHUXP 5f UDLVHG DJDLQVW SXULILHG SRUFLQH UHOD[LQ ZDV XWLOL]HG LQ WKHVH VWXGLHV )LHOGV DQG /DUNLQ f DOVR VKRZHG WKDW SODFHQWDH ZKLFK JDYH D SRVLWLYH VWDLQ IRU UHOD[LQ DOVR FRQWDLQHG ELRDVVD\DEOH UHOD[LQ
PAGE 22

UHTXLUH WKH RYDU\ IRU WKH PDLQWHQDQFH RI SUHJQDQF\ OLNH WKH UDEELW DQG WKH KXPDQ VHHP WR KDYH H[WUDRYDULDQ VRXUFHV RI UHOD[LQ 7KH YDOLGLW\ RI WKH DERYH JHQHUDOL]DWLRQ ZLOO EH WHVWHG DV IXWXUH VWXGLHV HQFRPSDVV D ODUJHU YDULHW\ RI VSHFLHV ,VRODWLRQ DQG &KDUDFWHUL]DWLRQ RI 5HOD[LQ 5HOD[LQ KDV EHHQ LVRODWHG DQG FKDUDFWHUL]HG IURP WKH RYDU\ RI WKH SUHJQDQW SLJ 6KHUZRRG DQG 2f%\PH 6FKZDEH HW DO f WKH RYDU\ RI WKH UDW )LHOGV DQG /DUNLQ 6KHUZRRG :DOVK DQG 1LDOO f WKH SODFHQWD RI WKH UDEELW )LHOGV HW DO f DQG WKH SODFHQWD DQG GHFLGXD RI WKH KXPDQ %LJD]]L HW DO )LHOGV DQG /DUNLQ
PAGE 23

DQG 2n%\UQH f XVHG DQ H[WUDFWLRQ SURFHGXUH VLPLODU WR WKDW RI 'RF]L f DQG *ULVV HW DO f DQG ZHUH WKH ILUVW WR IXOO\ FKDUDFWn HUL]H WKH SRUFLQH UHOD[LQ PROHFXOH 5HOD[LQ REWDLQHG E\ WKLV SURFHGXUH FRXOG EH VHSDUDWHG E\ FDUER[\PHWK\O FHOOXORVH &0&f LRQ H[FKDQJH FKURPDn WRJUDSK\ LQWR WKUHH IUDFWLRQV &0% &0D DQG &0Dn 7KHVH IUDFWLRQV KDG PZ DQG LVRHOHFWULF SRLQWV RI DQG S+ &0%f DQG S+ &0Df DQG DQG S+ &0Dnf 1RQH RI WKH IUDFWLRQV FRQWDLQHG DPLQR DFLG UHVLGXHV RI KLVWLGLQH W\URVLQH RU SUROLQH DQG DOO KDG HTXDO SRWHQF\ WR 8PJf DV GHWHUPLQHG E\ WKH PRXVH LQWHUSXELF OLJDPHQW ELRDVVD\ (DFK IUDFWLRQ FRQVLVWHG RI WZR VXEXQLWV DQ DOSKD DQG D EHWD FKDLQ OLQNHG E\ GLVXOILGH EULGJHV $PLQR DFLG DQDO\VHV RI WKH &0D DOSKD VXEXQLW VKRZHG LW WR FRQWDLQ DPLQR DFLG UHVLGXHV 7KH EHWD VXEXQLW FRQWDLQHG VRPH PLFURKHWHURJHQHLW\ ZLWK DPLQR DFLGV UDQJLQJ IURP WR LQ QXPEHU 6FKZDEH HW DO f XVLQJ WKH VDPH SXULILFDWLRQ VFKHPH DV 6KHUZRRG DQG 2n%\PH f VHTXHQFHG WKH SRUFLQH UHOD[LQ PROHFXOH 7KH\ VKRZHG WKH DOSKD DQG EHWD FKDLQV WR FRQWDLQ DQG DPLQR DFLG UHVLGXHV UHVSHFWLYHO\ DQG DOVR IRXQG WKDW SRUFLQH UHOD[LQ ODFNHG KLVWLGLQH W\URVLQH RU SUROLQH -DPHV HW DO f DOVR SXEOLVKHG WKH SULPDU\ VWUXFWXUH IRU SRUFLQH UHOD[LQ 7KHVH LQYHVWLJDWRUV XVHG WKH VDPH SXULILFDWLRQ VFKHPH DV 6KHUZRRG DQG 2n%\PH f EXW REWDLQHG DQ DPLQR DFLG VHTXHQFH GLIIHUHQW IURP WKDW REWDLQHG E\ 6FKZDEH HW DO f 7KH GLIIHUHQFH LQ WKH DOSKD FKDLQ ZDV PLQRU JOXWDPLQH LQVWHDG RI JOXWDPLF DFLG LQ WKH SRVLWLRQf 7KH EHWD FKDLQ ZDV IRXQG WR FRQWDLQ DPLQR DFLGV ZLWK DPLQR DFLGV IURP WKH WZHQW\WKLUG SRVLWLRQ WR WKH HQG WHUPLQXV EHLQJ RI D GLIIHUHQW

PAGE 24

VHTXHQFH WKDQ WKRVH IRXQG E\ 6FKZDEH HW DO f :DOVK DQG 1LDOO f XWLOL]HG D QRYHO DSSURDFK LQ WKH SXULILFDWLRQ RI SRUFLQH UHOD[LQ 7LVVXHV ZHUH LPPHGLDWHO\ IUR]HQ LQ OLTXLG QLWURJHQ XSRQ UHPRYDO IURP WKH DQLPDOV DQG KRPRJHQL]HG LQ D FROG VROXWLRQ FRQVLVWLQJ RI WULIOXRURDFHWLF DFLG IRUPLF DFLG K\GURFKORULF DFLG DQG VRGLXP FKORULGH $IWHU FHQWULIXJDWLRQ RI WKH KRPRJHQDWH WKH VXSHUQDWDQW ZDV SXPSHG WKURXJK DQ RFWDGHF\OVLOLFD 2'6f FROXPQ WR ZKLFK WKH UHOD[LQ DQG RWKHU SHSWLGHV DGKHUHG 7KH VROXWLRQ UHVXOWLQJ IURP WKLV SURFHGXUH ZDV WKHQ FKURPDWRJUDSKHG LQ JHO DQG &0& LRQ H[FKDQJH FROXPQV 7KH UHVXOWLQJ UHOD[LQ SUHSDUDWLRQ FRQVLVWHG RI RQH UHOD[LQ SHDN ZKLFK FRQWDLQHG DPLQR DFLGV LQ LWV EHWD FKDLQ DQG HOXWHG LQ WKH VDPH SRVLWLRQ DV &0D SRUFLQH UHOD[LQ DPLQR DFLGVf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f &OXHV WKDW WKH WKUHHGLPHQVLRQDO FRQILJXUDWLRQ RI SRUFLQH UHOD[LQ LV LPSRUWDQW WR LWV ELRORJLFDO DFWLYLW\ FDPH IURP WKH ZRUN RI 6FKZDEH DQG %UDGGRQ f ZKR VKRZHG WKDW SDUWLDO R[LGDWLRQ RI WKH WU\SWRSKDQ DW WKH SRVLWLRQ

PAGE 25

RI WKH EHWD FKDLQ OHG WR ELRORJLFDO LQDFWLYDWLRQ RI WKH PROHFXOH 5HGXFn WLRQ RI WKH GLVXOILGH ERQGV RI WKH UHOD[LQ PROHFXOH ZLWK GLWKLRWKULHWRO DOVR HOLPLQDWHG LWV ELRDFWLYLW\ 6FKZDEH HW DO f (YLGHQFH IRU D SURUHOD[LQ FRPSRXQG KDV EHHQ DFFXPXODWLQJ IURP VHYHUDO VRXUFHV -DPHV HW DO f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
PAGE 26

VWXGLHV WKDW UHOD[LQ OLNH LQVXOLQ PD\ EH FOHDYHG IURP D ODUJHU PZ SUHFXUVRU &RZ %RYLQH UHOD[LQ KDV EHHQ SXULILHG IURP RYDULHV RI WKH ODWH SUHJQDQW FRZV )LHOGV HW DO f ,Q WKLV VWXG\ FUXGH H[WUDFWV ZHUH SUHSDUHG IURP FRUSRUD OWHD E\ WKH WHFKQLTXH RI *ULVV HW DO f &KURPDWRJn UDSK\ RI WKH FUXGH H[WUDFW RQ D %LR*HO 3 FROXPQ GHPRQVWUDWHG WZR IUDFWLRQV KDYLQJ PZ RI DQG %RWK IUDFWLRQV ZHUH VKRZQ WR LQKLELW PRXVH XWHULQH FRQWUDFWLRQV LQ YLWUR DQG LQGXFH OHQJWKHQLQJ RI WKH PRXVH LQWHUSXELF OLJDPHQW ,PPXQRGLIIXVLRQ DQDO\VHV VKRZHG D FRQn WLQXRXV SUHFLSLWLQ OLQH EHWZHHQ WKH WZR FRZ UHOD[LQ IUDFWLRQV WKH 1,+ 53O SRUFLQH UHOD[LQ DQG DQ DQWLVHUXP 5f SURGXFHG DJDLQVW SXULILHG SRUFLQH UHOD[LQ 7KH PZ IUDFWLRQ JDYH EDQGV ZKHQ HOHFWURIRFXVHG S+ S+ DQG S+ 7KH S+ IRUP RI ERYLQH UHOD[LQ KDG WKH KLJKHVW ELRORJLFDO DFWLYLW\ 8PJf DFFRUGLQJ WR WKH PRXVH XWHULQH PRWLOLW\ DVVD\ 7KH ORZ PZ UHOD[LQ ORVW DFWLYLW\ LQ WKH SUHVHQFH RI GLWKLRWKULHWRO )LHOGV HW DO f 5DW ,Q 6KHUZRRG UHSRUWHG WKH SXULILFDWLRQ DQG FKDUDFWHUL]DWLRQ RI UDW UHOD[LQ 2YDULHV ZHUH KRPRJHQL]HG LQ D VDOLQH VROXWLRQ DQG WZR IRUPV RI UHOD[LQ ZHUH REWDLQHG DIWHU IUDFWLRQDWLRQ RI WKH FUXGH RYDULDQ H[WUDFW ZLWK 6HSKDGH[ JHO FKURPDWRJUDSK\ DQG &0& LRQ H[FKDQJH FKURPDWRJUDSK\ 7KH WZR IRUPV ZHUH GHVLJQDWHG DQG &0 DQG HDFK FRQWDLQHG FRPSDUDEOH VSHFLILF DFWLYLW\ ZKHQ DVVD\HG ZLWK WKH PRXVH LQWHUSXELF OLJDPHQW ELRn DVVD\ &0 DQG &0 KDG LVRHOHFWULF SRLQWV RI S+ DQG S+

PAGE 27

UHVSHFWLYHO\ DQG ERWK KDG UDZ RI DSSUR[LPDWHO\ 8QOLNH SLJ UHOD[LQ UDW UHOD[LQ FRQWDLQHG KLVWLGLQH SUROLQH DQG W\URVLQH $OVR DOWKRXJK JLYLQJ D OLQHDU ORJ GRVHUHVSRQVH FXUYH LQ WKH PRXVH LQWHUSXELF OLJDPHQW ELRDVVD\ WKH VORSH RI WKH OLQH ZDV QRW SDUDOOHO WR WKH DVVD\ VORSH RI WKH SXULILHG SLJ UHOD[LQ VWDQGDUG 7KHVH UHVXOWV VXSSRUWHG HDUOLHU ILQGLQJV E\ /DUNLQ f ZKR XVHG FUXGH SUHSDUDWLRQV IURP UDW DQG SLJ RYDULHV )LHOGV DQG /DUNLQ f DOVR UHSRUWHG RQ WKH LVRODWLRQ RI UDW RYDULDQ UHOD[LQ 7KH\ LVRODWHG D IUDFWLRQ IURP D %LR *HO 3 FROXPQ ZKLFK HOXWHG LQ WKH PZ UDQJH RI SRUFLQH UHOD[LQ DQG FRQWDLQHG D SRWHQF\ RI 8PJ LQ WKH PRXVH XWHUXV ELRDVVD\ (OHFWUR IRFXVLQJ RI WKH 6HSKDGH[ IUDFWLRQ \LHOGHG SHDNV ZLWK LVRHOHFWULF SRLQWV RI S+ DQG 7KHVH SHDNV KDG DFWLYLWLHV RI DQG 8PJ UHVSHFWLYHO\ :DOVK DQG 1LDOO f LVRODWHG RYDULDQ UHOD[LQ IURP SUHJQDQW UDWV XVLQJ WKH 2'6 WHFKQLTXH SUHYLRXVO\ PHQWLRQHG DQG REWDLQHG RQH PDMRU UHOD[LQ SHDN DIWHU SUHSDUDWLRQ RQ D &0& LRQ H[FKDQJH FKURPDWRJUDSK\ FROXPQ 1R LVRIRFXVLQJ GDWD ZHUH SUHVHQWHG IRU WKH UDW UHOD[LQ PROHFXOH LQ WKH :DOVK DQG 1LDOO f VWXG\ -RKQ HW DO f VWXGLHG WKH VHTXHQFH KRPRORJLHV EHWZHHQ UDW DQG SRUFLQH UHOD[LQV 7KH\ LVRODWHG UDW UHOD[LQ IURP ODWH SUHJQDQW UDW RYDULHV GD\V RI SUHJQDQF\f DFFRUGLQJ WR WKH WHFKQLTXH RI :DOVK DQG 1LDOO f $PLQR DFLG VHTXHQFLQJ VWXGLHV VKRZHG WKH DOSKD FKDLQ RI UDW UHOD[LQ WR EH UHVLGXHV ORQJ DQG WKH EHWD FKDLQ WR EH UHVLn GXHV ORQJ 7KH UDW UHOD[LQ PROHFXOH FRQWDLQHG W\URVLQH DQG KLVWLGLQH 2QO\ OLPLWHG KRPRORJ\ H[LVWHG EHWZHHQ UDW DQG SRUFLQH UHOD[LQ ZLWK DSSUR[LPDWHO\ b RI WKH DPLQR DFLG UHVLGXHV LQ FRUUHVSRQGLQJ SRVLWLRQV EHLQJ LGHQWLFDO $QWLJHQLF GLVVLPLODULWLHV EHWZHHQ WKH SRUFLQH DQG UDW

PAGE 28

UHOD[LQV ZHUH DOVR VKRZQ E\ WKH REVHUYDWLRQ WKDW RQO\ VOLJKW FURVVn UHDFWLYLW\ H[LVWHG EHWZHHQ DQWLVHUD SURGXFHG DJDLQVW SRUFLQH UHOD[LQ DQG UDW UHOD[LQ /DUNLQ HW DO )LHOGV DQG /DUNLQ 6KHUZRRG DQG &PHNRYLN f 5DEELW 7KH SODFHQWD RI WKH UDEELW KDG EHHQ UHSRUWHG WR FRQWDLQ UHOD[LQ E\ =DUURZ DQG 5RVHQEHUJ LQ 2I WKH WLVVXHV WHVWHG RYDU\ XWHUXV SODFHQWDf WKH PDWHUQDO SRUWLRQ RI WKH SODFHQWD VHHPHG WR FRQWDLQ WKH KLJKHVW OHYHOV RI ELRORJLFDOO\ DFWLYH UHOD[LQ /RZHU OHYHOV ZHUH VHHQ LQ WKH IHWDO SODFHQWD DQG XWHUXV )XUWKHU SURRI WKDW UDEELW SODFHQWDH FRQWDLQHG UHOD[LQ ZDV GHPRQVWUDWHG E\ /DUNLQ HW DO f ZKR VKRZHG WKDW DQWLVHUXP SURGXFHG DJDLQVW SRUFLQH UHOD[LQ LQKLELWHG WKH DFWLYLW\ RI UDEELW SODFHQWDO H[WUDFWV LQ WKH PRXVH XWHULQH PRWLOLW\ DVVD\ $OVR D UHDFWLRQ RI LGHQWLW\ ZDV REWDLQHG ZKHQ D %LR*HO 3 IUDFWLRQ RI UDEELW SODFHQWDO H[WUDFWV ZDV FRPSDUHG ZLWK SXULILHG SRUFLQH UHOD[LQ DQG DQ DQWLVHUXP PDGH DJDLQVW SRUFLQH UHOD[LQ LQ DQ DJDU GRXEOH LPPXQRn GLIIXVLRQ DVVD\ /DUNLQ HW DO f 7KLV SUHOLPLQDU\ ZRUN OHG )LHOGV HW DO f WR SXULI\ UHOD[LQ IURP WKH UDEELW SODFHQWD $IWHU H[WUDFWLRQ XVLQJ D PRGLILHG *ULVV PHWKRG *ULVV HW DO f VHSDUDWLRQ ZDV DFKLHYHG RQ D %LR*HO 3 FROXPQ 7KH %LR*HO 3 IUDFWLRQ HOXWLQJ DW GDOWRQV FRQWDLQHG ORZ ELRDFWLYLW\ LQ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ 8PJf :KHQ WKLV IUDFWLRQ ZDV FKURPDWRJUDSKHG LQ D &0& LRQ H[FKDQJH FROXPQ D VLQJOH SHDN FRQWDLQLQJ 8PJ ZDV HOXWHG 7KH PRXVH LQWHUSXELF OLJDn PHQW DVVD\ ZDV FRQGXFWHG RQ WKH &0& IUDFWLRQ 7KH GRVH UHVSRQVH FXUYH

PAGE 29

IRU UDEELW SODFHQWDO UHOD[LQ ZDV SDUDOOHO WR WKH GRVH UHVSRQVH FXUYH RI WKH SRUFLQH VWDQGDUG :LWK WKLV DVVD\ WKH &0& IUDFWLRQ ZDV FDOFXODWHG WR KDYH D ELRORJLFDO DFWLYLW\ RI 8PJ (OHFWURIRFXVLQJ RI WKH &0& SHDN UHVXOWHG LQ WKH VHSDUDWLRQ RI IRXU GLVWLQFW IUDFWLRQV +XPDQ :KHUHDV LW KDV EHHQ HVWDEOLVKHG WKDW WKH RYDU\ LV D VRXUFH RI UHOD[LQ LQ WKH SUHJQDQW KXPDQ :HLVV HW DO :HLVV HW DO 2n%\UQH HW DO 6]DOFKWHU HW DO f FKDUDFWHUL]DWLRQ RI UHOD[LQ IURP WKH RYDU\ KDV QRW EHHQ DFFRPSOLVKHG 2Q WKH RWKHU KDQG UHFHQW UHSRUWV RI LVRODWLRQ RI D SODFHQWDO UHOD[LQ KDYH EHHQ SXEOLVKHG )LHOGV DQG /DUNLQ
PAGE 30

VSXUULQJ LQ GRXEOH LPPXQRGLIIXVLRQ DQDO\VHV ZKHQ WHVWHG DJDLQVW SXULILHG SRUFLQH UHOD[LQ ,QFXEDWLRQ RI WKH KXPDQ UHOD[LQ ZLWK GLWKLRWKULHWRO LQDFWLYDWHG WKH KRUPRQH LQGLFDWLQJ WKDW GLVXOILGH ERQGV ZHUH QHFHVVDU\ IRU LWV ELRORJLFDO DFWLYLW\
PAGE 31

UHOD[LQ DQG KDG D WLVVXH OHYHO RI 8PJ RI IUHVK WLVVXH )XUWKHU SXULILFDWLRQ RI WKH H[WUDFW ZDV QRW UHSRUWHG 7KH UHOD[LQV VWXGLHG WR GDWH DSSHDU VLPLODU LQ WKDW WKH\ KDYH 66 OLQNDJHV DQG DQ DSSUR[LPDWH PZ RI 6RPH GLIIHUHQFHV KRZHYHU H[LVW DPRQJ SLJ UHOD[LQ DQG UHOD[LQV SXULILHG IURP RWKHU VSHFLHV f WKH SRUFLQH UHOD[LQ KDV WKH KLJKHVW VSHFLILF DFWLYLW\ LQ WKH ELRDVVD\V f WKH UHOD[LQV IURP YDULRXV VSHFLHV GLIIHU LQ LVRHOHFWULF SRLQWV f WKH WZR UHOD[LQV LQ ZKLFK DPLQR DFLG VHTXHQFLQJ KDV EHHQ GRQH DSSHDU WR EH GLIIHUHQW LH SRUFLQH UHOD[LQ FRQWDLQV QR W\URVLQH RU KLVWLGLQH ZKLOH UDW UHOD[LQ GRHV DQG f D ORZ PZ IRUP RI ERYLQH UHOD[LQ KDV EHHQ UHSRUWHG :KLOH QRW DOO UHOD[LQV KDYH EHHQ VWXGLHG ZLWK 5,$ WKH\ KDYH DOO EHHQ FKDUDFWHUL]HG XWLOL]LQJ ELRDVVD\ WHFKQLTXHV 5HOD[LQ LQ WKH *XLQHD 3LJ (DUO\ OLWHUDWXUH KDV VXJJHVWHG WKDW QRQRYDULDQ VRXUFHV PD\ EH YHU\ LPSRUWDQW LQ WKH SURGXFWLRQ RI UHOD[LQ LQ WKH JXLQHD SLJ +LVDZ f ZDV WKH ILUVW WR GLVFRYHU WKDW EORRG VHUXP IURP SUHJQDQW JXLQHD SLJV DQG UDEELWV ZKHQ LQMHFWHG VXEFXWDQHRXVO\ 6&f LQWR YLUJLQ JXLQHD SLJV GXULQJ HDUO\ SRVWHVWUXV FDXVHG SXELF V\PSK\VLV UHOD[DWLRQ VL[ KRXUV ODWHU +LVDZ HW DO f IXUWKHU GHPRQVWUDWHG WKDW WKH SXELF OLJDPHQWV RI FDVWUDWHG JXLQHD SLJV SUHWUHDWHG ZLWK HVWUDGLRO IRU GD\V FRXOG UHVSRQG WR D VLQJOH LQMHFWLRQ RI SURJHVWHURQH DQG H[KLELW LQFUHDVHG SHOYLF PRELOLW\ ZLWKLQ KRXUV &DVWUDWHG K\VWHUHFWRPL]HG DQG HVWURJHQWUHDWHG JXLQHD SLJV RQ WKH RWKHU KDQG GLG QRW UHVSRQG WR SURJHVWHURQH WUHDWPHQW UHJDUGOHVV RI WKH SURJHVWHURQH GRVH 7KHVH VDPH DQLPDOV FRXOG KRZHYHU UHVSRQG WR VPDOO TXDQWLWLHV RI UHOD[LQ ZLWKLQ

PAGE 32

VL[ KRXUV 7KHVH VWXGLHV LQGLFDWHG WKDW WKH HVWURJHQ SULPHG XWHUXV FRXOG EH LQGXFHG WR SURGXFH UHOD[LQ ZLWK SURJHVWHURQH WUHDWPHQW 7KH VWDWXV RI UHOD[LQ LQ WKH JXLQHD SLJ ZDV HTXLYRFDO EHFDXVH VRPH LQYHVWLJDWRUV REWDLQHG UHOD[DWLRQ RI WKH SHOYLV RI WKH JXLQHD SLJ E\ HVWURJHQ WKHUDS\ DORQH %URXKD f RU ZLWK FRPELQDWLRQV RI HVWURJHQ DQG SURJHVWHURQH )XJR f ,W VKRXOG EH SRLQWHG RXW WKDW LPSRUWDQW GLIIHUHQFHV H[LVWHG DPRQJ +LVDZnV REVHUYDWLRQV DQG WKRVH RI %URXKD DQG )XJR 7KH PRVW REYLRXV GLIIHUHQFH ZDV WKDW WKH WLPH UHTXLUHG WR SURGXFH D UHDFWLRQ LQ WKH UHOD[LQ WUHDWHG FDVWUDWHG DQLPDO SUHWUHDWHG ZLWK HVWURJHQ ZDV YHU\ VKRUW KUf 2Q WKH RWKHU KDQG HVWURJHQ DORQH GD\Vf RU FRPELQDWLRQV RI HVWURJHQ DQG SURJHVWHURQH GD\Vf WRRN PXFK ORQJHU WR HOLFLW WKHLU HIIHFW +LVDZnV HDUO\ ILQGLQJV ZHUH FRQILUPHG E\ =DUURZ f ZKR IRXQG ELRDVVD\DEOH UHOD[LQ LQ WKH EORRG RI JXLQHD SLJV GXULQJ PLGGOH DQG ODWH SUHJQDQF\ EXW QRW DIWHU SDUWXULWLRQ +H DOVR QRWHG UHOD[LQ DFWLYLW\ LQ H[WUDFWV RI WKH XWHUXV DQG SODFHQWDH RQ GD\V DQG RI SUHJQDQF\ 7KLV DGGHG FUHGHQFH WR +LVDZnV WKHRU\ WKDW WKH XWHUXV ZDV UHVSRQVLEOH IRU UHOD[LQ SURGXFWLRQ LQ WKH SUHJQDQW JXLQHD SLJ =DUURZ f IXUWKHU FRQILUPHG WKLV E\ VKRZLQJ WKDW SURJHVWHURQH FRXOG HOLFLW IRUPDn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

PAGE 33

5HFHQWO\ 2n%\UQH DQG 6WHLQHW] f DVVD\HG VHUD IURP SUHJQDQW JXLQHD SLJV DW GLIIHUHQW VWDJHV RI JHVWDWLRQ ZLWK 5,$ 7KH\ XVHG D KRPRORJRXV 5,$ HPSOR\LQJ DQWLERGLHV WR SRUFLQH UHOD[LQ ZKLFK ZDV DEOH WR GHWHFW DV OLWWOH DV QJ RI WKH JXLQHD SLJ UHOD[LQ 7KH\ IRXQG WKDW SHULSKHUDO EORRG OHYHOV RI UHOD[LQ JUDGXDOO\ LQFUHDVHG IURP DQ DYHUDJH RI OHVV WKDQ QJPO LQ WKH GD\ SUHJQDQW JXLQHD SLJV WR MXVW RYHU QJPO LQ WKH GD\ SUHJQDQW DQLPDOV 3RVWSDUWXP DQLPDOV KU DIWHU GHOLYHU\f VWLOO FRQWDLQHG KLJK UHOD[LQ OHYHOV DYHUDJH QJPOf 7KLV VWXG\ ZDV RQO\ FRQFHUQHG ZLWK RYHUDOO VHUXP OHYHOV RI LPPXQRUHDFWLYH UHOD[LQ DQG GLG QRW ORRN DW LQGLYLGXDO WLVVXH OHYHOV %LRDVVD\V ZHUH QRW FRQGXFWHG %R\G HW DO f XVHG D KRPRORJRXV SRUFLQH 5,$ WR DVVD\ SODVPD UHOD[LQ LPPXQRDFWLYLW\ LQ JXLQHD SLJV GXULQJ WKH HVWUXV F\FOH WKURXJKRXW PLG WR ODWH SUHJQDQF\ DQG SDUWXULWLRQ DQG GXULQJ ODFWDWLRQ $OWKRXJK YDULDELOLW\ DPRQJ DQLPDOV ZDV KLJK VHYHUDO PDMRU SRLQWV FRXOG EH GUDZQ IURP WKH VWXG\ f GXULQJ WKH HVWUXV F\FOH UHOD[LQ OHYHOV ZHUH ORZHVW GXULQJ HVWUXV QJPOf DQG KLJKHVW GXULQJ SRUWLRQV RI GLHVWUXV DQG SURHVWUXV QJPOf f GXULQJ WKH ODWWHU VWDJHV RI SUHJQDQF\ UHOD[LQ OHYHOV ZHUH KLJKHU QJPOf GHFUHDVLQJ WR EDVDO OHYHOV DIWHU SDUWXULWLRQ QJPOf DQG f GXULQJ ODFWDWLRQ VXFNOLQJ GLG QRW HOHYDWH UHOD[LQ OHYHOV LQ QXUVLQJ GDPV DQG LQ VRPH LQVWDQFHV DFWXDOO\ GHFUHDVHG WKHP ,Q VXPPDU\ ZRUN SUHYLRXV WR LQGLFDWHG WKDW UHOD[LQ ZDV SUHVHQW LQ WKH EORRG XWHUXV DQG SODFHQWD RI SUHJQDQW JXLQHD SLJV ,W DOVR VKRZHG WKDW SURJHVWHURQH VRPHKRZ VWLPXODWHG SURGXFWLRQ RI UHOD[LQ

PAGE 34

E\ WKH XWHUXV LQ HVWURJHQ SULPHG FDVWUDWHG JXLQHD SLJV 2QO\ UHFHQWO\ KDV 5,$ EHHQ HPSOR\HG WR GHWHFW WKH SUHVHQFH RI UHOD[LQ LQ VHUXP RI SUHJQDQW DQG F\FOLQJ JXLQHD SLJV ,Q WKH SDVW WKH JXLQHD SLJ ZDV XVHG H[WHQVLYHO\ DV DQ H[SHULPHQWDO DQLPDO LQ UHOD[LQ ZRUN 7KLV DQLPDO KRZHYHU KDV EHHQ QHJOHFWHG LQ UHFHQW UHVHDUFK GXH SRVVLEO\ WR VHYHUDO UHDVRQV f LQWHUHVW LQ JXLQHD SLJ UHOD[LQ GHFUHDVHG ZKHQ QHZHU IDVWHU DQG OHVV H[SHQVLYH ELRDVVD\ WHFKQLTXHV XVLQJ RWKHU DQLPDOV EHFDPH DYDLODEOH f FRUSRUD OWHD RI WKH SLJ EHFDPH HVWDEOLVKHG DV WKH PDLQ VRXUFH RI UHOD[LQ f FRVW RI NHHSLQJ JXLQHD SLJ FRORQLHV LQFUHDVHG FRPSDUHG WR RWKHU ODERUDWRU\ URGHQWV DQG f LQYHVWLJDWRUV IRFXVHG RQ WKH RYDU\ DV EHLQJ WKH RQO\ VRXUFH RI UHOD[LQ LQ PDQ\ PDPPDOV 7KHUH DUH RQ WKH RWKHU KDQG VHYHUDO FRPSHOOLQJ UHDVRQV WR VWXG\ UHOD[LQ LQ WKH JXLQHD SLJ 7KH JXLQHD SLJ LV TXLWH VLPLODU WR WKH KXPDQ LQ SODFHQWDWLRQ KRUPRQDO FKDQJHV ZKLFK RFFXU GXULQJ JHVWDWLRQ DQG WKH SUHVHQFH RI DQ H[WUD RYDULDQ VRXUFH RI UHOD[LQ =DUURZ 3DUGR HW DO f 6WDWHPHQW RI 3UREOHP 7KH SULPDU\ JRDO RI WKLV UHVHDUFK LV WR VWXG\ UHOD[LQ LQ WKH JXLQHD SLJ 6WXGLHV SURSRVHG DUH GHVLJQHG WR DQVZHU WKH IROORZLQJ TXHVWLRQV f ,V UHOD[LQ SURGXFHG E\ QRQRYDULDQ VRXUFHV LQ WKH JXLQHD SLJ" ,I VR ZKDW WLVVXH DQG FHOO W\SHV SURGXFH WKH KRUPRQH" f :KDW DUH WKH WLVVXH DQG VHUXP OHYHOV RI UHOD[LQ LQ WKH JXLQHD SLJ WKURXJKRXW SUHJQDQF\ DQG ODFWDWLRQ" f &DQ WKH ULVH DQG IDOO RI VHUXP DQG WLVVXH OHYHOV RI UHOD[LQ EH FRUUHODWHG ZLWK LPPXQRF\WRFKHPLFDO VWXGLHV" f :KDW DUH WKH SK\VLFDO DQG ELRFKHPLFDO FKDUDFWHULVWLFV RI WKH JXLH SLJ UHOD[LQ PROHFXOH"

PAGE 35

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n LQJ 1DQG ELODWHUDOO\ RYDULHFWRPL]HG WKURXJK WZR IODQN LQFLVLRQV 7ZR ZHHNV DIWHU WKH RSHUDWLRQ WKH DQLPDOV ZHUH VWDUWHG RQ D GDLO\ UHJLn PHQ RI KRUPRQH LQMHFWLRQV $QLPDOV ZHUH JLYHQ RQH RI WKH IROORZLQJ f HVWURJHQ DORQH \Jf f HVWURJHQ \Jf DQG SURJHVWHURQH PJf WRJHWKHU RU f QR LQMHFWLRQV 7KH KRUPRQHV ZHUH PL[HG LQ VHVDPH VHHG RLO DQG LQMHFWHG 6& DW WKH EDFN RI WKH QHFN (VWUDGLRO GLSURSLRQDWH ZDV REWDLQHG IURP &LED 3KDUPDFHXWLFDO 3URGXFWV ,QF 6XPPLW 13URn JHVWHURQH ZDV REWDLQHG IURP (OL /LOO\ DQG &R ,QGLDQDSROLV ,1 ,QMHFn WLRQV ZHUH JLYHQ GDLO\ DW DSSUR[LPDWHO\ $0 IRU GD\V WLPH QHHGHG IRU HVWURJHQSURJHVWHURQH WUHDWHG DQLPDOV WR XQGHUJR UHOD[DWLRQ RI WKH SHOYLF OLJDPHQWV =DUURZ ff 7ZR DQLPDOV ZHUH XVHG IRU HDFK RI WKH WKUHH WUHDWPHQWV DQG ZHUH PRQLWRUHG GDLO\ IRU SHOYLF IOH[LELOLW\ E\ PDQXDO SDOSDWLRQ

PAGE 36

$OO DQLPDOV ZHUH NLOOHG DW WKH VDPH WLPH RI WKH GD\ DP B KUf 7KH DQLPDOV ZHUH DQHVWKHWL]HG ZLWK SHQWREDUELWRO PJ J ERG\ ZHLJKWf DQG H[VDQJXLQDWHG YLD FDUGLDF SXQFWXUH 7KH UHSURGXFWLYH WUDFW ZDV UHPRYHG LPPHGLDWHO\ DQG SRUWLRQV RI WKH XWHUXV ZHUH IL[HG LQ %RXLQnV VROXWLRQ IRU KLVWRORJLF VWXG\ 7KLV WLVVXH ZDV SURFHVVHG IRU SDUDIILQ HPEHGGLQJ 7KH UHPDLQGHU RI WKH XWHUXV ZDV IUR]HQ DW r & DQG ODWHU XVHG LQ WKH H[WUDFWLRQ SURFHGXUH $QWLUHOD[LQ 6HUD $QWLVHUD DJDLQVW KLJKO\ SXULILHG SRUFLQH UHOD[LQ ZDV SURGXFHG LQ 1HZ =HDODQG ZKLWH UDEELWV DV GHVFULEHG E\ /DUNLQ HW DO f ,Q WKLV WHFKQLTXH PJ RI D SLJ UHOD[LQ SUHSDUDWLRQ :/ 8PJf REWDLQHG IURP :DUQHU /DPEHUW ,QF 0RUULV 3ODLQV 1ZHUH UXQ RQ 3$*( 7KH EDQGV ZHUH ORFDOL]HG E\ IL[LQJ WKHP LQ WULFKORURDFHWLF DFLG 7&$f bf DQG VWDLQLQJ LQ b &RRPDVVLH EOXH LQ b 7&$ 7KUHH EDQGV ZHUH SUHVHQW DQG ZHUH QDPHG &O & DQG & &O EHLQJ WKH FORVHVW WR WKH DQRGH 7KH & EDQGV ZHUH WKHQ FXW RXW RI WKH JHOV DQG KRPRJHQL]HG LQ DQ HTXDO YROXPH RI )UHXQGnV FRPSOHWH DGMXYDQW DQG LQMHFWHG LQWR 1HZ =HDODQG ZKLWH UDEELWV 6XEVHTXHQW LQMHFWLRQV ZHUH JLYHQ ZLWK WKH JHOV KRPRJHQL]HG LQ )UHXQGnV LQFRPSOHWH DGMXYDQW 7KH LQMHFWLRQ VFKHGXOH ZDV DV IROORZV 5DEELW ZDV JLYHQ RQH 6& LQMHFWLRQ SHU ZHHN IRU VL[ ZHHNV 7KH LQMHFWLRQV FRQn WDLQLQJ VL[& EDQGV ZHUH JLYHQ GRUVDOO\ EHWZHHQ WKH VFDSXODH %RRVWHU LQMHFWLRQV FRQVLVWLQJ RI VL[& EDQGV ZHUH JLYHQ DSSUR[LPDWHO\ HYHU\ WZR PRQWKV 5 DQWLVHUXP KDV EHHQ VKRZQ WR LQKLELW WKH ELRORJLFDO DFWLYLW\ RI SRUFLQH FRZ DQG UDEELW UHOD[LQV LQ YLWUR /DUNLQ HW DO f

PAGE 37

$OVR LW KDV EHHQ XVHG WR GHWHFW UHOD[LQ LPPXQRF\WRFKHPLFDOO\ LQ FHOOV RI WKH FRZ RYDU\ )LHOGV HW DO f DQG KXPDQ SODFHQWD )LHOGV DQG /DUNLQ f 7LVVXH ([WUDFWLRQ 3UHSDUDWLRQ RI FUXGH XWHULQH H[WUDFWV ZDV DFFRPSOLVKHG E\ XWLOL]LQJ RQH RI WZR PHWKRGV ,QLWLDOO\ WLVVXHV ZHUH H[WUDFWHG ZLWK WKH DFLG DFHWRQH SURFHGXUH RI *ULVV HW DO f 7KLV SURFHGXUH ZDV HPSOR\HG IRU WKH H[WUDFWLRQ RI XWHUL WDNHQ IURP LQGLYLGXDO DQLPDOV DQG WKH H[WUDFW ZDV XVHG IRU ELRDVVD\ DQG 5,$ H[SHULPHQWV 5HFHQWO\ D QHZ H[WUDFWLRQ SURFHGXUH IRU UHOD[LQ ZDV UHSRUWHG E\ :DOVK DQG 1LDOO f 7KLV QHZHU WHFKQLTXH ZDV HPSOR\HG WR H[WUDFW UHOD[LQ IURP XWHUL DQG WKH UHVXOWLQJ SUHSDUDWLRQV ZHUH XVHG LQ SXULILFDWLRQ DQG FKDUDFWHUL]DWLRQ VWXGLHV $ PRUH GHWDLOHG DFFRXQW RI WKHVH WHFKQLTXHV LV JLYHQ EHORZ *ULVV SURFHGXUHf§7KH H[WUDFWLRQ SURFHGXUH RI *ULVV HW DO f ZDV XVHG IRU H[WUDFWLRQ RI XWHUL XWLOL]HG LQ ELRDVVD\ DQG 5,$ H[SHULn PHQWV &ROG H[WUDFWLRQ VROXWLRQ DFHWRQHZDWHUK\GURFKORULF DFLG UDWLRf ZDV DGGHG WR PLQFHG IUR]HQ WLVVXHV DW D UDWLR RI POJf DQG KRPRJHQL]HG LQ D 6RUYDOO 2PQLPL[HU DW r & 7KH H[WUDFW ZDV LQFXEDWHG IRU KU DW r & DQG WKHQ FHQWULIXJHG DW 530nV r &f IRU PLQ LQ D %HFNPDQ -F FHQWULIXJH HTXLSSHG ZLWK D -$ URWRU )LYH YROXPHV RI DFHWRQH ZHUH DGGHG WR WKH VXSHUQDWDQW DQG WKH PL[n WXUH ZDV VWRUHG DW r & IRU KU 7KH PDMRULW\ RI WKH VXSHUQDWDQW ZDV GHFDQWHG DQG WKH SUHFLSLWDWH SHOOHWHG E\ FHQWULIXJDWLRQ DW 530nV IRU PLQ LQ D -$ URWRU DQG DLU GULHG 7KH GULHG SRZGHU ZDV ZHLJKHG DQG VWRUHG LQ D VHDOHG FRQWDLQHU DW URRP WHPSHUDWXUH

PAGE 38

:DOVK DQG 1LDOO SURFHGXUHf§7KH H[WUDFWLRQ SURFHGXUH RI :DOVK DQG 1LDOO f ZDV XWLOL]HG LQ WKH SXULILFDWLRQ DQG FKDUDFWHUL]DWLRQ VWDJHV RI WKH UHVHDUFK EHFDXVH LW KDG EHHQ UHSRUWHG WR \LHOG PRUH UHOD[LQ ZLWK OHVV SURWHRO\VLV 8WHUL ZHUH UHPRYHG IURP ODWH SUHJQDQW JXLQHD SLJV GD\Vf LPPHGLDWHO\ IUR]HQ LQ OLTXLG QLWURJHQ DQG VWRUHG DW r & LQ D 5HYHR IUHH]HU XQWLO H[WUDFWHG 7ZHQW\ JUDP DOLTXRWV RI PLQFHG IUR]HQ XWHUXV ZHUH SODFHG LQ PO RI D FROG VROXWLRQ RI b WULIOXRURDFHWLF DFLG b IRUPLF DFLG b 1D&O DQG 0 +& 7KH WLVVXH ZDV KRPRJHQL]HG IRU PLQ LQ D 6RUYDOO 2PQL PL[HU 7KH KRPRJHQn DWH ZDV FHQWULIXJHG r &f IRU PLQ DW 530nV LQ WKH -$ URWRU 7KH UHVXOWLQJ VXSHUQDWDQW ZDV ILOWHUHG WKURXJK :KDWPDQ ILOWHU SDSHU 1R f DQG D \P SRUH PLOOLSRUH ILOWHU 2FWDGHF\OVLOLFD FROXPQV SXUFKDVHG IURP :DWHUV $VVRFLDWHV 0LOOIRUG 0$ ZHUH SUHHTXLOLn EUDWHG E\ SDVVLQJ PO RI DQ b DFHWRQLWULOH b WULIOXRURDFHWLF DFLG VROXWLRQ IROORZHG E\ D PO ZDVK RI GLVWLOOHG ZDWHU 7KH UHOD[LQ FRQWDLQLQJ VXSHUQDWDQW ZDV SXPSHG WKURXJK WKUHH 2'6 FROXPQV WZLFH DQG WKH FROXPQV ZHUH ZDVKHG ZLWK PO RI D b DFHWRQLWULOH b WULn IOXRURDFHWLF DFLG VROXWLRQ 7KH HOXDWH ZDV HYDSRUDWHG WR QHDU GU\QHVV DW r & DQG UHVXVSHQGHG LQ D NQRZQ YROXPH RI 0 DPPRQLXP DFHWDWH EXIIHU S+ 'HWHFWLRQ RI 5HOD[LQ ,PPXQRF\WRFKHPLFDO /RFDOL]DWLRQ RI 5HOD[LQ ,PPXQRSHUR[LGDVH VWDLQLQJ ZDV FRQGXFWHG DV GHVFULEHG E\ 6WHPEHUJHU f DFFRUGLQJ WR WKH IROORZLQJ SURWRFRO $OO GLOXWLRQV RI DQWLVHUD ZHUH FDUULHG RXW ZLWK SKRVSKDWH EXIIHUHG VDOLQH 3%6f S+ DQG WKH LQFXEDWLRQV ZHUH FRQGXFWHG DW URRP WHPSHUDWXUH 3DUDIILQ VHFWLRQV

PAGE 39

SP LQ WKLFNQHVVf ZHUH GHSDUDIILQL]HG LPPHGLDWHO\ SULRU WR XVH 1RUPDO JRDW VHUXP GLOXWLRQf ZDV DSSOLHG WR WKH VHFWLRQV IRU PLQ 7KH VOLGHV ZHUH GUDLQHG EXW QRW ULQVHG DQG GURSV RI HLWKHU 5 DQWLVHUXP RU FRQWURO VROXWLRQV RI YDU\LQJ FRQFHQWUDWLRQV ZHUH DSSOLHG WR WKH VHFWLRQV IRU PLQ 7KH VOLGHV ZHUH ULQVHG ZLWK D VWUHDP RI 3%6 DQG SODFHG LQ &RSHODQG MDUV FRQWDLQLQJ 3%6 IRU WKUHH WKUHH PLQ ULQVHV 7KH VOLGHV ZHUH GUDLQHG RI H[FHVV 3%6 DQG EORWWHG WR DEVRUE H[FHVV 3%6 IURP DURXQG WKH VHFWLRQV )RXU GURSV RI JRDW DQWLUDEELW ,J* *$5f GLOXWLRQf SXUFKDVHG IURP 3RO\VFLHQFHV ,QF :DUULQJWRQ 3$ ZHUH DSSOLHG WR WKH VHFWLRQV IRU PLQ 7KH VHFWLRQV ZHUH ULQVHG GUDLQHG DQG EORWWHG DV GHVFULEHG SUHYLRXVO\ )RXU GURSV RI SHUR[LGDVHDQWLSHUR[LGDVH 3$3f GLOXWLRQ ZLWK 0 WULV VDOLQH S+ f SXUFKDVHG IURP 6WHP EHUJHU0H\HU ,PPXQRF\WRFKHPLFDOV -DUUHWVYLOOH 0' ZHUH DSSOLHG WR WKH VHFWLRQV IRU PLQ 7KH VHFWLRQV ZHUH ULQVHG GUDLQHG DQG EORWWHG DV GHVFULEHG SUHYLRXVO\ 7KH 3$3 ZDV YLVXDOL]HG E\ LQFXEDWLQJ WKH VOLGHV LQ D PJb '$% VROXWLRQ n GLDPLQREHQ]LGLQHf W\SH ,, SXUFKDVHG IURP 6LJPD 6W /RXLV 02 ZLWK b AA ARU PLQ 7KH VOLGHV ZHUH WKHQ ZDVKHG LQ GLVWLOOHG ZDWHU IRU PLQ EULHIO\ WUHDWHG ZLWK b A ULQVHG LQ GLVWLOOHG ZDWHU GHK\GUDWHG WKURXJK DOFRKROV DQG [\OHQH DQG FRYHUV OLSV ZHUH DSSOLHG 7KH IROORZLQJ FRQWUROV ZHUH FDUULHG RXW f VXEVWLWXWLRQ RI WKH 5 DQWLVHUXP ZLWK VHUXP IURP D PDOH UDEELW WKDW KDG QRW EHHQ LPPXQL]HG DJDLQVW UHOD[LQ 156f f RPLVVLRQ RI WKH 5 DQWLn VHUXP DQG UHSODFHPHQW ZLWK 3%6 S+ f f DEVRUSWLRQ RI WKH 5 DQWLn VHUXP ZLWK SRUFLQH UHOD[LQ VWDQGDUG 1,+5;13f DQG f VXFFHVVLYH GLOXWLRQV RI WKH 5 DQWLVHUXP

PAGE 40

%LRDVVD\ RI 5HOD[LQ &RQWDLQLQJ ([WUDFWV $OO PLFH XVHG LQ WKH ELRDVVD\V ZHUH IHPDOHV RI WKH ,&5 VWUDLQ ZKLFK ZHUH LQLWLDOO\ REWDLQHG IURP )ORZ /DERUDWRULHV 'XEOLQ 9$f 0RXVH XWHULQH PRWLOLW\7KH LQ YLWUR PRXVH XWHUXV ELRDVVD\ DV GHVFULEHG E\ .URF HW DO f DQG PRGLILHG E\ /DUNLQ HW DO f ZDV HPSOR\HG WR GHWHFW UHOD[LQ LQ WLVVXH H[WUDFWV )HPDOH PLFH Jf ZHUH SULPHG ZLWK PO RI HVWUDGLRO GLSURSLRQDWH \JPOf 7KH PLFH ZHUH NLOOHG RU GD\V ODWHU DQG WKHLU XWHUL UHPRYHG (DFK KRUQ RI WKH XWHUXV ZDV GLYLGHG LQ WR WZR SRUWLRQV DQG HDFK SRUWLRQ ZDV VXVSHQGHG LQ D WHVW WXEH FRQWDLQLQJ PO RI /RFNHnV VROXWLRQ DW r & 7KH XWHULQH VHJPHQWV ZHUH DWWDFKHG WR D KHDUW OHYHU DJDLQVW J WHQVLRQ DQG FRQn WUDFWLRQV ZHUH UHFRUGHG RQ DQ LQN ZULWLQJ N\PRJUDSK 6SHFLILF YROXPHV RI HLWKHU 1,+5;13 VWDQGDUG UHOD[LQ SUHSDUDWLRQ RU XQNQRZQ VROXWLRQV RI NQRZQ FRQFHQWUDWLRQV ZHUH DGGHG WR WKH WXEHV VR WKDW WKH EDWK FRQFHQn WUDWLRQV ZHUH GRXEOHG HYHU\ PLQ 6SHFLILF DFWLYLWLHV ZHUH FDOFXODWHG XVLQJ WKH IROORZLQJ HTXDWLRQ 6SHFLILF $FWLYLW\ RI 8QNQRZQ [ f§ [ A3A6 98 &8 ZKHUH 96 LV WKH YROXPH LQ \Of RI VWDQGDUG UHOD[LQ SUHSDUDWLRQ QHHGHG WR UHGXFH WKH XWHULQH FRQWUDFWLRQV E\ KDOI 98 LV WKH YROXPH LQ \Of RI XQNQRZQ UHOD[LQ SUHSDUDWLRQ QHHGHG WR UHGXFH WKH XWHULQH FRQWUDFWLRQV E\ KDOI &6 LV WKH FRQFHQWUDWLRQ RI WKH VWDQGDUG UHOD[LQ SUHSDUDWLRQ LQ \JPOf &8 LV WKH FRQFHQWUDWLRQ RI WKH XQNQRZQ UHOD[LQ SUHSDUDWLRQ LQ \JPOf DQG 6S$6 LV WKH VSHFLILF DFWLYLW\ RI WKH 1,+5;13 UHOD[LQ VWDQGn DUG 8PJ SURWHLQf 0RXVH LQWHUSXELF OLJDPHQW7KH LQ YLYR DVVD\ IRU UHOD[LQ DFWLYLW\ ZDV HPSOR\HG DFFRUGLQJ WR WKH WHFKQLTXH RI 6WHLQHW] HW DO f 7KH

PAGE 41

OHQJWK RI WKH LQWHUSXELF OLJDPHQW ZDV GHWHUPLQHG LQ D WKUHH SRLQW SDUDOOHO OLQH DVVD\ HPSOR\LQJ PLFH DW HDFK GRVH OHYHO RI WKH UHOD[LQ VWDQGDUG DQG PLFH DW HDFK GRVH OHYHO RI WKH XQNQRZQ $W GD\ YLUJLQ SUHSXEHUDO IHPDOH PLFH J ZHLJKWf ZHUH SULPHG ZLWK DQ 6& LQMHFWLRQ RI \J HVWUDGLRO F\SLRQDWH SXUFKDVHG IURP WKH 8SMRKQ &R .DODPD]RR 0, LQ PO RI VHVDPH VHHG RLO 2Q GD\ WKH UHOD[LQ VWDQGDUG 1,+5;13f DQG XQNQRZQV RI FRPn SDUDEOH OHYHOV RI DFWLYLW\ DV GHWHUPLQHG E\ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\f ZHUH LQMHFWHG 6& LQ PO RI D b VROXWLRQ RI EHQ]RSXUSXULQH % &RQWURO PLFH UHFHLYHG PO RI HVWUDGLRO F\SLRQDWH DQG PO b EHQ]RSXUSXULQH% 7KH GRVH OHYHOV IRU WKH 1,+5;13 VWDQGDUG ZHUH \J \J DQG \J SHU PRXVH 7KH GRVH OHYHOV RI WKH JXLQHD SLJ 6HSKDGH[ IUDFWLRQ ZHUH PJ PJ DQG PJ (LJKWHHQ WR WZHQW\IRXU KRXUV ODWHU WKH PLFH ZHUH NLOOHG LQ D &&A FKDPEHU WKH DEGRPn LQDO FDYLWLHV RSHQHG DQG WKH XWHUL H[DPLQHG IRU HYLGHQFH RI HVWURJHQ SULPn LQJ 1R PLFH H[KLELWHG WKUHDGOLNHf XWHUL GXH WR ODFN RI SULPLQJ 7KH DQDO DQG YXOYDO DUHDV DQG XSSHU KDOI RI WKH WUXQN ZHUH GLVVHFWHG DZD\ ZLWK VFLVVRUV WKHUHE\ UHPRYLQJ WKH VNLQ DQG DOO SHOYLF RUJDQV VXUURXQGn LQJ WKH SXELF V\PSK\VLV 7KH SHOYLV ZDV SRVLWLRQHG XQGHU D OLJKW VRXUFH DOORZLQJ D EHDP RI OLJKW WR SDVV WKURXJK WKH SXELF OLJDPHQW 7KH VKRUWn HVW GLVWDQFH EHWZHHQ WKH HGJHV RI WKH SXELF ERQHV ZDV PHDVXUHG ZLWK D GLVVHFWLQJ PLFURVFRSH ILWWHG ZLWK DQ RFFXODU PLFURPHWHU 5HVXOWV ZHUH HYDOXDWHG E\ WKH PHWKRG RI OHDVWVTXDUHV RI YDULDQFH WKH FRPSXWHU SURJUDP ZDV 352& &/0 RI 6WDWLVWLFDO $QDO\VLV 6\VWHP %DUU DQG *RRGQLJKW f 7KH PDWKHPDWLFDO PRGHO ZDV SUHSDUDWLRQ 1,+ YHUVXV JXLQHD SLJf

PAGE 42

DQG GRVH OHYHOVf 'RVH HIIHFWV ZHUH H[DPLQHG IXUWKHU E\ SRO\QRPLDO UHJUHVVLRQ 'LIIHUHQFHV LQ GRVHWUHQGV EHWZHHQ SUHSDUDWLRQV 1,+ YHUVXV JXLQHD SLJf ZHUH H[DPLQHG E\ WHVWV RI KHWHURJHQHLW\ RI UHJUHVVLRQ $ YDOLG DVVD\ LV RQH LQ ZKLFK WKHUH LV D VLJQLILFDQW 3!f OLQHDU UHJUHVVLRQ RI UHVSRQVH WR ORJ GRVH QR GLYHUJHQFH IURP SDUDOOHOLVP WR WKH 1,+5;13 VWDQGDUG QR TXDGUDWLF UHJUHVVLRQ FRPSRQHQWV DQG D ODPEGD YDOXH RI OHVV WKDQ 7KH UHVXOWV ZHUH H[SUHVVHG DV 8PJ UHODWLYH WR WKH 1,+5;13 SRUFLQH UHOD[LQ VWDQGDUG 5DGLRLPPXQRDVVD\ 7KUHH GLIIHUHQW LRGLQDWLRQ PHWKRGV ZHUH DWWHPSWHG LQ WKH GHYHOn RSPHQW RI WKH KRPRORJRXV SRUFLQH 5,$ IRU JXLQHD SLJ UHOD[LQ 6LQFH VXIILFLHQW DPRXQWV RI SXULILHG JXLQHD SLJ UHOD[LQ ZHUH QRW DYDLODEOH IRU WKH 5,$ H[SHULPHQWV LW EHFDPH QHFHVVDU\ WR HPSOR\ SRUFLQH UHOD[LQ IRU ERWK WKH LPPXQL]DWLRQ SURFHGXUH DQG IRU LRGLQDWLRQ 7KH ILUVW WZR SURFHGXUHV LQYROYHG WKH XVH RI WKH %ROWRQ DQG +XQWHU UHDJHQW IRU WKH LRGLQDWLRQ RI SRUFLQH UHOD[LQ 1,+5;13f %ROWRQ DQG +XQWHU f 7KHVH WZR SURFHGXUHV ZHUH QRW XWLOL]HG LQ WKH UHVHDUFK UHSRUWHG DQG VSHFLILF LQIRUPDWLRQ DERXW WKHVH DVVD\V ZLOO EH IRXQG LQ $SSHQGLFHV DQG ,Q WKH WKLUG SURFHGXUH SRO\W\URV\O UHOD[LQ ZDV LRGLQDWHG E\ WKH PHWKRG RI 2n%\UQH DQG 6WHLQHW] f ,RGLQDWLRQ RI SRO\W\URV\O UHOD[LQ7KH LRGLQDWLRQ ZDV FRQGXFWHG DFFRUGLQJ WR WKH WHFKQLTXH RI 2n%\UQH DQG 6WHLQHW] f ZLWK VRPH PRGLILFDWLRQV 3RO\W\URV\O UHOD[LQ \Jf GRQDWHG E\ 'U % 6WHLQHW] RI WKH &LED *HLJ\ &RUS $UGV OH\ 1< ZDV GLVVROYHG LQ \O RI 0 VRGLXP SKRVSKDWH EXIIHU S+ 7KH SRO\W\URV\O VROXWLRQ DQG P &L SXUFKDVHG IURP WKH $PHUVKDP &RUS ZHUH DGGHG WR D [ PP WHVW

PAGE 43

WXEH FRDWHG ZLWK \J GULHG LRGRJHQ ,RGLQDWLRQ ZDV DFKLHYHG XWLOn L]LQJ WKH WHFKQLTXH RI 0DUNZHOO DQG )R[ f ,RGRJHQ WHWUDFKORURGLSKHQ\OJO\FRXULOf ZDV SXUFKDVHG IURP WKH 3LHUFH &KHPLFDO &RUS 5RFNIRUG ,/ 7KH UHDFWLRQ VROXWLRQ ZDV PL[HG DW URRP WHPSHUDWXUH IRU PLQ ZLWK LQWHUPLWWHQW VKDNLQJ DQG WKHQ WUDQVIHUUHG WR D [ FP FROXPQ RI 6HSKDGH[ SUHHTXLOLEUDWHG ZLWK 0 VRGLXP SKRVSKDWH EXIIHU S+ )UDFWLRQV GURSVWXEHf IURP WKH JUDYLW\ IHG FROXPQ ZHUH FROOHFWHG LQ [ PP WXEHV FRQWDLQLQJ PO RI 3%6 b RYDOEXPLQ S+ 7HQ PLFUROLWHUV RI WKH SRROHG DVVD\ WXEHV RI WKH SRO\W\URV\O UHOD[LQ SHDN FRQWDLQHG FSP RI UDGLRDFWLYLW\ $SSUR[LPDWHO\ b RI WKH A,ODEHOOHG SRO\W\URV\O UHOD[LQ ZDV SUHFLSL WDEOH LQ DQWLERG\ H[FHVV 7KH ,ODEHOOHG SRO\W\URV\O UHOD[LQ ZDV XVHG IRU IRXU ZHHNV DIWHU LRGLQDWLRQ EHIRUH D QRWLFHDEOH GURS LQ VHQVLn WLYLW\ ZDV QRWLFHG LQ WKH 5,$ QU 'HYHORSPHQW RI 5,$ XWLOL]LQJ ,ODEHOOHG SRO\W\URV\O UHOD[LQ )UDFWLRQV FRQWDLQLQJ WKH ‘f‘fA,ODEHOOHG UHOD[LQ ZHUH SRROHG DQG HPSOR\HG LQ WKH GHYHORSPHQW RI WKH 5,$ XVHG WR GHWHFW JXLQHD SLJ UHOD[LQ )RU WKH GHWHFWLRQ RI UHOD[LQ LQ FUXGH XWHULQH H[WUDFWV PJ RI WKH DFLG DFHWRQH H[WUDFWHG SRZGHU ZDV VXVSHQGHG LQ PO RI 3%6b RYDOEXPLQ S+ DQG WKH UHVXOWLQJ VXVSHQVLRQ FHQWULIXJHG WR UHPRYH QRQVROXELOL]HG PDWHULDO 7KH VXSHUQDWDQW ZDV WKHQ GLOXWHG ZLWK 3%6b RYDOEXPLQ DQG WHVWHG LQ WKH 5,$ 'RXEOH DQWLERG\ 5,$V ZHUH FRQGXFWHG LQ [ PP GLVSRVDEOH JODVV FXOWXUH WXEHV 4XDQWLWLHV RI UHOD[LQ VWDQGDUG VROXWLRQV 1,+5;13f FRQWDLQLQJ SJ RI UHOD[LQ LQ 3%6b RYDOEXPLQ RU YROXPHV RI XWHULQH H[WUDFW VXSHUQDWDQWV ZHUH DGGHG WR WKH FXOWXUH WXEHV 6XIILFLHQW

PAGE 44

TXDQWLWLHV RI 3%6b RYDOEXPLQ ZHUH DGGHG WR HDFK WXEH WR EULQJ WKH YROXPH WR \O 2QH KXQGUHG PLFUROLWHUV RI 5 DQWLVHUXP ILQDO GLOXWLRQf LQ 0 HWK\OHQH GLDPLQH WHWUDDFHWLF DFLG3%6 FRQn WDLQLQJ b PDOH UDEELW VHUXP ZHUH DGGHG WR HDFK WXEH 7KH WXEHV ZHUH YRUWH[HG DQG WKHQ LQFXEDWHG DW r & IRU KU 2QH KXQGUHG PLFUROLWHUV RI ,ODEHOOHG UHOD[LQ &30f LQ 3%6b RYDOEXPLQ ZHUH DGGHG WR HDFK WXEH WKH WXEHV ZHUH YRUWH[HG DQG WKHQ LQFXEDWHG IRU KU DW r & 7KH WXEHV ZHUH WKHQ FHQWULIXJHG DW 530nV IRU PLQ GUDLQHG RI VXSHUQDWDQW DQG WKH SHOOHWV FRXQWHG LQ D 6HDUOH DQDO\WLF JDPPD FRXQWHU $ VWDQGDUG FXUYH HPSOR\LQJ 1,+5;13 UHOD[LQ ZDV UXQ FRQFXUUHQW ZLWK HYHU\ DVVD\ 5DGLRDFWLYLW\ H[SUHVVHG DV b ERXQG ZDV SORWWHG LQ D b ERXQG YHUVXV ORJ GLOXWLRQ FXUYH DQG XQNQRZQ JXLQHD SLJ YDOXHV ZHUH UHDG RII WKH VWDQGDUG FXUYHV DQG H[SUHVVHG DV SRUFLQH UHOD[LQ HTXLYDOHQWV 7KH IROORZLQJ FRQWUROV ZHUH HPSOR\HG f 7RWDO FRXQW WXEH L RU 7f \O RI LnL,ODEHOOHG SRO\W\URV\O UHOD[LQ JLYHV WKH WRWDO DPRXQW RI LVRWRSH DGGHG WR HDFK WXEH f 1RQVSHFLILF ELQGLQJ 16%f SULPDU\ DQWLVHUXP 5f ZDV RPLWWHG WR GHWHUPLQH ZKHWKHU WKHUH ZDV DQ\ QRQVSHFLILF ELQGLQJ RI WKH ,ODEHOOHG UHOD[LQ WR RWKHU DVVD\ FRPSRQHQWV f =HUR FRXQW WXEH %Rf UDGLRLQHUW UHOD[LQ ZDV QRW DGGHG WR GHWHUPLQH WKH PD[LPXP DPRXQW RI SRVVLEOH ELQGLQJ RI WKH -, ODEHOOHG UHOD[LQ WR WKH DQWLUHOD[LQ VHUXP 3HUFHQW ELQGLQJ b %f ZDV GHWHUPLQHG E\ GLYLGLQJ WKH UDGLRDFWLYLW\ RI WKH VWDQGDUG RU XQNQRZQ WXEHV ERXQGf E\ WKH ]HUR FRXQW WXEH %Rf 1RQVSHFLILF UDGLRDFWLYH ELQGLQJ ZDV VXEWUDFWHG IURP DOO YDOXHV EHIRUH FDOFXODWLRQV ZHUH PDGH R Q ERXQG16% n2 f§ %R16%

PAGE 45

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n LQJ D SJ VDPSOH RI SRUFLQH 1,+5;13 UHOD[LQ LQ WKH VDPH DVVD\ GLIIHUHQW WLPHV 3XULILFDWLRQ DQG &KDUDFWHUL]DWLRQ RI *XLQHD 3LJ 5HOD[LQ 3XULILFDWLRQ *HO ILOWUDWLRQf§7KH :DOVK DQG 1LDOO f SURFHGXUH ZDV XWLOL]HG WR H[WUDFW J RI XWHULQH WLVVXH IURP ODWH SUHJQDQW JXLQHD SLJV DQG WKH FUXGH H[WUDFW IURP WKH 2'6 H[WUDFWLRQ PJ SURWHLQf ZDV VXVSHQGHG LQ PO DPPRQLXP DFHWDWH EXIIHU S+ 3URWHLQ ZDV GHWHUPLQHG E\ WKH PHWKRG RI /RZU\ HW DO f 7KH SURWHLQ VROXWLRQ ZDV OD\HUHG RQ D 6HSKDGH[ ILQHf FROXPQ [ FPf SXUFKDVHG IURP 3KDUPDFLD )LQH &KHPLFDOV 8SSVDOD 6ZHGHQ DQG HTXLOLEUDWHG ZLWK WKH VDPH EXIIHU 7KH FROXPQ ZDV GHYHORSHG DW URRP WHPSHUDWXUH DW D UDWH RI POKU 0DWHULDO HOXWLQJ IURP WKH FROXPQV ZDV PRQLWRUHG DW PP ZDYHOHQJWK ZLWK D %HFNPDQ $FWD ,,, 6SHFWURSKRWRPHWHU )UDFWLRQV ZHUH FROOHFWHG HYHU\ PLQ )UDFWLRQV FRQWDLQLQJ WKH SURWHLQ SHDNV ZHUH

PAGE 46

SRROHG O\RSKLOL]HG DQG DVVD\HG XVLQJ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ &ROXPQV ZHUH FDOLEUDWHG E\ XVLQJ D VHULHV RI ORZ PZ PDUNHUV DQG E\ XVLQJ D SRUFLQH UHOD[LQ VWDQGDUG %XIIHU FRQWDLQLQJ VRGLXP D]LGH bf ZDV SXPSHG WKURXJK WKH FROXPQ EHWZHHQ H[SHULPHQWV WR HOLPLQDWH EDFWHULDO JURZWK %LR*HO 3 SXUFKDVHG IURP %LR5DG /DERUDWRULHV 5LFKPRQG &$ ZDV XWLOL]HG DV WKH JHO FKURPDWURJUDSK\ UHVLQ WR SUHSDUH WKH UHOD[LQ IUDFWLRQ XVHG LQ WKH GRXEOH LPPXQRGLIIXVLRQ H[SHULPHQWV 7KLV JHO ZDV HTXLOLEUDWHG DQG UXQ LQ H[DFWO\ WKH VDPH PDQQHU DV WKH 6HSKDGH[ UHVLQ ,RQ H[FKDQJH FKURPDWRJUDSK\7KH DFWLYH IUDFWLRQ IURP WKH 6HSKDGH[ FROXPQ PJf ZDV DSSOLHG WR D [ FP &0& FROXPQ &0f SXUFKDVHG IURP :KDWPDQ /WG 6SULQJILHOG (QJODQG DQG WKHQ HTXLOLEUDWHG ZLWK 0 DPPRQLXP DFHWDWH EXIIHU S+ XQWLO DOO XQDGVRUEHG PDWHULDO ZDV UHPRYHG 7KH FROXPQ ZDV GHYHORSHG DW D UDWH RI POKU ZLWK D OLQHDU 1D&O JUDGLHQW 0 WR 0f LQ 0 DPPRQLXP DFHWDWH EXIIHU S+ WR D ILQDO FRQGXFWLYLW\ RI P 0KR )UDFWLRQV POf ZHUH FROOHFWHG HYHU\ PLQ &KDUDFWHUL]DWLRQ 'RXEOH LPPXQRGLIIXVLRQ VWXGLHV'RXEOH LPPXQRGLIIXVLRQ SODWHV ZHUH HPSOR\HG DV GHVFULEHG E\ &ODXVHQ f IRU WKH PLFURWHFKQLTXH 3HWUL GLVKHV RI [ PP b DJDU LQ b VDOLQH ZLWK b VRGLXP D]LGHf ZHUH XVHG $ FHQWHU ZHOO ZDV ILOOHG ZLWK \O RI 5 DQWLVHUXP DQG SHULSKHUDO ZHOOV ZHUH ILOOHG ZLWK SL RI D %LR*HO 3 PZ IUDFWLRQ IURP JXLQHD SLJ XWHUXV PJPOf DQG 1,+5;13 SRUFLQH UHOD[LQ 7KH VXEVWDQFHV ZHUH DOORZHG WR GLIIXVH DW URRP WHPSHUDWXUH IRU KU

PAGE 47

7ZR GLPHQVLRQDO JHO HOHFWURSKRUHVLV7ZR GLPHQVLRQDO JHO HOHFWURn SKRUHVLV ZDV FRQGXFWHG XVLQJ D PRGLILFDWLRQ RI WKH +RUVW HW DO f WHFKQLTXH 7KH ILUVW GLPHQVLRQ HPSOR\HG D 1(3+*( QRQHTXLOLEULXP S+ JUDGLHQW HOHFWURSKRUHVLVf V\VWHP XVLQJ WXEHV FP ORQJ ZLWK DQ LQQHU GLDPHWHU RI PP 7KH 1(3+*( V\VWHP ZDV PRGLILHG WR DFFRPPRGDWH PRUH EDVLF SRO\SHSWLGHV DV GHVFULEHG E\ 6DQGHUV HW DO f UHVXOWLQJ LQ DQ HIIHFWLYH S+ JUDGLHQW RI WR 7KH ORZHU FKDPEHU RI WKH HOHFWURSKRUHVLV DSSDUDWXV ZDV ILOOHG ZLWK 0 1D2+ DQG WKH XSSHU FKDPEHU ZLWK +A62A 2QH KXQGUHG PLFURJUDPV RI HLWKHU JXLQHD SLJ &0& SXULILHG UHOD[LQ RU 1,+5;13 UHOD[LQ ZHUH OD\HUHG RQ WKH JHOV 7KH JHOV ZHUH DOORZHG WR VWDFN DW 9 IRU PLQ DQG WKHQ UXQ IRU KU DW 9 7KH SURWHLQV ZHUH VHSDUDWHG LQ WKH VHFRQG GLPHQVLRQ RQ b VRGLXP GRGHF\O VXOIDWH 6'6f SRO\DFU\ODPLGH VODE JHOV (OHFWURSKRUn HVLV EXIIHU J WULV J JO\FLQH DQG J SHU OLWHU 6'6f ZDV SODFHG LQ WKH UHVHUYRLUV DQG WKH JHOV ZHUH UXQ WRZDUG WKH DQRGH )LIWHHQ PDPS VODE ZHUH XVHG DV WKH VWDFNLQJ FXUUHQW IRU KU 7KH FXUUHQW ZDV WXUQHG XS WR PDPSVODE DQG WKH JHOV ZHUH UXQ IRU KU 7KH JHOV ZHUH IL[HG ZLWK b DFHWLF DFLGb HWK\O DOFRKRO DQG VWDLQHG ZLWK b &RRPDVVLH EOXH 5 7KH JHOV ZHUH GHVWDLQHG LQ b DFHWLF DFLGb HWK\O DOFRKRO 5HGXFWLRQ ZLWK GLWKLRWKULHWRO)LYH KXQGUHG PLFUROLWHUV RI DQ 2'6 FUXGHr UHOD[LQ IUDFWLRQ PJPO +2f IURP ODWH SUHJQDQW JXLQHD SLJ r'6 FUXGH UHOD[LQ LV D SDUWLDOO\ SXULILHG XWHULQH H[WUDFW WDNHQ DIWHU WKH LQLWLDO SXULILFDWLRQ VWHS LQ WKH 2'6 SURFHGXUH 7KLV H[WUDFW ZDV WHVWHG LQ WKH PRXVH XWHULQH PRWLOLW\ DVVD\ ZLWKRXW EHLQJ DOWHUHG FRQWUROf DQG DIWHU WKH DGGLWLRQ RI GLIIHUHQW DJHQWV H[SHULPHQWDOf $ UDWLR ZDV GHWHUPLQHG E\ GLYLGLQJ WKH ILQDO YROXPH RI WKH H[SHULPHQWDO E\ WKH ILQDO YROXPH RI WKH FRQWURO 7KH DVVD\V ZHUH UXQ WZLFH DQG DQ DYHUDJH YDOXH ZDV FRPSXWHG 7KH JUHDWHU WKH H[SHULPHQWDO WR FRQWURO UDWLR WKH JUHDWHU WKH DELOLW\ RI WKH DJHQW WR LQKLELW WKH DFWLRQ RI UHOD[LQ

PAGE 48

XWHUXV ZDV UHGXFHG E\ WKH DGGLWLRQ RI GLWKLRWKULHWRO ILQDO FRQFHQWUDn WLRQ 0f 7KH VROXWLRQ ZDV LQFXEDWHG IRU KU DW URRP WHPSHUDWXUH $ QRQWUHDWHG VDPSOH IURP WKH VDPH IUDFWLRQ ZDV WHVWHG DV D FRQWURO DQG SRWHQFLHV ZHUH FRPSDUHG XVLQJ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ +HDWLQJ2QH KXQGUHG PLFUROLWHUV RI DQ 2'6 FUXGH UHOD[LQ IUDFWLRQ PJPO +"f IURP ODWH SUHJQDQW JXLQHD SLJ XWHUXV ZDV KHDWHG DW r & IRU KU $ QRQWUHDWHG VDPSOH IURP WKH VDPH IUDFWLRQ ZDV WHVWHG DV D FRQWURO DQG SRWHQFLHV ZHUH FRPSDUHG XVLQJ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ 7U\SVLQ GLJHVWLRQ6HYHQ KXQGUHG PLFUROLWHUV RI DQ 2'6 FUXGH UHOD[LQ IUDFWLRQ PJPO +A2f IURP ODWH SUHJQDQW JXLQHD SLJ XWHUXV ZDV LQFXEDWHG ZLWK WKH WU\SVLQ DW D ILQDO FRQFHQWUDWLRQ RI PJ WU\SVLQPJ SURWHLQ 7KH VROXWLRQ ZDV LQFXEDWHG IRU KU DW URRP WHPSHUDWXUH S+ f $ QRQWUHDWHG VDPSOH IURP WKH VDPH IUDFWLRQ ZDV WHVWHG DV D FRQWURO DQG SRWHQFLHV ZHUH FRPSDUHG XVLQJ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ ,Q YLWUR DVVD\ RI DQWLVHUD /DUNLQ HW DO f)LIW\ PLFURn OLWHUV RI 5 DQWLVHUXP ZHUH DGGHG WR RQH RI WKH WXEHV LQ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ ZKLOH WKH RWKHU WXEH UHFHLYHG SL RI 156 DV D FRQWURO $Q 2'6 UHOD[LQ SUHSDUDWLRQ RI ODWH SUHJQDQW JXLQHD SLJ XWHUXV PJPO +Af ZDV WKHQ DGGHG WR HDFK WXEH LQ HTXDO FRQFHQWUDWLRQV DQG WKH YROXPHV UHTXLUHG WR LQKLELW WKH XWHULQH FRQWUDFWLRQV ZHUH FRPn SDUHG

PAGE 49

5(68/76 'HWHFWLRQ RI *XLQHD 3LJ 5HOD[LQ ,PPXQRF\WRFKHPLFDO /RFDOL]DWLRQ RI 8WHULQH 5HOD[LQ 7KH 3$3 LPPXQRF\WRFKHPLFDO WHFKQLTXH ZDV XVHG WR H[DPLQH WKH RYDU\ SODFHQWD XWHUXV VSOHHQ DQG OLYHU RI JXLQHD SLJV 7KH HQGRn PHWULDO JODQG FHOOV (*&f RI WKH XWHUXV ZDV WKH RQO\ FHOO W\SH WKDW VKRZHG KHDY\ GHSRVLWLRQ RI SHUR[LGDVH UHDFWLRQ SURGXFW 53f LQGLFDWLQJ WKH SUHVHQFH RI UHOD[LQ 7KH RYDU\ GHPRQVWUDWHG ZHDN VWDLQLQJ ZKLOH WKH OLYHU VSOHHQ DQG SODFHQWDH GLG QRW VWDLQ 7KHUHIRUH VXEVHTXHQW VWXGLHV HPSOR\HG WKH XWHUXV 7LVVXH VDPSOHV ZHUH WDNHQ IURP JXLQHD SLJV DW GLIIHUHQW VWDJHV RI SUHJQDQF\ GXULQJ ODFWDWLRQ DQG LQ RYDUL HFWRPL]HG DQLPDOV XQGHUJRLQJ HVWURJHQSURJHVWHURQH WUHDWPHQWV WR GHWHUn PLQH SHULRGV RI UHOD[LQ SURGXFWLRQ 5HOD[LQ ZDV QRW GHWHFWHG LQ VHFWLRQV RI XWHUL WDNHQ IURP QRQSUHJn QDQW RYDULHFWRPL]HG FRQWURO QR KRUPRQH WUHDWPHQW RU HVWURJHQ WUHDWHG DQLPDOVf RU GD\ SUHJQDQW DQLPDOV 'D\ ZDV WKH HDUOLHVW VWDJH RI SUHJQDQF\ VWXGLHG WKDW VKRZHG DFFXPXODWLRQ RI UHOD[LQ LQ WKH (*& $W WKLV VWDJH RQO\ D IHZ JODQGV FRQWDLQHG UHOD[LQ )LJXUH f &RQWURO 156 WUHDWHGf VHFWLRQV GLG QRW H[KLELW 53 )LJXUH f +HPDWR[\OLQ DQG HRVLQ + (f VWDLQHG VHFWLRQV YLHZHG DW KLJKHU PDJQLILFDWLRQ UHYHDOHG WKDW WKH (*& ZHUH ORZ FROXPQDU FHOOV ZLWK EDVDOO\ ORFDWHG QXFOHL DQG FRXOG EH HDVLO\ GLVWLQJXLVKHG IURP XWHULQH VXUIDFH HSLWKHOLXP 6(f )LJXUH f $ VHFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH VWDLQHG ZLWK 5 DQWLVHUXP DQG WKH 3$3 WHFKQLTXH GHPRQVWUDWHG ƒ

PAGE 50

HYHQ GHSRVLWLRQ RI 53 LQ WKH F\WRSODVP RI (*& ZLWK QR QXFOHDU VWDLQLQJ )LJXUH f 1RW DOO (*& ZLWKLQ D VLQJOH JODQG H[KLELWHG 53 )LJXUH f 6HFWLRQV RI XWHUL WDNHQ RQ GD\ RI SUHJQDQF\ VKRZHG WKDW D KLJKHU SHUFHQWDJH RI HQGRPHWULDO JODQGV (*f FRQWDLQHG UHOD[LQ WKDQ GLG GD\ WLVVXH )LJXUH f &RQWURO 156 WUHDWHGf VHFWLRQV GLG QRW H[KLELW 53 )LJXUH f 1R UHPDUNDEOH IHDWXUHV ZHUH QRWHG DW D KLJKHU PDJQLILFDWLRQ LQ + i ( VWDLQHG VHFWLRQV EH\RQG WKRVH PHQWLRQHG IRU WKH GD\ (*& )LJXUH f $ VHFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK 5 DQWLVHUXP DQG WKH 3$3 WHFKQLTXH VKRZHG 53 LQ PRVW EXW QRW DOO RI WKH (*& )LJXUH f 7KH (*& WKDW ZHUH VWDLQHG KDG 53 HYHQO\ GLVWULEXWHG WKURXJKRXW WKH F\WRSODVP )LJXUH f $OO (* LQ VHFWLRQV RI WKH GD\ XWHUL GHPRQVWUDWHG GHQVH DFFXPXn ODWLRQV RI 53 )LJXUH f &RQWURO 156 WUHDWHGf VHFWLRQV GLG QRW KDYH 53 )LJXUH f $W KLJKHU PDJQLILFDWLRQV + ( VWDLQHG (*& DSSHDUHG WR FRQWDLQ JUDQXODU DFFXPXODWLRQV RI DFLGRSKLOLF PDWHULDO LQ WKH OXPLQDO SRUWLRQV RI WKH F\WRSODVP )LJXUH f ,QGLYLGXDO JUDQXOHV FRXOG QRW EH FOHDUO\ UHVROYHG LQ WKHVH FHOOV DOWKRXJK XQGHU KLJKHU PDJQLILFDWLRQ VWUXFWXUHV UHVHPEOLQJ JUDQXOHV FRXOG EH GHWHFWHG LQ WKH OXPLQDO SRUWLRQ RI WKH FHOOV (OHFWURQ PLFURJUDSKV RI (*& IURP GD\ SUHJQDQW DQLPDOV VKRZHG GHQVH DFFXPXODWLRQV RI JUDQXOHV ORFDWHG LQ WKH DSLFDO DUHDV RI WKH FHOOV GDWD QRW VKRZQf $ VHFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK 5 DQWLVHUXP DQG WKH 3$3 WHFKQLTXH VKRZHG WKDW HYHU\ (*& LQ HYHU\ JODQG H[KLELWHG 53 )LJXUH f :KLOH WKH SDWWHUQ RI VWDLQLQJ YDULHG VRPHZKDW EHWZHHQ DQLPDOV WKH PRVW FKDUDFWHULVWLF IHDWXUH ZDV D GHQVH DFFXPXODWLRQ RI 53 LQ WKH DSLFDO SRUWLRQ RI WKH FHOOV ZLWK OLWWOH RU QR VWDLQ REVHUYHG LQ WKH EDVDO F\WRSODVPLF UHJLRQV

PAGE 51

$V LQ GD\ WLVVXH VHFWLRQV RI XWHUL WDNHQ IURP WKH ODWH SUHJn QDQW JURXS RI DQLPDOV UHYHDOHG WKDW DOO JODQGV JDYH D SRVLWLYH UHDFWLRQ IRU UHOD[LQ )LJXUH f &RQWURO 156 WUHDWHGf VHFWLRQV GLG QRW H[KLELW 53 ORFDOL]DWLRQ )LJXUH f +LJK PDJQLILFDWLRQ RI + i ( VWDLQHG WLVVXH VKRZHG OLWWOH GLIIHUHQFHV EHWZHHQ LQGLYLGXDO (*& )LJXUH f $ VHFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH VWDLQHG ZLWK 5 DQWLVHUXP DQG WKH 3$3 WHFKQLTXH VKRZHG D VWULNLQJ SDWWHUQ RI 53 GHSRVLWLRQ LQ VRPH (*& ZKLFK GLIIHUHG PDUNHGO\ IURP WKH GD\ WLVVXH )LJXUH f 6RPH FHOOV GHPRQVWUDWHG 53 WKURXJKRXW WKH F\WRSODVP 2WKHU FHOOV KDG D GHQVH DFFXPXODWLRQ RI ODEHOLQJ ORFDOL]HG LQ D MX[WDQXFOHDU UHJLRQ LQ WKH DSLFDO SRUWLRQ RI WKH (*& ZLWK D FRQVSLFXRXV DEVHQFH RI VWDLQ IURP WKH RWKHU DUHDV RI WKH FHOO F\WRSODVP )LJXUH f 6HFWLRQV RI XWHUL IURP WKH ODFWDWLQJ JURXS RI JXLQHD SLJV GHPRQn VWUDWHG D ORZ SHUFHQWDJH RI JODQGV WKDW JDYH D SRVLWLYH UHDFWLRQ IRU UHOD[LQ )LJXUH f &RQWURO 156 WUHDWHGf VHFWLRQV GLG QRW H[KLELW GHSRVLWLRQ RI 53 )LJXUH f +LJK PDJQLILFDWLRQ RI + i ( WUHDWHG VHFWLRQV VKRZHG WKH (*& WR EH WDOO FROXPQDU W\SH FHOOV ZLWK D ODUJH QXPEHU RI PLWRWLF ILJXUHV )LJXUH f )HZ HQGRPHWULDO JODQGV VKRZHG GHSRVLWLRQ RI 53 DQG WKRVH WKDW GLG KDG 53 HYHQO\ GLVWULEXWHG WKURXJKRXW WKH F\WRSODVP RI WKH FHOOV )LJXUH f 7KH JURXS RI DQLPDOV RYDULHFWRPL]HG DQG WUHDWHG ZLWK HVWUDGLRO DQG SURJHVWHURQH SURGXFHG SUHSDUDWLRQV WKDW PRVW UHVHPEOHG WLVVXH WDNHQ IURP DQLPDOV RQ GD\ RI SUHJQDQF\ :KHQ D VHFWLRQ RI XWHUXV IURP WKLV JURXS RI DQLPDOV ZDV WUHDWHG ZLWK 5 DQWLVHUXP DQG WKH 3$3 WHFKQLTXH WKH PDMRULW\ RI WKH (* JDYH D SRVLWLYH UHDFWLRQ IRU UHOD[LQ )LJXUH f

PAGE 52

&RQWURO 156 WUHDWHGf WLVVXH GLG QRW VKRZ GHSRVLWLRQ RI 53 )LJXUH f +LJK PDJQLILFDWLRQ RI + i ( WUHDWHG VHFWLRQV UHYHDOHG WKH (*& WR EH FXERLGDO FHOOV ZLWK IHZ GLVWLQJXLVKLQJ IHDWXUHV )LJXUH f $ VHFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK 5 VHUXP DQG WKH 3$3 WHFKQLTXH VKRZHG WKDW WKH GHSRVLWLRQ RI 53 LQ WKLV XWHUXV ZDV OHVV GHQVH WKDQ LQ JODQGV RI XWHUL WDNHQ IURP DQLPDOV LQ WKH ODWWHU VWDJHV RI SUHJQDQF\ )LJXUH f ,Q DOO VWDJHV VWXGLHG 53 ZDV IRXQG RQO\ LQ (*& WKDW LV QR 53 ZDV QRWHG LQ WKH XWHULQH VWURPD OXPLQDO HSLWKHOLXP RU XWHULQH FHUYLFDO JODQGV 7KH IROORZLQJ UHSUHVHQW UHVXOWV RI FRQWURO VWXGLHV XWLOL]HG LQ WKH 3$3 SURFHGXUH f LPPXQRSHUR[LGDVH ODEHOLQJ ZDV DEROLVKHG ZKHQ DQWLVHUXP 5 ZDV DEVRUEHG ZLWK SXULILHG SRUFLQH 1,+5;13f UHOD[LQ SULRU WR LQFXEDWLRQ ZLWK WLVVXH VHFWLRQV f VXFn FHVVLYH GLOXWLRQV f RI DQWLVHUXP 5 HYHQWXDOO\ DEROLVKHG WLVVXH LPPXQRODEHOLQJ DQG f VWDLQLQJ ZDV HOLPLQDWHG ZKHQ 156 ZDV VXEn VWLWXWHG IRU 5 *$5 RU 3$3 LQ WKH LPPXQRODEHOLQJ SURFHGXUH 2YHUDOO WKHVH UHVXOWV VXSSRUW HYLGHQFH REWDLQHG E\ RWKHUV WKDW WKH XWHUXV LV D VRXUFH RI UHOD[LQ LQ WKH JXLQHD SLJ %LRORJLFDOO\ $FWLYH 8WHULQH 5HOD[LQ 'XULQJ 3UHJQDQF\ DQG /DFWDWLRQ 7KH SUHFHGLQJ F\WRORJLFDO HYLGHQFH LV H[WHQGHG E\ ELRDVVD\V ZKLFK VKRZ WKDW ELRORJLFDOO\ DFWLYH UHOD[LQ LV RQO\ GHWHFWHG LQ WKH XWHUXV DQG WKDW WKH DFWLYLW\ SHDNV LQ WKH ODWHU VWDJHV RI SUHJQDQF\ 7DEOH )LJn XUHV DQG f 8WHUL IURP GD\ SUHJQDQW DQLPDOV FRQWDLQHG ORZ ELRn ORJLFDO DFWLYLW\ A XQLWV SHU JUDP ZHW ZHLJKW 8JZZf B 8 WRWDOf $W GD\ RI SUHJQDQF\ D VLJQLILFDQW LQFUHDVH LQ XWHULQH UHOD[LQ ELRORJLFDO DFWLYLW\ ZDV QRWHG B 8JZZ A 8 WRWDOf 8WHULQH UHOD[LQ OHYHOV IXUWKHU LQFUHDVHG DW GD\ RI SUHJQDQF\

PAGE 53

MA 8JZZ B 8 WRWDOf 8WHUL IURP ODWH SUHJQDQW DQLPDOV VKRZHG WKH KLJKHVW ELRORJLFDO DFWLYLW\ OHYHOV 8JZZ B 8 WRWDOf 5HOD[LQ OHYHOV GURSSHG LQ ODFWDWLQJ DQLPDOV B 8JZZ B 8 WRWDOf 6WDWLVWLFDO $QDO\VHV RI %LRDVVD\ 'DWD 7RWDO DFWLYLW\f§$ UHJUHVVLRQ DQDO\VLV RI WKH ELRDVVD\ GDWD 7DEOH )LJXUH f VKRZHG WKDW WRWDO ELRORJLFDO DFWLYLW\ RI WKH XWHULQH H[WUDFWV ZDV GLIIHUHQW RYHU WLPH RI SUHJQDQF\ ZLWK D KLJK OHYHO RI VLJQLILFDQFH Sf DQG D TXDGUDWLF FRPSRQHQW ZLWK D KLJK FRU UHODWLRQ FRHIILFLHQW U f $ 'XQFDQnV PXOWLSOH UDQJH WHVW IRU WRWDO ELRORJLFDO DFWLYLW\ YDOXHV VKRZHG WKH IROORZLQJ UHVXOWV DOSKD OHYHO f ,S B ODF 6WDJHV LQWHUFRQQHFWHG E\ EDUV DUH VWDWLVWLFDOO\ LQGLVWLQJXLVKDEOH IURP HDFK RWKHU DFFRUGLQJ WR WKH 'XQFDQnV PXOWLSOH UDQJH WHVW 6SHFLILF DFWLYLW\f§$ UHJUHVVLRQ DQDO\VHV RI ELRDVVD\ GDWD 7DEOH )LJXUH f VKRZHG WKDW VSHFLILF DFWLYLW\ 8JZZf RI WKH XWHULQH H[WUDFWV ZDV GLIIHUHQW RYHU WLPH RI SUHJQDQF\ ZLWK D KLJK OHYHO RI VLJQLILFDQFH Sf DQG D TXDGUDWLF FRPSRQHQW U f $ 'XQFDQnV PXOWLSOH UDQJH WHVW IRU VSHFLILF ELRORJLFDO DFWLYLW\ YDOXHV VKRZHG WKH IROORZLQJ UHVXOWV DOSKD OHYHO f OS e ODF &UXGH H[WUDFWV RI OLYHU DQG SODFHQWD RI ODWH SUHJQDQW JXLQHD SLJV VKRZHG QR UHOD[LQ DFWLYLW\ LQ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ ,Q DQ DWWHPSW WR ILQG VRPH UHOD[LQ DFWLYLW\ WKH SODFHQWDO H[WUDFW ZDV IXUWKHU SXULILHG E\ SDVVLQJ LW WKURXJK D %LR*HO 3 FROXPQ 7KH

PAGE 54

IUDFWLRQ HOXWLQJ LQ WKH PZ UDQJH FRQWDLQHG YHU\ ORZ DFWLYLW\ 8PJf GDWD QRW VKRZQf 7KH OLYHU H[WUDFW ZDV QRW SXULILHG RU WHVWHG IXUWKHU ,W VKRXOG EH HPSKDVL]HG WKDW QHLWKHU RI WKHVH WZR WLVVXHV H[KLELWHG DQ\ GHPRQVWUDEOH LPPXQRODEHOLQJ ZKHQ WHVWHG ZLWK 5 DQWLVHUXP DQG WKH 3$3 LPPXQRF\WRFKHPLFDO WHFKQLTXH 7KHUHIRUH WKH UHOD[LQ DFWLYLW\ LQ WKH SODFHQWDO H[WUDFW PD\ KDYH EHHQ WKH UHVXOW RI EORRG ERUQH UHOD[LQ 5DGLRLPPXQRDVVD\ RI 8WHULQH 5HOD[LQ 'XULQJ 3UHJQDQF\ DQG /DFWDWLRQ 5,$ FKDUDFWHUL]DWLRQ7KH LRGLQDWLRQ FXUYH )LJXUH f DQWLVHUXP WLWUDWLRQ FXUYH )LJXUH f DQG GLOXWLRQ FXUYHV )LJXUH f REWDLQHG ZLWK 1,+5;13 UHOD[LQ VXSSRUW WKH YDOLGLW\ RI WKH 5,$ HPSOR\HG LQ WKH SUHVHQW VWXG\ 7KH GLOXWLRQ FXUYHV RI WKH 1,+5;13 UHOD[LQ DQG WKH WZR JXLQHD SLJ UHOD[LQ SUHSDUDWLRQV ZHUH RI VLPLODU VORSHV )LJXUH f 7KHUHIRUH LW ZDV GHHPHG IHDVLEOH WR XWLOL]H 1,+5;13 IRU WKH GHYHORSn PHQW RI VWDQGDUG FXUYHV 7KH 5,$ GLG QRW GHWHFW UHOD[LQ LQ VHUXP RI SUHJQDQW JXLQHD SLJV 7KH LQWHUDVVD\ FRHIILFLHQW RI YDULDWLRQ IRU WKH 5,$ DVVD\Vf ZDV b DW SJ DQG WKH LQWUDDVVD\ FRHIILFLHQW RI YDULDWLRQ VDPSOHVf DW SJ ZDV b 7KH 5,$ ZDV FDSDEOH RI GHWHFWn LQJ OHYHOV RI UHOD[LQ WKDW UDQJHG IURP SJ WR SJ 5,$ RI XWHULQH H[WUDFWVf§ f 7RWDO DPRXQW RI LPPXQRUHDFWLYH UHOD[LQ SHU XWHUXV7KH 5,$ RI FUXGH H[WUDFWV VKRZHG WKDW XWHUL IURP GD\ SUHJQDQW JXLQHD SLJV 7DEOH )LJXUH f FRQWDLQHG WKH OHDVW DPRXQW RI UHOD[LQ B QJf 'DWD DUH H[SUHVVHG DV QDQRJUDP QJf SRUFLQH UHOD[LQ HTXLYDOHQWV $PRXQWV RI UHOD[LQ LQFUHDVHG LQ XWHULQH H[WUDFWV IURP GD\ SUHJQDQW DQLPDOV B QJf $W GD\ RI SUHJQDQF\ XWHULQH UHOD[LQ

PAGE 55

OHYHOV LQFUHDVHG WR B QJ DQG ZHUH KLJKHVW E\ GD\ RI SUHJQDQF\ B QJf %\ ODWH SUHJQDQF\ WRWDO XWHULQH UHOD[LQ OHYHOV KDG GHFUHDVHG WR B QJ DQG D ZLGH YDULDELOLW\ H[LVWHG EHWZHHQ DQLPDOV ,Q WKH ODFWDWLQJ DQLPDOV WKH XWHULQH UHOD[LQ OHYHOV KDG GHFUHDVHG WR A QJ $ UHJUHVVLRQ DQDO\VLV RI WKH 5,$ GDWD 7DEOH )LJXUH f VKRZHG WKDW WRWDO DPRXQWV RI LPPXQRUHDFWLYH UHOD[LQ ZHUH GLIIHUHQW Sf RYHU WLPH RI SUHJQDQF\ ZLWK D TXDGUDWLF FRP SRQHQW UA f $ 'XQFDQnV PXOWLSOH UDQJH WHVW IRU WRWDO UHOD[LQ LPPXQRDFWLYLW\ VKRZHG WKH IROORZLQJ UHVXOWV DOSKD OHYHO f OS ODF f &RQFHQWUDWLRQ RI LPPXQRUHDFWLYH UHOD[LQ8WHULQH H[WUDFWV IURP GD\ SUHJQDQW DQLPDOV )LJXUH f FRQWDLQHG ORZ FRQFHQWUDWLRQV RI UHOD[LQ QJJZZf ZKLFK LQFUHDVHG DW GD\ RI SUHJQDQF\ A QJJZZf 7KH KLJKHVW VSHFLILF DFWLYLW\ ZDV IRXQG LQ WKH XWHUL RI GD\ SUHJQDQW DQLPDOV B QJJZZf 8WHUL RI GD\ DQG ODWH SUHJQDQW DQLPDOV KDG ORZHU UHOD[LQ FRQFHQWUDWLRQV B QJJZZ DQG B QJJZZ UHVSHFWLYHO\f :KLOH XWHUL IURP ODFWDWLQJ DQLPDOV DJDLQ FRQWDLQHG YHU\ ORZ FRQFHQWUDWLRQV RI UHOD[LQ B QJJZZf $ UHJUHVVLRQ DQDO\VLV RI WKH 5,$ GDWD 7DEOH DQG )LJXUH f VKRZHG WKDW VSHFLILF UHOD[LQ LPPXQRDFWLYLW\ QJJZZf RI WKH FUXGH XWHULQH H[WUDFWV ZDV GLIIHUHQW Sf RYHU WLPH RI SUHJQDQF\ ZLWK D TXDG UDWLF FRPSRQHQW U f $ 'XQFDQnV PXOWLSOH UDQJH WHVW IRU VSHFLILF LPPXQRDFWLYLW\ YDOXHV VKRZHG WKH IROORZLQJ UHVXOWV DOSKD OHYHO f B Oe ODF

PAGE 56

3XULILFDWLRQ DQG &KDUDFWHUL]DWLRQ RI *XLQHD 3LJ 8WHULQH 5HOD[LQ 3XULILFDWLRQ 236 3URFHGXUH 8WHUL RI WKH ODWH SUHJQDQW JXLQHD SLJV ZHUH SXULILHG ZLWK WKH H[WUDFWLRQ SURFHGXUH RI :DOVK DQG 1LDOO f )LYH JXLQHD SLJV LQ WKH ODWH VWDJHV RI SUHJQDQF\ GD\V f ZHUH NLOOHG DQG J ZHW ZHLJKW RI XWHUL ZHUH XWLOL]HG 7DEOH f 7KH 2'6 H[WUDFWHG PDWHULDO PJf FRQWDLQHG ORZ EXW GHWHFWDEOH DFWLYLW\ LQ WKH PRXVH XWHULQH PRWLOLW\ DVVD\ 8PJf 7KH 2'6 PDWHULDO ZDV IUDFWLRQDWHG LQ D 6HSKDGH[ FROXPQ )LJXUH f DQG WKH PZ IUDFWLRQ KDG DQ DFWLYLW\ RI 8PJ 7KLV DFWLYH IUDFWLRQ IURP WKH 6HSKDGH[ FROXPQ PJf ZDV IXUWKHU FKURPDWRJUDSKHG LQ D &0& LRQ H[FKDQJH FROXPQ 7KH PRVW DFWLYH IUDFWLRQV IURP WKH &0& FROXPQ WXEHV f FRQWDLQHG PJ SURWHLQ KDG DQ DFWLYLW\ RI 8PJ DQG WKH SHDN SURWHLQ IUDFWLRQ WXEH f HOXWHG LQ WKH P 0KR FRQGXFWDQFH UDQJH )LJXUH f 6LPLODUO\ UXQ 1,+5;13 SRUFLQH UHOD[LQ HOXWHG LQ WKH P 0KR FRQGXFWDQFH UDQJH GDWD QRW VKRZQf 5,$ RI HYHU\ WHQWK WXEH RI WKH &0& FROXPQ UXQ VKRZHG WKDW LPPXQRUHDFWLYH UHOD[LQ ZDV SUHVHQW LQ WKH HOXDWH 7KH UHJLRQV FRQWDLQLQJ WKH KLJKHVW LPPXQRUHDFWLYH UHOD[LQ A QJPO2' WXEHV f DOVR H[KLELWHG ELRDFWLYLW\ )LJXUH f &KDUDFWHUL]DWLRQ 'RXEOH LPPXQRGLIIXVLRQ VWXGLHV$QDO\VHV RI D %LR*HO 3 UHOD[LQ IUDFWLRQ PJPOf IURP WKH JXLQHD SLJ XWHUXV GD\ RI SUHJQDQF\f WHVWHG DJDLQVW 5 DQWLVHUXP WR SRUFLQH UHOD[LQ DQG 1,+ 5;13 SRUFLQH UHOD[LQ VKRZHG D VLQJOH SUHFLSLWLQ OLQH ZLWK QR VSXUULQJ )LJXUH f

PAGE 57

7ZR GLPHQVLRQDO JHO HOHFWURSKRUHVLV&DUER[\PHWK\O FHOOXORVH SXULILHG JXLQHD SLJ UHOD[LQ WHVWHG LQ D WZR GLPHQVLRQDO JHO HOHFWURn SKRUHVLV V\VWHP VKRZHG WKDW JXLQHD SLJ UHOD[LQ PLJUDWHG LQ WKH VDPH PZ UDQJH DV GLG 1,+5;13 UHOD[LQ ZKLFK LV NQRZQ WR KDYH D PZ RI )LJXUH f $Q HTXLOLEULXP LVRHOHFWULF SRLQW RI D PROHFXOH FDQQRW EH UHVROYHG ZLWK 1(3+*( 1HYHUWKHOHVV LW ZDV DSSDUHQW WKDW WKH JXLQHD SLJ UHOD[LQ PROHFXOH GLG QRW PLJUDWH DV IDU LQ WKH ILUVW GLPHQVLRQ S+ f DV GLG 1,+5;13 SRUFLQH UHOD[LQ S+ f LQGLFDWLQJ WKDW WKH JXLQHD SLJ UHOD[LQ PROHFXOH KDG D ORZHU S+ WKDQ WKH SRUFLQH PROHFXOH )LJXUH f 7KLV REVHUYDWLRQ VXSSRUWHG GDWD IURP WKH &0& FROXPQ UXQ ZKLFK VKRZHG JXLQHD SLJ UHOD[LQ HOXWLQJ HDUOLHU P 0KR UDQJHf WKDQ 1,+5;13 SRUFLQH UHOD[LQ P 0KR UDQJH )LJXUH ff )LJXUH LV D UHSUHVHQWDWLRQ RI D VODE JHO PDGH XS RI D FRPSRVLWH RI WZR VHSDUDWHO\ UXQ JHOV 0RXVH LQWHUSXELF OLJDPHQW DVVD\$ 6HSKDGH[ PZ IUDFWLRQ IURP JXLQHD SLJ XWHULQH H[WUDFWV ZDV WHVWHG LQ WKH PRXVH LQWHUSXELF OLJDPHQW DVVD\ )LJXUH f $ SRVLWLYH UHVSRQVH ZDV REWDLQHG DV QRWHG E\ OLQHDU UHVSRQVH WR ORJGRVH RI WKH 6HSKDGH[ IUDFWLRQ 7KH GDWD LQGLFDWH D YDOLG DVVD\ DFFRUGLQJ WR WKH IROORZLQJ FULWHULD f SDUDOOHOn LVP H[LVWHG EHWZHHQ WKH JXLQHD SLJ UHOD[LQ IUDFWLRQ DQG WKH 1,+5;13 SRUFLQH UHOD[LQ VWDQGDUG DQG f D ODPEGD YDOXH VWDQGDUG HUURUVORSHf RI OHVV WKDQ ZDV REWDLQHG 7KH VSHFLILF DFWLYLW\ RI WKH 6HSKDGH[ IUDFWLRQ ZDV FDOFXODWHG WR EH 8PJ SURWHLQ 7KH PRXVH XWHULQH PRWLOn LW\ DVVD\ RI WKH VDPH IUDFWLRQ JDYH D ELRORJLFDO DFWLYLW\ RI 8PJ 7KH EHVW ILW FXUYH IRU WKH 1,+5;13 SRUFLQH UHOD[LQ ZDV \ ORJ [f ZLWK D VWDQGDUG HUURU 6(f DQG D ODPEGD YDOXH RI

PAGE 58

7KH JXLQHD SLJ &0& SXULILHG UHOD[LQ KDG D EHVW ILW FXUYH RI \ ORJ [f ZLWK DQ 6( DQG D ODPEGD YDOXH RI &RQWURO HVWURJHQ WUHDWHGf DQLPDOV KDG D PHDQ LQWHUSXELF OLJDPHQW OHQJWK RI ; 6(0f 3K\VLRFKHPLFDO FKDUDFWHULVWLFVf§7UHDWPHQW RI WKH 2'6 FUXGH H[WUDFW ZLWK GLWKLRWKULHWRO WU\SVLQ DQG 5 UHGXFHG LWV DELOLW\ WR LQKLELW XWHULQH FRQWUDFWLRQV 7DEOH f 7KHVH UHVXOWV LQGLFDWH WKDW WKH JXLQHD SLJ UHOD[LQ PROHFXOH GHSHQGV RQ LQWDFW GLVXOILGH OLQNDJHV DQG VWUXFWXUDO LQWHJULW\ IRU LWV ELRORJLFDO DFWLYLW\ $OVR EORFNLQJ WKH LPPXQRORJLFDOO\ DFWLYH VLWHV RI WKH UHOD[LQ PROHFXOH ZLWK DQWL UHOD[LQ DQWLERGLHV LQKLELWV WKH KRUPRQHnV ELRORJLFDO DFWLYLW\ +HDW r &f GLG QRW DGYHUVHO\ DIIHFW WKH JXLQHD SLJ UHOD[LQ PROHFXOH 'LWKLRWKULHWRO DQG WU\SVLQ WHVWHG E\ WKHPVHOYHV GLG QRW DOWHU WKH DPSOLn WXGH RU IUHTXHQF\ RI WKH FRQWUDFWLRQV

PAGE 59

',6&866,21 5 $QWLVHUXP 'HWHFWLRQ RI *XLQHD 3LJ 5HOD[LQ $ PDMRU SRUWLRQ RI WKLV GLVVHUWDWLRQ XWLOL]HG WHFKQLTXHV ZKLFK HPSOR\HG DQWLUHOD[LQ VHUXP 5f 5 ZDV SURGXFHG LQ UDEELWV DJDLQVW KLJKO\ SXULILHG SRUFLQH UHOD[LQ /DUNLQ HW DO f 7KLV DQWLVHUXP KDV EHHQ VKRZQ WR FURVVUHDFW ZLWK UHOD[LQ IURP GLIIHUHQW VSHFLHV LH FRZ )LHOGV HW DO f KXPDQ )LHOGV DQG /DUNLQ f DQG UDEELW )LHOGV HW DO f 5 DOVR KDV WKH DELOLW\ WR GHWHFW JXLQHD SLJ UHOD[LQ DV GHPRQVWUDWHG E\ WKH IROORZLQJ REVHUYDWLRQV IURP WKH FXUUHQW VWXG\ f 'RXEOH LPPXQRGLIIXVLRQ DJDU SODWH DVVD\V VKRZHG D UHDFWLRQ RI LGHQWLW\ ZKHQ SDUWLDOO\ SXULILHG XWHULQH UHOD[LQ IURP GD\ SUHJn QDQW JXLQHD SLJV ZDV WHVWHG DJDLQVW KLJKO\ SXULILHG SLJ UHOD[LQ DQG 5 f 5 ZDV VKRZQ WR EH HIIHFWLYH LQ LQKLELWLQJ WKH DFWLRQ RI JXLQHD SLJ UHOD[LQ XVLQJ WKH LQ YLWUR PRXVH XWHULQH PRWLOLW\ DQWLn VHUXP DVVD\ f 7KH LPPXQRSHUR[LGDVH ODEHOLQJ GDWD IURP GLIIHUHQW VWDJHV RI JHVWDWLRQ FRUUHODWHG ZHOO ZLWK ELRDVVD\ DQG UDGLRLPPXQRDVVD\ GDWD RI FUXGH XWHULQH H[WUDFWV IURP WKHVH VWDJHV WKDW LV WKH VWDJHV GHPRQVWUDWLQJ WKH JUHDWHVW GHJUHH RI ODEHOLQJ ZHUH DOVR WKH VWDJHV ZLWK WKH KLJKHVW UHOD[LQ OHYHOV GD\V RI SUHJQDQF\ DQG ODWH SUHJQDQW DQLPDOVf f $OO WKH FRQWUROV LQ WKH LPPXQRORFDOL]DWLRQ H[SHULPHQWV ZHUH QHJDWLYH LQFOXGLQJ WKH REVHUYDWLRQ WKDW LPPXQRSHUR[Ln GDVH ODEHOLQJ ZDV HOLPLQDWHG ZKHQ WKH 5 DQWLVHUXP ZDV DEVRUEHG ZLWK SXULILHG SRUFLQH UHOD[LQ VWDQGDUG 1,+5;13f SULRU WR WLVVXH LQFXEDWLRQ

PAGE 60

'HWHFWLRQ RI *XLQHD 3LJ 5HOD[LQ ZLWK WKH 3$3 7HFKQLTXH 7KH 5 DQWLVHUXP GLVFXVVHG LQ WKH SUHYLRXV VHFWLRQ ZDV HPSOR\HG LQ LPPXQRF\WRFKHPLFDO VWXGLHV RQ VHFWLRQV RI XWHUL IURP SUHJQDQW DQG ODFWDWLQJ JXLQHD SLJV DV ZHOO DV LQ XWHUL RI JXLQHD SLJV LQGXFHG WR XQGHUJR SHOYLF UHOD[DWLRQ ZLWK HVWURJHQ DQG SURJHVWHURQH WUHDWPHQWV 7KH LPPXQRF\WRFKHPLFDO VWXGLHV VKRZ DQ LQFUHDVH GXULQJ SUHJQDQF\ LQ 53 ORFDOL]HG LQ WKH (* 7KLV VXJJHVWV WKDW WKHUH LV DQ LQFUHDVH LQ WKH DFFXPXODWLRQ RI UHOD[LQ LQ WKHVH FHOOV VXSSRUWLQJ WKH FRQWHQWLRQ WKDW WKH (* DUH WKH VLWHV IRU UHOD[LQ SURGXFWLRQ 2I WKH LQWHUYDOV HYDOXn DWHG GD\ ZDV WKH ILUVW VWDJH RI SUHJQDQF\ ZKHUH D VPDOO DPRXQW RI LPPXQRSHUR[LGDVH ODEHOLQJ UHOD[LQf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n SDUWXPf UHOD[LQ ZDV DEVHQW IURP PRVW RI WKH (* DQG D KLJK LQFLGHQFH RI PLWRWLF ILJXUHV ZHUH QRWLFHG LQ WKH JODQGXODU HSLWKHOLXP 5HVXOWV IURP H[SHULPHQWV LQYROYLQJ QRQSUHJQDQW RYDULHFWRPL]HG KRUPRQH WUHDWHG DQLPDOV SURYLGH VWURQJ HYLGHQFH IRU D QRQRYDULDQ VRXUFH RI UHOD[LQ :KHQ RYDULHFWRPL]HG DQLPDOV ZHUH WUHDWHG ZLWK HVWURJHQ QR 53 ZDV VHHQ ORFDOL]HG RYHU WKH (*& :KHQ RYDULHFWRPL]HG DQLPDOV ZHUH WUHDWHG ZLWK HVWURJHQ DQG SURJHVWHURQH 53 ZDV VHHQ ORFDOL]HG RYHU

PAGE 61

WKH (*& 7KHVH UHVXOWV DJUHH ZLWK DQG H[WHQG WKH UHVXOWV RI =DUURZ f ZKR QRWHG WKDW K\VWHUHFWRPL]HG DQG RYDULHFWRPL]HG JXLQHD SLJV GLG QRW XQGHUJR SHOYLF UHOD[DWLRQ RU FRQWDLQ VHUXP UHOD[LQ ZKHQ WUHDWHG ZLWK D VLPLODU FRXUVH RI VWHURLG LQMHFWLRQV ,W LV QRW NQRZQ KRZ HVWURJHQ DQG SURJHVWHURQH LQGXFH WKH DFFXPXODWLRQ RU V\QWKHVLV RI UHOD[LQ LQ WKH (*& 'HWHFWLRQ RI *XLQHD 3LJ 5HOD[LQ ZLWK 5DGLRLPPXQRDVVD\ 7KHUH KDYH EHHQ QR SXEOLFDWLRQV WR GDWH WKDW UHSRUW RQ WKH SURGXFn WLRQ RI DQWLVHUD WR SXULILHG JXLQHD SLJ UHOD[LQ $OO VWXGLHV GHDOLQJ ZLWK UDGLRLPPXQRORJLFDO GHWHFWLRQ RI UHOD[LQ LQ WKH JXLQHD SLJ KDYH HPSOR\HG DQWLSRUFLQH UHOD[LQ VHUD 6KHUZRRG HW DO 2n%\UQH DQG 6WHLQHW] %U\DQW*UHHQZRRG DQG *UHHQZRRG %R\G HW DO 1DJDR DQG %U\DQW*UHHQZRRG f ,W VHHPV ORJLFDO WKDW GLIIHUHQFHV LQ WKH VSHFLILFLW\ DQG VHQVLWLYLW\ RI WKH YDULRXV DQWLVHUD FRXOG EH UHVSRQVLEOH IRU WKH ZLGH YDULDWLRQV LQ UHOD[LQ OHYHOV UHSRUWHG EHWZHHQ GLIIHUHQW ODERUDWRULHV ZKLFK KDYH VWXGLHG UHOD[LQ LQ WKH JXLQHD SLJ )RU H[DPSOH WKH SUHVHQW 5,$ HPSOR\LQJ 5 DQWLVHUXPf GHWHFWHG SRUFLQH UHOD[LQ DV ZHOO DV UHOD[LQ IURP JXLQHD SLJ XWHULQH H[WUDFWV EXW QRW JXLQHD SLJ VHUXP UHOD[LQ 7KLV DQWLVHUXP ZDV SURGXFHG LQ UDEELWV WR KLJKO\ SXULILHG SRUFLQH UHDO[LQ 6KHUZRRG HW DO f DOVR UHSRUWHG WKDW DQ DQWLVHUXP SURGXFHG LQ UDEELWV WR KLJKO\ SXULILHG SRUFLQH UHOD[LQ GHWHFWHG RQO\ SRUFLQH UHOD[LQ 7KHVH LQYHVWLJDWRUV ZHUH QRW DEOH WR GHWHFW UHOD[LQ LQ VHUD RI SUHJQDQW JXLQHD SLJV 7KLV LV LQ DJUHHPHQW ZLWK WKH SUHVHQW VWXG\ +RZHYHU 6KHUZRRG HW DO f GLG QRW UHSRUW DWWHPSWV WR GHWHFW UHOD[LQ LQ H[WUDFWV RI UHOD[LQ FRQWDLQLQJ WLVVXHV

PAGE 62

2n%\UQH DQG 6WHLQHW] f RQ WKH RWKHU KDQG GHPRQVWUDWHG WKDW DQL VHUXP 5 FURVVUHDFWHG ZLWK UHOD[LQ LQ VHUXP IURP D YDULHW\ RI SUHJQDQW DQLPDOV KXPDQV EDERRQV UKHVXV PRQNH\V GRJV FDWV JXLQHD SLJV UDWV DQG PLFH 7KH 5 DQWLVHUXP ZDV SURGXFHG LQ UDEELWV WR D UHOD[LQ IUDFWLRQ 6HSKDGH[ 8PJf 2n%\PH DQG 6WHLQHW] f GLG QRW SXEOLVK ELRDVVD\ UHVXOWV 7KH 5,$ HPSOR\HG LQ WKH SUHVHQW VWXGLHV DV ZHOO DV LQ WKH H[SHULPHQWV RI 6KHUZRRG HW DO f DQG 2n%\PH DQG 6WHLQHW] f XVHG SRO\W\URV\O UHOD[LQ ,W VHHPV WKDW WKH GLIIHUHQFH LQ LPPXQRORJLFDO VSHFLILFLW\ HQFRXQWHUHG E\ WKH WKUHH ODERUDWRULHV VWHPPHG QRW IURP WKH SRO\W\URV\O UHOD[LQ EXW IURP WKH XVH RI GLIIHUHQW DQWLVHUD RU SRVVLEO\ IURP WKH WHFKQLTXH HPSOR\HG 1DJDR DQG %U\DQW *UHHQZRRG f IRXQG WKDW DQ DQWLVHUXP SURGXFHG WR D UHODWLYHO\ LPSXUH SRUFLQH UHOD[LQ IUDFWLRQ 6HSKDGH[ FROXPQ FXWf DSSHDUHG FDSDEOH RI GHWHFWLQJ D JUHDWHU UDQJH RI UHOD[LQ LPPXQRDFWLYLW\ WKDQ DQ DQWLVHUXP SURGXFHG DJDLQVW D SXULILHG UHOD[LQ IUDFWLRQ SRUFLQH &0Dnf 3HUKDSV WKLV H[WHQGHG UDQJH LV GXH WR WKH DELOLW\ RI WKH PRUH LPSXUH DQWLVHUD WR FURVVUHDFW ZLWK PHWDEROLWHV RI UHOD[LQ RU ZLWK QRQUHOD[LQ FRPSRQHQWV RI VHUXP 7KLV LV QRW XQUHDVRQDEOH WR SURSRVH VLQFH LW KDV EHHQ VKRZQ E\ LQYHVWLJDWRUV ZRUNLQJ LQ 'U %U\DQW*UHHQZRRGnV ODERUDWRU\ $UDNDUL HW DO f WKDW WKH DQWLVHUXP WR LPSXUH UHOD[LQ UHFRJQL]HV FRQQHFWLYH WLVVXH HOHPHQWV LQ LPPXQRIOXRUHVFHQFH VWXGLHV LQYROYLQJ WKH SUHJQDQW VRZ RYDU\ 1DJDR DQG %U\DQW*UHHQZRRG f XWLOL]HG WKH DQWLVHUXP UDLVHG DJDLQVW &0Dn WR DVVD\ XWHULQH H[WUDFWV WDNHQ IURP JXLQHD SLJV LQ GLIIHUHQW VWDJHV RI JHVWDWLRQ 7KH KLJKHVW OHYHOV HQFRXQWHUHG E\ WKHVH LQYHVWLJDWRUV ZHUH RI DQ RUGHU RI PDJQLWXGH WR WLPHV JUHDWHU WKDQ

PAGE 63

WKH OHYHOV HQFRXQWHUHG LQ WKH SUHVHQW VWXG\ ,I WKH ELRDVVD\ OHYHOV RI WKH SUHVHQW VWXG\ DUH FRQYHUWHG WR QJ SRUFLQH UHOD[LQ HTXLYDOHQWVr WKH\ IDOO LQ WKH UDQJH RI UHOD[LQ OHYHOV IRXQG E\ 1DJDR DQG %U\DQW *UHHQZRRG f XWLOL]LQJ 5,$ ,W DSSHDUV WKDW WKH DQWLVHUXP XVHG E\ %U\DQW*UHHQZRRG GHWHFWHG JUHDWHU DPRXQWV RI UHOD[LQ WKDQ WKH 5 DQWLVHUXP XVHG LQ WKH SUHVHQW VWXG\ ,W VHHPV REYLRXV IURP WKHVH FRPn SDULVRQV WKDW D GLUHFW FRUUHODWLRQ FDQQRW EH PDGH EHWZHHQ 5,$ GDWD REWDLQHG IURP GLIIHUHQW ODERUDWRULHV LI GLIIHUHQW ODEHOLQJ WHFKQLTXHV DQG DQWLVHUD ZHUH XVHG ,W LV UHDVRQDEOH KRZHYHU WR FRPSDUH UHODWLYH GDWD IURP WKH VDPH V\VWHP LI WKH DSSURSULDWH FRQWUROV DUH FDUULHG RXW $Q LPSRUWDQW DVSHFW RI WKH SUHVHQW LQYHVWLJDWLRQ ZDV WKH FRUUHODn WLRQ RI 5,$ VWXGLHV ZLWK ELRDVVD\ DQG LPPXQRORJLFDO ORFDOL]DWLRQ VWXGLHV 6WDJHV RI SUHJQDQF\ ZKLFK GHPRQVWUDWHG KLJK OHYHOV RI ELRORJLFDOO\ DQG LPPXQRORJLFDOO\ DFWLYH UHOD[LQ LQ XWHULQH H[WUDFWV ZHUH DOVR WKH VWDJHV ZKLFK GLVSOD\HG LQFUHDVHG LPPXQRSHUR[LGDVH ODEHOLQJ LQ WKH XWHULQH WLVVXH VHFWLRQV $ FRPSDULVRQ RI UHOD[LQ OHYHOV GHWHFWHG ZLWK ELRDVVD\ DQG UDGLRLPPXQRDVVD\ DW WKH GLIIHUHQW VWDJHV RI SUHJQDQF\ UDLVHV DQ LQWHUHVWn LQJ SRLQW %LRDVVD\ RI FUXGH XWHULQH H[WUDFWV GHWHFWHG WKH KLJKHVW FRQn FHQWUDWLRQ RI UHOD[LQ LQ ODWH SUHJQDQW DQLPDOV 2Q WKH RWKHU KDQG FRQFHQWUDWLRQV RI LPPXQRUHDFWLYH UHOD[LQ ZHUH KLJKHVW LQ WKH GD\ SUHJQDQW DQLPDOV EXW GHFUHDVHG E\ GD\ RI SUHJQDQF\ DQG FRQWLQXHG WR GHFOLQH GXULQJ ODWH SUHJQDQF\ ,W VHHPV WKDW WKH ELRDVVD\ GHWHFWHG OHYHOV r7KLV FRPSDULVRQ LV PDGH E\ XVLQJ WKH IROORZLQJ FRQYHUVLRQ QJ HTXLYDOHQW UHOD[LQ ELRORJLFDO DFWLYLW\ 8QLWVf [ QJ RI SRUFLQH UHOD[LQ 8QLWV

PAGE 64

RI UHOD[LU WKDW ZHUH QRW GHWHFWHG E\ WKH 5,$ 2QH PD\ VSHFXODWH RQ WKH SRVVLELOLW\ WKDW DFFXPXODWLRQV RI UHOD[LQ SURUHOD[LQf WKDW DUH ELRORJLFDOO\ DFWLYH EXW QRW LPPXQRUHDFWLYH DUH SUHVHQW LQ WKRVH ODWWHU VWDJHV RI SUHJQDQF\ 7KH SUHVHQW VWXG\ GLG QRW UHSRUW RQ VHUXP UHOD[LQ OHYHOV 6HUXP VWXGLHV XQGHUWDNHQ LQ RWKHU ODERUDWRULHV KRZHYHU VKRZHG WKDW ELRORJLFDOO\ DFWLYH =DUURZ f DQG LPPXQRUHDFWLYH 2n%\UQH DQG 6WHLQHW] %R\G HW DO f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n WLRQ HPEU\R )LQQ f ,Q WKH PRXVH WKH HQG UHVXOW RI JODQGXODU GLIIHUHQWLDWLRQ LV WKH VHFUHWLRQ RI SHULRGLF DFLG 6FKLII SRVLWLYH PDWHULDO IURP WKH (* LQWR WKH XWHULQH OXPHQ DQG LW KDV EHHQ VKRZQ WKDW HVWURJHQ DQG SURJHVWHURQH WRJHWKHU FDQ LQGXFH XWHULQH JODQGXODU VHFUHn WLRQV )LQQ )LQQ DQG 0DUWLQ f ,Q WKH SLJ %D]HU f KDV VKRZQ WKDW D XWHULQH VSHFLILF SXUSOH SURWHLQ LV VHFUHWHG E\ WKH JODQGXODU HQGRPHWULDO HSLWKHOLXP WKURXJKRXW SUHJQDQF\ 0RVW DQLPDOV VWXGLHG KDYH EHHQ VKRZQ WR SURGXFH XWHULQH VSHFLILF SURWHLQV HVSHFLDOO\ GXULQJ WKH HDUO\ VWDJHV RI SUHJQDQF\ $LWNHQ f $QLPDOV ZLWK DQ

PAGE 65

HSLWKHOLRFKRULDO RU V\QGHVPRFKRULDO W\SH RI SODFHQWDWLRQ PD\ SURYLGH D VRXUFH RI QXWULWLRQ WR WKH GHYHORSLQJ HPEU\R WKURXJK WKH SURGXFWLRQ RI XWHULQH VSHFLILF SURWHLQV ZKLFK GLIIXVH WKURXJK WKH SODFHQWD $QLPDOV ZLWK D KHPRFKRULDO W\SH RI SODFHQWDWLRQ OLNH WKH KXPDQ DQG WKH JXLQHD SLJ GHULYH PRVW RI WKHLU HPEU\RQLF QXWULWLRQ IURP WKH PDWHUQDO EORRGn VWUHDP ,W KDV QHYHUWKHOHVV EHHQ VKRZQ WKDW DPQLRWLF IOXLG IURP KXPDQV LQ WKH VHFRQG WULPHVWHU RI SUHJQDQF\ FRQWDLQV XWHULQH VSHFLILF SURWHLQV 6XWFOLIIH HW DO f 7KH (* DQGRU WKH 6( DUH PRVW OLNHO\ DFWLYH GXULQJ SUHJQDQF\ DQG SURGXFH SURWHLQV ZKLFK SRVVLEO\ FRPH LQWR FRQWDFW ZLWK WKH HPEU\R DQG IHWXV 'LUHFW DQG LQGLUHFW HYLGHQFH IURP RWKHU ODERUDWRULHV KDV EHHQ DFFXPXODWHG ZKLFK LPSOLFDWHV WKH XWHUXV DV DQ LPSRUWDQW VRXUFH RI UHOD[LQ LQ WKH JXLQHD SLJ f )ULHGHQ DQG $GDPV f KDYH VKRZQ WKDW VRIWHQLQJ RI WKH SHOYLF OLJDPHQWV FDQ EH GHWHFWHG E\ SDOSDWLRQ DV HDUO\ DV PLGSUHJQDQF\ LQ WKH JXLQHD SLJ ZKLFK LV WKH DSSUR[LPDWH WLPH GD\ f ZKHQ DFFXPXODWLRQ RI UHOD[LQ ZDV LQLWLDOO\ GHWHFWHG LQ WKH (*& ZLWK LPPXQRSHUR[LGDVH ODEHOLQJ 5,$ DQG ELRDVVD\ f 3RUWHU f KDV VKRZQ WKDW D XWHULQH TXLHWLQJ VXEVWDQFH PRVW OLNHO\ UHOD[LQ LV SUHVHQW LQ WKH EORRG RI SUHJQDQW JXLQHD SLJV f =DUURZ f DQG 1DJDR DQG %U\DQW*UHHQZRRG f KDYH GHWHFWHG WKH SUHVHQFH RI UHOD[LQ LQ VHUD RI RYDULHFWRPL]HG HVWURJHQSURJHVWHURQH WUHDWHG JXLQHD SLJV f &DWFKSROH f KDV VKRZQ WKDW WKH JXLQHD SLJ LV DEOH WR SURFHHG WKURXJK D QRUPDO SUHJQDQF\ DQG SDUWXULWLRQ DIWHU RYDULHFWRP\ DV HDUO\ DV GD\ RI SUHJQDQF\ WKXV VWURQJO\ LQGLFDWLQJ D QRQRYDULDQ VRXUFH RI UHOD[LQ IRU WKLV VSHFLHV

PAGE 66

7KH XWHUXV RI WKH JXLQHD SLJ KDV EHHQ NQRZQ WR EH D VRXUFH RI UHOD[LQ IRU VRPH WLPH =DUURZ f 'D\ RI SUHJQDQF\ VHHPV WR EH WKH DSSUR[LPDWH WLPH ZKHQ PHDVXUDEOH OHYHOV RI UHOD[LQ ILUVW DSSHDU LQ WKH XWHUXV DQG EORRG RI JXLQHD SLJV 2n%\PH DQG 6WHLQHW] %R\G HW DO 1DJDR DQG %U\DQW*UHHQZRRG f 7KLV LV DOVR DSSUR[LPDWHO\ GD\V DIWHU WKH ILUVW GHWHFWDEOH ULVH LQ VHUXP HVWURJHQ DQG SURJHVWHURQH &KDOOLV HW DO f $V VKRZQ E\ =DUURZ f GD\V LV WKH WLPH UHTXLUHG IRU LQMHFWLRQV RI HVWURJHQ DQG SURJHVWHURQH WR HYRNH WKH V\QWKHVLV RI UHOD[LQ E\ WKH XWHUXV RI QRQn SUHJQDQW RYDULHFWRPL]HG JXLQHD SLJV 7KLV ODWHQF\ SHULRG DSSHDUV WR EH VKRUWHU WKDQ GD\V VLQFH E\ WKLV WLPH WUHDWPHQW ZLWK HVWURJHQ DQG SURJHVWHURQH KDG HYRNHG D FKDQJH LQ WKH LQWHUSXELF OLJDPHQW OHQJWK RI WKH JXLQHD SLJV 1DJDR DQG %U\DQW *UHHQZRRG f GHWHFWHG D ULVH LQ XWHULQH UHOD[LQ GD\V DIWHU RYDULn HFWRPL]HG JXLQHD SLJV ZHUH SULPHG ZLWK HVWURJHQ DQG SURJHVWHURQH +RZHYHU QR RQH KDV UHSRUWHG D V\VWHPDWLF VWXG\ WR GHWHUPLQH ZKHQ UHOD[LQ LV ILUVW SURGXFHG E\ WKH XWHUXV IROORZLQJ HVWURJHQ DQG SURJHVWHURQH VWLPXODWLRQ $XWRUDGLRJUDSKLF HYLGHQFH LQGLFDWHV WKDW HVWURJHQ SULPHG RYDULn HFWRPL]HG JXLQHD SLJV FRQWDLQ SURJHVWHURQH UHFHSWRUV LQ WKH HQGRPHWULDO JODQGV DV HYLGHQFHG E\ DFFXPXODWLRQ RI + SURJHVWHURQH LQ WKH F\WRSODVP DQG QXFOHL RI WKH (*& 6WXPSI 6DU DQG 6WXPSI :DUHPERXUJ f 7KLV LV FRQVLVWHQW ZLWK WKH FRQWHQWLRQ WKDW WKH (* DUH D WDUJHW WLVVXH IRU WKH VWHURLG KRUPRQHVDQG DOO HYLGHQFH LQGLFDWHV WKDW HVWURJHQ DQG SURJHVWHURQH DUH QHFHVVDU\ IRU UHOD[LQ V\QWKHVLV E\ WKH (*

PAGE 67

3RVVLEOH $FWLRQV RI 8WHULQH 5HOD[LQ LQ WKH *XLQHD 3LJ $V HYLGHQFH DFFXPXODWHV WKDW WKH FRUSXV OXWHXP RI WKH SUHJQDQW IHPDOH LV QRW WKH RQO\ VRXUFH RI UHOD[LQ LW EHFRPHV DSSDUHQW WKDW UHOD[LQ PD\ EH SURGXFHG E\ RWKHU WLVVXHV DQG DFW ORFDOO\ DV ZHOO DV V\VWHPLFDOO\ 3HUKDSV WKLV LV EHVW LOOXVWUDWHG E\ WKH JXLQHD SLJ ZKLFK PD\ KDYH DQ RYDULDQ VRXUFH RI UHOD[LQ 1DJDR DQG %U\DQW*UHHQZRRG f EXW DOVR FRQWDLQV D XWHULQH VRXUFH DV ZHOO 5HOD[LQ SURGXFHG E\ WKH XWHUXV RI WKLV DQLPDO PD\ KDYH V\VWHPLF DV ZHOO DV ORFDO HIIHFWV 5HOD[LQ KDV EHHQ VKRZQ WR EH SUHVHQW LQ WKH V\VWHPLF EORRG RI SUHJQDQW JXLQHD SLJV ZLWK ELRDVVD\ =DUURZ f DQG 5,$ 2n%\UQH DQG 6WHLQHW] %R\G HW DO f $OVR =DUURZ f KDV VKRZQ WKDW UHOD[LQ LV SUHVHQW LQ V\VWHPLF EORRG RI RYDULHFWRPL]HGHVWURJHQ DQG SURJHVWHURQH WUHDWHG JXLQHD SLJV (IIHFWV VXFK DV FKDQJHV ZKLFK RFFXU LQ WKH LQWHU SXELF OLJDPHQW SULRU WR SDUWXULWLRQ LQ PDPPDU\ JODQG GHYHORSPHQW DV ZHOO DV LQ WKH PDLQWHQDQFH RI XWHULQH TXLHVFHQFH FRXOG EH SURSRVHG DV SRVVLEOH V\VWHPLF HIIHFWV RI UHOD[LQ $Q LQFUHDVLQJ ERG\ RI HYLGHQFH RQ WKH RWKHU KDQG LQGLFDWHV WKDW UHOD[LQ PD\ DFW DV D ORFDO KRUPRQH LQ WKH JXLQHD SLJ ,PPXQRORFDOL]DWLRQ VWXGLHV IURP WKLV ODERUDWRU\ LOOXVWUDWH D GLVWLQFW VWDLQLQJ SDWWHUQ LQ VRPH RI WKH (*& LQ XWHULQH WLVVXH WDNHQ IURP JXLQHD SLJV LQ WKH ODWWHU VWDJHV RI SUHJQDQF\ ,Q GD\ DQG ODWH SUHJQDQW DQLPDOV 53 ZDV ORFDOL]HG DSLFDOO\ LQ VRPH (*& LH LPPXQRODEHOLQJ ZDV QRW VHHQ LQ WKH EDVDO DUHDV RI WKH F\WRSODVP LQ WKHVH FHOOV $OVR HOHFWURQ PLFURJUDSKV RI (* WDNHQ IURP WKH VDPH VWDJHV VKRZHG D ODUJH QXPEHU RI DSLFDO JUDQn XOHV 7KHVH REVHUYDWLRQV PD\ VXJJHVW WKDW UHOD[LQ LV EHLQJ UHOHDVHG LQWR WKH XWHULQH OXPHQ RI WKH HQGRPHWULDO JODQGV 5HOD[LQ V\QWKHVL]HG

PAGE 68

DQG UHOHDVHG IURP WKH (* LQWR WKH XWHULQH OXPHQ FRXOG f +DYH IUHH DFFHVV WR IHWDOPDWHUQDO WLVVXHV GXULQJ SUHJQDQF\ +DUNQHVV DQG +DUN QHVV f KDYH VKRZQ WKDW WKH WHQVLOH VWUHQJWK RI UDW IHWDO PHPEUDQHV LV JUHDWO\ UHGXFHG GXULQJ WKH ELUWK SURFHVV 5HOD[LQ PD\ EH UHVSRQVLEOH IRU WKLV SKHQRPHQRP f 0DLQWDLQ XWHULQH TXLHVFHQFH GXULQJ SUHJQDQF\ $OWKRXJK WKHUH LV QR GLUHFW HYLGHQFH IRU WKLV DVVXPSn WLRQ WKH SUR[LPLW\ RI WKH XWHULQH HQGRPHWULXP WR WKH P\RPHWULXP FRXOG SRVVLEO\ DOORZ IRU ORFDO GLIIXVLRQ RI UHOD[LQ WR RFFXU 3RUWHU f KDV VKRZQ WKDW UHOD[LQ LV PRVW OLNHO\ WKH KRUPRQH UHVSRQVLEOH IRU NHHSLQJ WKH XWHUXV TXLHVFHQW LQ WKH JXLQHD SLJ GXULQJ SUHJQDQF\ f (IIHFW FHUYLFDO VRIWHQLQJ DW WHUP 0DF/HQQDQ HW DO f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f VXJJHVWHG DQ HQGRFULQHH[RFULQH PHFKDQLVP IRU WKH UHOHDVH RI SURVWDJODQGLQ ) DOSKD

PAGE 69

3*) DOSKDf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n%\UQH )LHOGV DQG /DUNLQ 6KHUZRRG )LHOGV DQG /DUNLQ )LHOGV HW DO 5HLQLJ HW DO f DFFRUGLQJ WR WKH IROORZLQJ FULWHULD f PZ RI DSSUR[LPDWHO\ f EDVLF LVRHOHFWULF SRLQW f DELOLW\ WR LQKLELW XWHULQH FRQWUDFWLRQV PRXVH XWHULQH PRWLOLW\ ELRDVVD\f f DELOLW\ WR LQGXFH LQWHUSXELF OLJDPHQW IRUPDWLRQ LQ HVWURJHQ SULPHG PLFH PRXVH LQWHUSXELF OLJDPHQW DVVD\f f VXVFHSWLELOLW\ WR HQ]\PH GLJHVWLRQ ZLWK WU\SVLQ DQG WR WKH VWURQJ UHGXFLQJ DJHQW GLWKLR WKULHWRO LQGLFDWLQJ LWV SURWHLQDFHRXV QDWXUH DQG LWV UHOLDQFH RQ GLVXOILGH ERQGV IRU LWV ELRORJLFDO DFWLYLW\ DQG f UHVLVWDQFH WR PRGHUDWH KHDW *XLQHD SLJ UHOD[LQ ZDV DQWLJHQLFDOO\ VLPLODU WR SRUFLQH UHOD[LQ LQ WKDW f D UHDFWLRQ RI LGHQWLW\ ZDV REWDLQHG ZKHQ DQ H[WUDFW RI JXLQHD SLJ XWHUXV IURP D GD\ SUHJQDQW DQLPDO ZDV UHDFWHG DJDLQVW DQWLVHUXP WR SXULILHG SRUFLQH UHOD[LQ DQG SRUFLQH UHOD[LQ 1,+5;13f f DQ DQWLVHUXP SURGXFHG DJDLQVW SRUFLQH UHOD[LQ LQKLELWHG WKH DELOLW\ RI JXLQHD SLJ H[WUDFWV WR UHWDUG VSRQWDQHRXV XWHULQH FRQWUDFWLRQV f SDUDOOHO GLOXWLRQ FXUYHV ZHUH REWDLQHG EHWZHHQ UHOD[LQ FRQWDLQLQJ XWHULQH H[WUDFWV RI ODWH SUHJQDQW JXLQHD SLJV DQG SRUFLQH UHOD[LQ

PAGE 70

1,+5;13f LQ D KRPRORJRXV SRUFLQH UHOD[LQ 5,$ DQG f WKH ELRORJLFn DOO\ DFWLYH JXLQHD SLJ UHOD[LQ &0& SHDN )LJXUH f ZDV DOVR LPPXQRn ORJLFDO O\ DFWLYH LQ WKH KRPRORJRXV SRUFLQH 5,$ *XLQHD SLJ UHOD[LQ GLVSOD\HG D YHU\ ORZ VSHFLILF ELRORJLFDO DFWLYLW\ ZKHQ FRPSDUHG WR SRUFLQH UHOD[LQ 7KH SUHVHQW VWXGLHV FRQILUPHG SUHOLPLQDU\ ZRUN GRQH E\ 3DUGR HW DO XWLOL]LQJ D GLIIHUHQW H[WUDFn WLRQ SURFHGXUH ZKR VKRZHG WKDW FUXGH XWHULQH H[WUDFWV RI GD\ SUHJn QDQW JXLQHD SLJV FRQWDLQHG ORZ ELRORJLFDO DFWLYLW\ 8PJf ZKHQ WHVWHG ZLWK WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ +LJKHU DFWLYLW\ OHYHOV ZHUH UHSRUWHG IRU UHOD[LQ VHSDUDWHG RQ D FROXPQ RI %LR*HO 8PJf 3DUGR HW DO f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f 6KDUN UHOD[LQ KDV EHHQ IRXQG WR EH LQHIIHFWLYH LQ WKH PRXVH ELRDVVD\V XWHULQH PRWLOLW\ DQG LQWHUSXELF OLJDPHQW IRUPDWLRQf +RZHYHU VKDUN UHOD[LQ LV DFWLYH ZKHQ JXLQHD SLJV ZHUH HPSOR\HG IRU WKH XWHULQH PRWLOLW\ DQG LQWHUSXELF OLJDPHQW DVVD\V ,W LV FOHDU KRZHYHU PRUH WKDQ RQH ELRDVVD\ VKRXOG EH HPSOR\HG ZKHQ FRQVLGHULQJ ZKHWKHU D SUHSDUDWLRQ FRQWDLQV UHOD[LQ *XLQHD SLJ UHOD[LQ ZDV HIIHFWLYH LQ ERWK WKH PRXVH

PAGE 71

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

PAGE 72

%,%/,2*5$3+< $EUDPRZLW] $ $ : / 0RQH\ 0 ; =DUURZ 5 9 1 7DOPDJH / + .OHLQKRO] DQG ) / +LVDZ f 3UHSDUDWLRQ ELRORJLFDO DVVD\ DQG SURSHUWLHV RI UHOD[LQ (QGRFULQRORJ\ $EUDPVRQ ( +XUZLWW DQG /HVQLN f 5HOD[LQ LQ KXPDQ VHUXP DV D WHVW RI SUHJQDQF\ 6XUJ *\QHFRO 2EVWHW $LWNHQ 5 f 8WHULQH SURWHLQV ,Q 2[IRUG 5HYLHZV RI 5HSURGXFWLYH %LRORJ\ $ $ )LQQ HGf &ODUHQGRQ 3UHVV 2[IRUG (QJODQG $QGHUVRQ 0 / DQG $ /RQJ f /RFDOL]DWLRQ RI UHOD[LQ LQ WKH SUHJQDQW UDW %LRDVVD\ RI WLVVXH H[WUDFWV DQG FHOO IUDFWLRQDWLRQ VWXGLHV %LRO 5HSURG $QGHUVRQ 0 / $ /RQJ DQG 7 +D\DVKLGD f ,PPXQRIOXRUHVFHQFH VWXGLHV RQ WKH ORFDOL]DWLRQ RI UHOD[LQ LQ WKH FRUSXV OXWHXP RI WKH SUHJQDQW UDW %LRO 5HSURG $UDNDUL 5 ) 5 .OHLQIHOG DQG %U\DQW*UHHQZRRG f ,PPXQRIOXRUHVFHQFH VWXGLHV XVLQJ DQWLVHUD WR FUXGH DQG WR SXULILHG SRUFLQH UHOD[LQ %LRO 5HSURG %DUU $ DQG + $ *RRGQLJKW f 8VHUV *XLGH WR WKH 6WDWLVWLn FDO $QDO\VLV 6\VWHP 1RUWK &DUROLQD 6WDWH 8QLYHUVLW\ 5DOHLJK 1 & %D]HU ) : f 8WHULQH SURWHLQ VHFUHWLRQV 5HODWLRQVKLS WR GHYHORSPHQW RI WKH FRQFHSWXV $QLP 6FL %D]HU ) : DQG : : 7KDWFKHU f 7KHRU\ RI PDWHUQDO UHFRJQLWLRQ RI SUHJQDQF\ LQ VZLQH EDVHG RQ HVWURJHQ FRQWUROOHG HQGRFULQH YHUVXV H[RFULQH VHFUHWLRQ RI 3URVWDJODQGLQ )DOSKD E\ WKH XWHULQH HQGRPHWULXP 3URVWDJODQGLQV %HOW : / / $QGHUVRQ / ) &DYD]RV DQG 5 0 0HODPS\ f &\WRSODVPLF JUDQXOHV DQG UHOD[LQ OHYHOV LQ SRUFLQH FRUSRUD OWHD (QGRFULQRORJ\ %LJD]]L 0 ( 1DUGL 3 %UXQL DQG ) 3HWUXFFL f 5HOD[LQ LQ KXPDQ GHFLGXD &OLQ (QGRFULQRO 0HWDE %OXQGHOO 7 f &RQIRUPDWLRQ DQG PROHFXODU ELRORJ\ RI SRO\SHSWLGH KRUPRQHV ,QVXOLQ LQVXOLQOLNH JURZWK IDFWRU DQG UHOD[LQ 7,%6 0DUFK

PAGE 73

%ROWRQ $ ( DQG : 0 +XQWHU f 7KH ODEHOOLQJ RI SURWHLQV WR KLJK VSHFLILF UDGLRDFWLYLWLHV E\ FRQMXJDWLRQ WR D 6LBFRQWDMQMQJ DF\ODWLQJ DJHQW %LRFKHP %R\G 6 = .HQGDOO 1 0HQW DQG %U\DQW*UHHQZRRG f 5HOD[LQ LPPXQRDFWLYLW\ LQ SODVPD GXULQJ WKH UHSURGXFWLYH F\FOH RI WKH IHPDOH JXLQHD SLJ %LRO 5HSURG %URXKD / f 5HFKHUFKHV VXU OD PRELOLVDWLRQ GH OD V\PSK\VH SXELHQQH FKH] OH FRED\H LPSXEHUH &RPSWH 5HQGX 6RF %LRO %U\DQW f 7KH GHWHFWLRQ RI UHOD[LQ LQ SRUFLQH RYLQH DQG KXPDQ SODVPD E\ UDGLRLPPXQRDVVD\ (QGRFULQRORJ\ %U\DQW DQG : $ &KDPOH\ f &KDQJHV LQ UHOD[LQ DQG SURn ODFWLQ LPPXQRDFWLYLWLHV LQ RYLQH SODVPD IROORZLQJ VXFNOLQJ 5HSURG )UWLO %U\DQW 0 ( $ 3DQWHU DQG 7 6WHOPDVLDN f ,PPXQRUH DFWLYH UHOD[LQ LQ KXPDQ VHUXP GXULQJ WKH PHQVWUXDO F\FOH &OLQ (QGRFULQRO 0HWDE %U\DQW ) 6DVVLQ ( :HLW]PDQ 6 .DSHQ DQG $ )UDQW] f 5HOD[LQ LPPXQRDFWLYLW\ LQ KXPDQ SODVPD GXULQJ D KRXU SHULRG 5HSURG )UWLO %U\DQW DQG 7 6WHOPDVLDN f 7KH VSHFLILFLW\ RI UDGLRn LPPXQRDVVD\ IRU UHOD[LQ (QGR 5HV &RPPXQ %U\DQW*UHHQZRRG DQG ) & *UHHQZRRG f 6SHFLILFLW\ RI UDGLRLPPXQRDVVD\V IRU UHOD[LQ (QGRFULQRO &DVWUR+HUQDQGH] $ f ,VRODWLRQ DQG SXULILFDWLRQ RI ERYLQH OXWHDO SRO\SHSWLGHV ZLWK UHOD[LQ KRUPRQH DFWLYLW\ 'RFWRUDO 'LVVHUWDWLRQ 8QLYHUVLW\ RI )ORULGDf &DWFKSROH + 5 f +RUPRQDO PHFKDQLVPV GXULQJ SUHJQDQF\ DQG SDUWXULWLRQ ,Q 5HSURGXFWLRQ LQ 'RPHVWLF $QLPDOV + + &ROH DQG 3 7 &XSSV HGVf $FDGHPLF 3UHVV 1HZ
PAGE 74

'DOOHQEDFK ) DQG 'DOOHQEDFK+HOOZHJ f ,PPXQRKLVWRORJLVFKH XQWHUVXFKXQJHQ ]XU ORNDOLVLHUXQJ GHV UHOD[LQV LQ PHQVFKOLFKHU SOD]HQWD DQG GHFLGXD 9LUFK $UFK 3DWK $QDW 'DOOHQEDFK+HOOZHJ 9 %DWWLVWD DQG ) 'DOOHQEDFK f ,PPXQRKLVWRORJLFDO DQG KLVWRFKHPLFDO ORFDOL]DWLRQ RI UHOD[LQ LQ WKH PHWULDO JODQG RI WKH SUHJQDQW UDW $P $QDW )HYROG + ) / +LVDZ DQG 5 0H\HU f 7KH UHOD[DWLYH KRUn PRQH RI WKH FRUSXV OXWHXP LWV SXULILFDWLRQ DQG FRQFHQWUDWLRQ $P &KHP 6RF )LHOGV 0 3 $ )LHOGV $ &DVWUR+HUQDQGH] DQG / + /DUNLQ f (YLGHQFH IRU UHOD[LQ LQ FRUSRUD OWHD RI ODWH SUHJQDQW FRZV (QGRFULQRORJ\ )LHOGV 3 $ DQG / + /DUNLQ f ,VRODWLRQ RI UDW UHOD[LQ $QDW 5HF )LHOGV 3 $ DQG / + /DUNLQ f 3XULILFDWLRQ DQG LPPXQRKLVWR FKHPLFDO ORFDOL]DWLRQ RI UHOD[LQ LQ WKH KXPDQ WHUP SODFHQWD &OLQ (QGRFULQRO 0HWDE )LHOGV 3 $ / + /DUNLQ DQG 5 3DUGR f 3XULILFDWLRQ RI UHOD[LQ IURP WKH SODFHQWD RI WKH UDEELW $QQ 1 < $FDG 6FL )LQQ & $ f 7KH LPSODQWDWLRQ UHDFWLRQ ,Q %LRORJ\ RI WKH 8WHUXV 5 0 :\QQ HGf 3OHQXP 3UHVV 1HZ
PAGE 75

*ULVV .HFN 5 (QJHOKRP DQG + 7XSS\ f 7KH LVRODWLRQ DQG SXULILFDWLRQ RI DQ RYDULDQ SRO\SHSWLGH RI XWHULQHUHOD[LQJ DFWLYLW\ %LRFKLP %LRSK\V $FWD +DOO f 5HOD[LQ 5HSURG )UWLO +DUNQHVV 0 / 5 DQG 5 +DUNQHVV f &KDQJHV LQ WKH IRHWDO PHPEUDQH GXULQJ SUHJQDQF\ LQ WKH UDW 3K\VLRO +DUNQHVV 0 / 5 DQG 5 +DUNQHVV f &KDQJHV LQ WKH SK\VLFDO SURSHUWLHV RI WKH XWHULQH FHUYL[ RI WKH UDW GXULQJ SUHJQDQF\ 3K\VLRO +LVDZ ) / f ([SHULPHQWDO UHOD[DWLRQ RI WKH SXELF OLJDPHQW RI WKH JXLQHD SLJ 3URF 6RF ([S %LRO 0HG +LVDZ ) / f ([SHULPHQWDO UHOD[DWLRQ RI WKH V\PSK\VLV SXELV RI WKH JXLQHD SLJ $QDW 5HF +LVDZ ) / DQG 0 ; =DUURZ f 7KH SK\VLRORJ\ RI UHOD[LQ 9LW DQG +RUP +LVDZ ) / 0 ; =DUURZ : / 0RQH\ 5 9 1 7DOPDJH DQG $ $ $EUDPRZLW] f ,PSRUWDQFH RI WKH IHPDOH UHSURGXFWLYH WUDFW LQ WKH IRUPDWLRQ RI UHOD[LQ (QGRFULQRORJ\ +RUVW 0 1 6 0 0 %DVKD $ %DXPEDFK ( + 0DQVILHOG DQG 5 0 5REHUWV f $ONDOLQH XUHD VROXELOL]DWLRQ WZRGLPHQVLRQDO HOHFWURSKRUHVLV DQG OHFWLQ VWDLQLQJ RI PDPPDOLDQ FHOO SODVPD PHPEUDQH DQG SODQW VHHG SURWHLQV $QDO %LRFKHP +XQWHU : 0 DQG ) & *UHHQZRRG f 3UHSDUDWLRQ RI LRGLQH ODEHOOHG KXPDQ JURZWK KRUPRQH RI KLJK VSHFLILF DFWLYLW\ 1DWXUH ,VDDFV 1 5 -DPHV + 1LDOO %U\DQW*UHHQZRRG 'RGVRQ $ (YDQV DQG $ & 7 1RUWK f 5HOD[LQ DQG LWV VWUXFWXUDO UHODWLRQn VKLS WR LQVXOLQ 1DWXUH -DPHV 5 + 1LDOO 6 .ZRN DQG %U\DQW*UHHQZRRG f 3ULPDU\ VWUXFWXUH RI SRUFLQH UHOD[LQ +RPRORJ\ ZLWK LQVXOLQ DQG UHODWHG JURZWK IDFWRUV 1DWXUH -RKQ 0 % : %RUMHVVRQ 5 :DOVK DQG + 1LDOO f /LPLWHG VHTXHQFH KRPRORJ\ EHWZHHQ SRUFLQH DQG UDW UHOD[LQV ,PSOLn FDWLRQV IRU SK\VLRORJLFDO VWXGLHV (QGRFULQRORJ\ .HQGDOO = & 3ORSSHU DQG %U\DQW*UHHQZRRG f 8OWUDVWUXFWXUDO LPPXQRSHUR[LGDVH GHPRQVWUDWLRQ RI UHOD[LQ LQ FRUSRUD OWHD IURP D SUHJQDQW VRZ %LRO 5HSURG

PAGE 76

.UDQW] & + + %U\DQW DQG & &DUU f 7KH DFWLRQ RI DTXHRXV FRUSXV OXWHXP H[WUDFW XSRQ XWHULQH DFWLYLW\ 6XUJ *\QHFRO 2EVWHW .URF 5 / % 6WHLQHW] DQG 9 / %HDFK f 7KH HIIHFWV RI HVWURJHQV SURJHVWDJHQV DQG UHOD[LQ LQ SUHJQDQW DQG QRQSUHJQDQW ODERUDWRU\ URGHQWV $QQ 1 < $FDG 6FL /DUNLQ / + f %LRDVVD\ RI UDW PHWULDO JODQG H[WUDFWV IRU UHOD[LQ XVLQJ WKH PRXVH LQWHUSXELF OLJDPHQW WHFKQLTXH (QGRFULQn RORJ\ /DUNLQ / + 3 $ )LHOGV DQG 5 0 2OLYHU f 3URGXFWLRQ RI DQWLVHUD DJDLQVW HOHFWURSKRUHWLFDOO\ VHSDUDWHG UHOD[LQ DQG LPPXQR IOXRUHVFHQW ORFDOL]DWLRQ RI UHOD[LQ LQ WKH SRUFLQH FRUSXV OXWHXP (QGRFULQRORJ\ /DUNLQ / + 3 $ )LHOGV DQG 5 3DUGR f 0RXVH XWHUXV ELRn DVVD\ IRU UHOD[LQ ,Q 5HOD[LQ %U\DQW*UHHQZRRG + 1LDOO DQG ) & *UHHQZRRG HGVf (OVHYLHU 1RUWK +ROODQG /DUNLQ / + & $ 6XDUH]4XLDQ DQG 3 $ )LHOGV f ,Q YLWUR DQDO\VHV RI DQWLVHUD WR UHOD[LQ $FWD (QGRFULQRO /RXPD\H ( % 7HXZLVVHQ DQG 7KRPDV f &KDUDFWHUL]DWLRQ RI UHOD[LQ UDGLRLPPXQRDVVD\ XVLQJ %ROWRQ+XQWHU UHDJHQW *\QHFRO 2EVWHW ,QYHVW /RZU\ + 1 5RVHERURXJK $ / )DUU DQG 5 5DQGDOO f 3URWHLQ PHDVXUHPHQW ZLWK WKH IROLQ SKHQRO UHDJHQW %LRO &KHP 0DF/HQQDQ $ + 5 & *UHHQ %U\DQW*UHHQZRRG ) & *UHHQZRRG DQG 5 ) 6HDPDUN f 5LSHQLQJ RI WKH KXPDQ FHUYL[ DQG LQGXFWLRQ RI ODERU ZLWK SXULILHG SRUFLQH UHOD[LQ /DQFHW )HE SS 0DUFXV f 0LWRVLV LQ WKH UDW XWHUXV GXULQJ WKH HVWUXV F\FOH HDUO\ SUHJQDQF\ DQG HDUO\ SVHXGRSUHJQDQF\ %LRO 5HSURG 0DUNZHOO 0 $ DQG & ) )R[ f 6XUIDFHVSHFLILF LRGLQDWLRQ RI PHPEUDQH SURWHLQV RI YLUXVHV DQG HXNDU\RWLF FHOOV XVLQJ WHWUDFKORUR DOSKD DOSKDGLSKHQ\OJO\FROXULO %LRFKHP 0DWKLHX 3 + 5DWKLHU DQG 7KRPDV f /RFDOL]DWLRQ RI UHOD[LQ LQ KXPDQ JHVWDWLRQDO FRUSXV OXWHXP &HOO 7LVV 5HV

PAGE 77

1DJDR 5 DQG %U\DQW*UHHQZRRG f (YLGHQFH IRU D XWHULQH UHOD[LQ LQ WKH JXLQHD SLJ ,Q 5HOD[LQ %U\DQW*UHHQZRRG + 1LDOO DQG ) & *UHHQZRRG HGVf (OVHYLHU 1RUWK +ROODQG 1RDOO 0 : DQG ( + )ULHGHQ f 9DULDWLRQV RI VHQVLWLYLW\ RI RYDULHFWRPL]HG JXLQHD SLJV WR UHOD[LQ (QGRFULQRORJ\ 2n%\UQH ( 0 ) ) )OLWFUDIW : 6DZ\HU +RFKPDQ :HLVV DQG % 6WHLQHW] f 5HOD[LQ ELRDFWLYLW\ DQG LPPXQRDFWLYLW\ LQ KXPDQ FRUSRUD OWHD (QGRFULQRORJ\ 2n%\UQH ( 0 : 6DZ\HU 0 & %XWOHU DQG % 6WHLQHW] f 6HUXP LPPXQRUHDFWLYH UHOD[LQ DQG VRIWHQLQJ RI WKH XWHULQH FHUYL[ LQ SUHJQDQW KDPVWHUV (QGRFULQRORJ\ 2n%\UQH ( 0 DQG % 6WHLQHW] f 5DGLRLPPXQRDVVD\ 5,$f RI UHOD[LQ LQ VHUD RI YDULRXV VSHFLHV XVLQJ DQ DQWLVHUXP WR SRUFLQH UHOD[LQ 3URF 6RF ([S %LRO 0HG 3DUGR 5 / + /DUNLQ DQG 3 $ )LHOGV f ,PPXQRF\WRFKHPLFDO ORFDOL]DWLRQ RI UHOD[LQ LQ HQGRPHWULDO JODQGV RI WKH SUHJQDQW JXLQHD SLJ (QGRFULQRORJ\ 3RUWHU f 0\RPHWULXP RI WKH SUHJQDQW JXLQHD SLJ 7KH SUREDEOH LPSRUWDQFH RI UHOD[LQ %LRO 5HSURG 3RUWHU f 5HOD[LQ 2OG +RUPRQH QHZ SURVSHFW ,Q 2[IRUG 5HYLHZV RI 5HSURGXFWLYH %LRORJ\ 9RO & $ )LQQ HGf &ODUHQGRQ 3UHVV 2[IRUG (QJODQG 5HLQLJ : 1 /DPEHUW & 6FKZDEH / *RZDQ % 6WHLQHW] DQG ( 0 2n%\UQH f ,VRODWLRQ DQG FKDUDFWHUL]DWLRQ RI UHOD[LQ IURP WKH VDQG WLJHU VKDUN RGRQWDVSLV WDXUXVf (QGRFULQn RORJ\ 6DQGHUV 0 0 9 ( *URSSL -U DQG ( 7 %URZQLQJ f 5HVROXn WLRQ RI EDVLF FHOOXODU SURWHLQV LQFOXGLQJ KLVWRQH YDULDQWV E\ WZRGLPHQVLRQDO JHO HOHFWURSKRUHVLV (YDOXDWLRQ RI O\VLQH WR DUJLQLQH UDWLRV DQG SKRVSRU\ODWLRQ $QDO %LRFKHP 6DU 0 DQG : ( 6WXPSI f &HOOXODU DQG VXEFHOOXODU ORFDOL]DWLRQ RI A+SURJHVWHURQH RU LWV PHWDEROLWHV LQ WKH RYLGXFW XWHUXV YDJLQD DQG OLYHU RI WKH JXLQHD SLJ (QGRFULQRORJ\ 6FKZDEH & DQG 6 $ %UDGGRQ f (YLGHQFH IRU RQH HVVHQWLDO WU\SWRSKDQ UHVLGXH DW WKH DFWLYH VLWH RI UHOD[LQ %LRFKHP %LRSK\V 5HV &RPPXQ

PAGE 78

6FKZDEH & 0F'RQDOG DQG % 6WHLQHW] f 3ULPDU\ VWUXFWXUH RI WKH $FKDLQ RI SRUFLQH UHOD[LQ %LRFKHP %LRSK\V 5HV &RPPXQ 6FKZDEH & 0F'RQDOG DQG % 6WHLQHW] f 3ULPDU\ VWUXFWXUH RI WKH %FKDLQ RI SRUFLQH UHOD[LQ %LRFKHP %LRSK\V 5HV &RPPXQ 6FKZDEH & % 6WHLQHW] :HLVV $ 6HJDORII 0F'RQDOG ( 2n%\UQH +RFKPDQ % &DUULHUH DQG / *ROGVPLWK f 5HOD[LQ 5HF 3URJ +RUP 5HV 6KHUZRRG f 3XULILFDWLRQ DQG FKDUDFWHUL]DWLRQ RI UDW UHOD[LQ (QGRFULQRORJ\ 6KHUZRRG DQG 9 ( &UQHNRYLF f 'HYHORSPHQW RI D KRPRn ORJRXV UDGLRLPPXQRDVVD\ IRU UDW UHOD[LQ (QGRFULQRORJ\ 6KHUZRRG 3 $ 0DUWLQ & & &KDQJ DQG 3 ']LXN Df 3ODVPD UHOD[LQ OHYHOV LQ SLJV ZLWK FRUSRUD OWHD LQGXFHG GXULQJ ODWH SUHJQDQF\ %LRO 5HSURG 6KHUZRRG 3 $ 0DUWLQ & & &KDQJ DQG 3 ']LXN Ef 3ODVPD UHOD[LQ OHYHOV GXULQJ ODWH SUHJQDQF\ DQG DW SDUWXULWLRQ LQ SLJV ZLWK DOWHUHG XWHURRYDULDQ FRQQHFWLRQV %LRO 5HSURG 6KHUZRRG DQG ( 0 2n%\UQH f 3XULILFDWLRQ DQG FKDUDFWHUn L]DWLRQ RI SRUFLQH UHOD[LQ $UFK %LRFKP %LRSK\V 6KHUZRRG 5 5RVHQWUHWHU DQG 0 / %LUNKLPHU f 'HYHORSn PHQW RI D UDGLRLPPXQRDVVD\ IRU SRUFLQH UHOD[LQ XVLQJ E, ODEHOOHG SRO\W\URV\OUHOD[LQ (QGRFULQRORJ\ 6WHLQHW] % 9 / %HDFK 5 / .URF 1 5 6WDVLOOL 5 ( 1XVVEDXP 3 1HPLWK DQG 5 'XQ f %LRDVVD\ RI UHOD[LQ XVLQJ D UHIHUHQFH VWDQGDUG $ VLPSOH DQG UHOLDEOH PHWKRG XWLOL]LQJ GLUHFW PHDVXUHPHQW RI LQWHUSXELF OLJDPHQW IRUPDWLRQ LQ PLFH (QGRFULQRORJ\ 6WHUQEHUJHU / $ f ,PPXQRF\WRFKHPLVWU\ -RKQ :LOH\ DQG 6RQV 1HZ
PAGE 79

6XWFOLIIH 5 + %URFN / % 9 1LFKROVRQ DQG ( 'XQQ f )HWDO DQG XWHULQHVSHFLILF DQWLJHQV LQ KXPDQ DPQLRWLF IOXLG 5HSURG )UWLO 6]DOFKWHU 1 ( 2n%\UQH / *ROGVPLWK % 6WHLQHW] DQG :HLVV f 0\RPHWULDO LQKLELWLQJ DFWLYLW\ RI UHOD[LQFRQWDLQLQJ H[WUDFWV RI KXPDQ FRUSRUD OWHD $P 2EVWHW &\QHFRO :DOVK 5 DQG + 1LDOO f 8VH RI DQ RFWDGHF\OVLOLFD SXULILFDWLRQ PHWKRG PLQLPL]HV SURWHRO\VLV GXULQJ LVRODWLRQ RI SRUFLQH DQG UDW UHOD[LQV (QGRFULQRORJ\ :DUHPERXUJ 0 f 5DGLRJUDSKLF VWXG\ RI WKH JXLQHD SLJ XWHUXV DIWHU LQMHFWLRQ DQG LQFXEDWLRQ ZLWK A+SURJHVWHURQH (QGRFULQRORJ\ :HLVV ( 0 2n%\UQH $ +RFKPDQ / 7 *ROGVPLWK 5LINLQ DQG % 6WHLQHW] f 6HFUHWLRQ RI SURJHVWHURQH DQG UHOD[LQ E\ WKH KXPDQ FRUSXV OXWHXP DW PLGSUHJQDQF\ DQG DW WHUP 2EVWHW *\QHFRO :HLVV ( 0 2n%\UQH $ +RFKPDQ % 6WHLQHW] / *ROGVPLWK DQG )OLWFUDIW f 'LVWULEXWLRQ RI UHOD[LQ LQ ZRPHQ GXULQJ SUHJQDQF\ 2EVWHW *\QHFRO :HLVV ( 0 2n%\PH DQG % 6WHLQHW] f 5HOD[LQ $ SURGXFW RI WKH FRUSXV OXWHXP RI SUHJQDQF\ 6FLHQFH
PAGE 80

=DUURZ 0 ; DQG : % 2n&RQQRU f /RFDOL]DWLRQ RI UHOD[LQ LQ WKH FRUSXV OXWHXP RI WKH UDEELW 3URF 6RF ([S %LRO 0HG =DUURZ 0 ; DQG % 5RVHQEHUJ f 6RXUFHV RI UHOD[LQ LQ WKH UDEELW (QGRFULQRORJ\

PAGE 81

$33(1',; 7$%/(6

PAGE 82

7DEOH %LRDFWOYH UHOD[LQ FRQWHQW RI XWHUL IURP JXLQHD SLJV LQ GLIIHUHQW VWDJHV RI SUHJQDQF\ DQG ODFWDWLRQ 67$*( 2) $1,0$/ *(67$7,21 12 Jf PJf :(7 :7 '5< :7 727$/ 81,76 2) 5(/$;,1 3(5 87(586 81,76 2) 5(/$;,1 7(5 *5$0 :(7 :(,*+7 '$< f f frr f f f '$< f f f f f f '$< f f f f f f /DWH f 3UHJQDQW f f f f f f /DFWDWLQJ f f f f f f r 7ZHQW\ PLOOLJUDPV PJf RI $FLG $FHWRQH H[W UDFWHG SRZGHU ZDV KRPRJHQL]HG LQ PO RI GLVWLOOHG ZDWHU rr 8QLWV RI DFWLYLW\ DUH H[SUHVVHG DV SRUFLQH 1,85;1 3, VWDQGDUG UHOD[LQ HTXLYDOHQWV ; 6(0f GHWHUPLQHG E\ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ f GHQRWHV WKH QXPEHU RI ELRDVVD\V FRQGXFWHG

PAGE 83

7DEOH ,PPXQRUHDFWLYH UHOD[LQ FRQWHQW RI XWHUL WDNHQ IURP JXLQHD SLJV LQ GLIIHUHQW VWDJHV RI SUHJQDQF\ DQG ODFWDWLRQ QJf QJf 67$*( 2) $1,0$/ r! IDJf 727$/ *3 5(/$;,1 $07 5(/$;,1 35(*1$1&< 12 :(7 :7 '5< :7 3(5 87(586 3(5 *5$0 :(7 :(,*+7 '$< frr 2L '$< +n f '$< f '$< A G s f n /$7( 35(*1$17 f O $&7$7,1* $ ff B f R f7ZHQW\ PLOOLJUDPV RI $FLG $FHWRQH H[WUDFWHG SRZGHU ZDV KRPRJHQL]HG ,Q PO RI GLVWLOOHG ZDWHU rr$FWLYOW\ H[SUHVVHG DV QJ SRUFLQH 1,+5;13 VWDQGDUG UHOD[LQ HTXLYDOHQWV ; 6)+f DV GHWHUPLQHG E\ D KRPRORJRXV SRUFLQH UDGLRLPPXQRDVVD\ $OO DVVD\V ZHUH FRQGXFWHG ,Q GXSOLFDWH nr

PAGE 84

7DEOH 3URWHLQ \LHOG DQG SRWHQFLHV RI ODWH SUHJQDQW JXLQHD SLJ XWHUXV WKURXJKRXW WKH 2'6 SXULILFDWLRQ SURFHGXUH )UDFWLRQ 5HFRYHU\ PJf 3URWHLQ
PAGE 85

7DEOH 3K\VLRFKHPLFDO FKDUDFWHULVWLFV RI JXLQHD SLJ XWHULQH UHOD[LQ 7KH VRXUFH RI UHOD[LQ ZDV DQ 2'6r SXULILHG XWHULQH SUHSDUDWLRQ 5HGXFHG $FWLYLW\ 1R &KDQJH 'LWKLRWKULHWRO [ +HDWLQJ DW r & [ 7U\SVLQ [ 5 DQWLVHUXP O[ r2'6 FUXGH UHOD[LQ LV D SDUWLDOO\ SXULILHG XWHULQH H[WUDFW WDNHQ DIWHU WKH LQLWLDO SXULILFDWLRQ VWHS LQ WKH 2'6 SURn FHGXUH 7KLV H[WUDFW ZDV WHVWHG LQ WKH PRXVH XWHULQH PRWLOLW\ DVVD\ ZLWKRXW EHLQJ DOWHUHG FRQWUROf DQG DIWHU H[SHULPHQWDO WUHDWPHQWV $ UDWLR ZDV GHWHUPLQHG E\ GLYLGLQJ WKH ILQDO YROXPH RI WKH H[SHULPHQWDO E\ WKH ILQDO YROXPH RI WKH FRQWURO 7KH DVVD\V ZHUH UXQ WZLFH DQG DQ DYHUDJH YDOXH ZDV FRPSXWHG 7KH JUHDWHU WKH H[SHULPHQWDO WR FRQWURO UDWLR WKH JUHDWHU WKH DELOLW\ RI WKH DJHQW WR LQKLELW WKH DFWLRQ RI UHOD[LQ

PAGE 86

$33(1',; ),*85(6

PAGE 87

)LJXUHV UHSUHVHQW VHFWLRQV RI XWHUL WDNHQ IURP JXLQHD SLJV RQ GD\ RI SUHJQDQF\ 7UDQVYHUVH VHFWLRQ RI XWHUXV VWDLQHG XVLQJ WKH 3$3 WHFKn QLTXH ZLWK 5 DQWLVHUXP GLOXWLRQf $UURZ HQGRPHWULDO JODQGV H[KLELWLQJ 53 DUURZKHDG HQGRPHWULDO JODQGV ODFNLQJ 53 ; 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK QRUPDO UDEELW VHUXP GLOXWLRQf 1RWH ODFN RI 53 RYHU HQGRn PHWULDO JODQGV DUURZVf ; +HPDWR[\OLQ DQG HRVLQ VWDLQHG VHFWLRQ ; 6HFWLRQ VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLn VHUXP GLOXWLRQf 1RWH WKDW QRW DOO HQGRPHWULDO JODQG FHOOV GHPRQVWUDWH 53 &OHDU DUHDV LQ EDVDO UHJLRQV RI WKH HQGRPHWULDO JODQG FHOOV UHSUHVHQW XQVWDLQHG QXFOHDU SURILOHV ;

PAGE 89

)LJXUHV UHSUHVHQW VHFWLRQV RI XWHUL WDNHQ IURP JXLQHD SLJV RQ GD\ RI SUHJQDQF\ 7UDQVYHUVH VHFWLRQ RI WKH JXLQHD SLJ XWHUXV WDNHQ RQ GD\ RI SUHJQDQF\ DQG VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLVHUXP GLOXWLRQf $UURZ HQGRPHWULDO JODQGV H[KLELWLQJ 53 DUURZKHDG HQGRPHWULDO JODQGV ODFNLQJ 53 $ KLJKHU SHUFHQWDJH RI JODQGV DUH ODEHOHG WKDQ LQ GD\ WLVVXH KRZHYHU QRW DOO JODQGV KDYH 53 DW WKLV VWDJH ; 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK QRUPDO UDEELW VHUXP GLOXWLRQf 1RWH ODFN RI 53 RYHU HQGRPHWULDO JODQGV DUURZVf ; +HPDWR[\OLQ DQG HRVLQ VWDLQHG VHFWLRQ ; 6HFWLRQ VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLn VHUXP GLOXWLRQf :KLOH QRW DOO FHOOV LQ HDFK JODQG VKRZ WKH SUHVHQFH RI 53 QRWH WKDW WKH PDMRULW\ RI FHOOV LQ HDFK JODQG VKRZ D KHDY\ DFFXPXODWLRQ RI 53 ;

PAGE 91

)LJXUHV UHSUHVHQW VHFWLRQV RI XWHUL WDNHQ IURP JXLQHD SLJV RQ GD\ RI SUHJQDQF\ 7UDQVYHUVH VHFWLRQ RI WKH JXLQHD SLJ XWHUXV WDNHQ RQ GD\ RI SUHJQDQF\ VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLn VHUXP GLOXWLRQf 1RWH WKDW DOO RI WKH HQGRPHWULDO JODQGV H[KLELW 53 ; 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK QRUPDO UDEELW VHUXP GLOXWLRQf 1RWH ODFN RI 53 RYHU HQGRn PHWULDO JODQGV ; +HPDWR[\OLQ DQG HRVLQ VWDLQHG VHFWLRQ 1RWH ZKDW DSSHDU WR EH GHQVH DJJUHJDWHV RI PDWHULDO QHDU WKH OXPLQDO VXUIDFH RI WKH HQGRPHWULDO JODQG FHOOV ; 6HFWLRQ VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLn VHUXP GLOXWLRQf 'HQVH DJJUHJDWHV RI 53 VLPLODU WR WKDW GHPRQVWUDWHG LQ )LJXUH DUH VKRZQ LQ WKH OXPLQDO VXUIDFHV RI WKH (*& &OHDU DUHDV LQ EDVH RI (*& UHSUHVHQW XQVWDLQHG QXFOHDU SURILOHV ;

PAGE 93

)LJXUHV UHSUHVHQW VHFWLRQV RI XWHUL WDNHQ IURP ODWH SUHJQDQW JXLQHD SLJV 7UDQVYHUVH VHFWLRQ RI WKH JXLQHD SLJ XWHUXV IURP ODWH SUHJQDQW DQLPDOV GD\ RU RI SUHJQDQF\f DQG VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLVHUXP GLOXWLRQf 1RWH DOO RI WKH HQGRPHWULDO JODQGV H[KLELW 53 ; 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK QRUPDO UDEELW VHUXP GLOXWLRQf 1RWH ODFN RI 53 RYHU HQGRn PHWULDO JODQGV ; +HPDWR[\OLQ DQG HRVLQ VWDLQHG VHFWLRQ &RQWLQXLW\ EHWZHHQ WKH OXPHQ RI DQ HQGRPHWULDO JODQG DQG WKH OXPHQ RI WKH XWHUXV FDQ EH VHHQ LQ WKH ORZHVW RI WKH WKUHH JODQG SURILOHV 1RWH WKH GLIIHUHQFH LQ F\WRSODVPLF DQG QXFOHDU VWDLQLQJ GHQVLWLHV EHWZHHQ WKH (*& DQG FHOOV RI WKH XWHULQH OXPHQ HSLWKHOLXP ; 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLVHUXP GLOXWLRQf 1RWH WKDW VRPH RI WKH HQGRPHWULDO JODQG FHOOV KDYH 53 GLVWULEXWHG WKURXJKn RXW WKH F\WRSODVP VRPH KDYH QR 53 DQG LQ VRPH FHOOV ORZHU JODQGf WKH 53 LV ORFDOL]HG LQ D VSHFLILF VXSUDQXFOHDU UHJLRQ 1RWH DOVR WKDW WKH H[WHQW RI WKH JODQG FDQ EH GHWHUPLQHG E\ WKH UHJLRQ ZKHUH GHSRVLWLRQ RI 53 FHDVHV LQ FHOOV WKDW DUH FRQWLQXRXV ZLWK WKH XWHULQH OXPHQ HSLWKHOLXP 7KLV SDWWHUQ RI GHSRVLWLRQ RI 53 FRUUHVSRQGV ZLWK GLIIHUHQFHV LQ VWDLQLQJ QRWHG LQ + i ( VWDLQHG WLVVXH )LJXUH f ;

PAGE 95

)LJXUHV UHSUHVHQW VHFWLRQV RI XWHUL WDNHQ IURP ODFWDWLQJ JXLQHD SLJV 7UDQVYHUVH VHFWLRQ RI JXLQHD SLJ XWHUXV WDNHQ IURP ODFn WDWLQJ DQLPDOV GD\V SRVWSDUWXPf DQG VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLVHUXP GLOXWLRQf $UURZ HQGRPHWULDO JODQGV H[KLELWLQJ 53 DUURZKHDG HQGRPHWULDO JODQGV ODFNLQJ 53 ; 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK QRUPDO UDEELW VHUXP GLOXWLRQf 1RWH ODFN RI 53 RYHU HQGRn PHWULDO JODQGV DUURZVf ; +HPDWR[\OLQ DQG HRVLQ VWDLQHG VHFWLRQ 1RWH WKH ODUJH QXPEHU RI PLWRVHV LQ WKH JODQGV DUURZVf ; 6HFWLRQ VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLn VHUXP GLOXWLRQf 1RWH WKDW 53 LV ORFDWHG LQ RQO\ D IHZ FHOOV RI WKH JODQG DQG WKDW WKH SDWWHUQ RI GHSRVLWLRQ RI 53 LV YDULDEOH IURP FHOO WR FHOO ;

PAGE 97

)LJXUHV UHSUHVHQW VHFWLRQV RI XWHUL WDNHQ IURP RYDUL HFWRPL]HG KRUPRQH WUHDWHG DQLPDOV 7UDQVYHUVH VHFWLRQ RI JXLQHD SLJ XWHUXV WDNHQ IURP RYDULHFWRPL]HG DQLPDOV WUHDWHG ZLWK HVWURJHQ \Jf DQG SURJHVWHUn RQH PJf GDLO\ IRU GD\V VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLVHUXP GLOXWLRQf $UURZ HQGRPHWULDO JODQGV H[KLELWn LQJ 53 ; 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ )LJXUH WUHDWHG ZLWK QRUPDO UDEELW VHUXP GLOXWLRQf 1RWH ODFN RI 53 RYHU HQGRn PHWULDO JODQGV DUURZVf ; +HPDWR[\OLQ DQG HRVLQ VWDLQHG VHFWLRQ *ODQGXODU FHOOV DUH FXERLGDO ZLWK GHQVHO\ VWDLQLQJ QXFOHL ; 6HFWLRQ VWDLQHG XVLQJ WKH 3$3 WHFKQLTXH ZLWK 5 DQWLn VHUXP GLOXWLRQf 2QO\ D IHZ HQGRPHWULDO JODQG FHOOV GR QRW VKRZ WKH SUHVHQFH RI UHOD[LQ /RFDWLRQ RI 53 YDULHV IURP FHOO WR FHOO KRZHYHU WKH PDMRULW\ RI FHOOV DSSHDU WR KDYH 53 GLVWULEXWHG WKURXJKRXW WKH F\WRSODVP ;

PAGE 99

)LJXUH %LRORJLFDOO\ DFWLYH UHOD[LQ FRQWHQW RBI XWHUL WDNHQ IURP JXLQHD SLJV GXULQJ SUHJQDQF\ DQG ODFWDWLRQ ; B6(0f 'DWD H[SUHVVHG DV WRWDO XQLWV SHU XWHUXV DV GHWHUPLQHG E\ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ RI XWHULQH H[WUDFWV

PAGE 100

67$*( 2) 35(*1$1&< /$&7$7,21 81,76 2) 5(/$;,1 $&7,9,7< WRWDO XQLWV SHU XWHUXVf B WY! RL Z M! R R R R R R L L L L WL L U L U L L L L L L L L L L L L YYnLLf L L L L 92

PAGE 101

)LJXUH %LRORJLFDOO\ DFWLYH UHOD[LQ FRQWHQW LQ WKH JXLQHD SLJ XWHUXV GXULQJ SUHJQDQF\ DQG ODFWDWLRQ ; B 6(0f 'DWD H[SUHVVHG DV XQLWV SHU JUDP ZHW ZHLJKW RI XWHUXV DV GHWHUPLQHG E\ WKH PRXVH XWHULQH PRWLOLW\ ELRDVVD\ RI XWHULQH H[WUDFWV

PAGE 102

67$*( 2) 35(*1$1&< /$&7$7,21 81,76 2) 5(/$;,1 $&7,9,7< XQLWV SHU JUDP ZHW ZHLJKWf WR /W$

PAGE 103

)LJXUH 6HSDUDWLRQ RI SRO\W\URV\O UHOD[LQ IURP XQERXQG FRPSRQHQWV XWLOL]LQJ D 6HSKDGH[ FKURPDWRJUDSK\ FROXPQ [ FPf 7XEHV ZHUH LPPXQRUHDFWLYH ZLWK DQWLVHUXP 5

PAGE 104

& 30 92 FQ

PAGE 105

)LJXUH $QWLVHUXP WLWUDWLRQ FXUYH 3RROHG WXEHV ZHUH WHVWHG ZLWK GHFUHDVLQJ GLOXWLRQV RI 5 DQWLVHUXP DQG b DQWLJHQ ERXQG ZDV GHWHUPLQHG $ GLOXWLRQ RI ZDV HPSOR\HG LQ WKH 5,$ DSSUR[LPDWHO\ b ELQGLQJf b %RXQG DPRXQW RI M UHOD[LQ SUHFLSLWDWHG E\ WKH 5 DQWLVHUXP

PAGE 106

b %281' ,-%22 ),1$/ $17,6(580 &21& /2 ,

PAGE 107

)LJXUH ,QKLELWLRQ E\ ODWH SUHJQDQW JXLQHD SLJ DFLG DFHWRQH FUXGH H[WUDFWV DQG 2'6 FUXGH H[WUDFWV LQ D 5,$ XVLQJ SRO\W\URV\O SRUFLQH UHOD[LQ 5 DQWLSRUFLQH UHOD[LQ VHUXP GLOXWLRQf DQG 1,+5;13 SRUFLQH UHOD[LQ VWDQGDUG 6WDQGDUG SRUFLQH UHOD[LQ 1,+5;13f ff 2'6 FUXGH H[WUDFWV rf DQG DFLG DFHW!QH FUXGH H[WUDFWV ‘f b %RXQG %RXQG16% %R16%

PAGE 108

, R 1_+ SJ 5;13, *3 SJ 2'6&58'( *3 MMJ $&,' $&(721( &58'( $02817 2) 0$7(5,$/

PAGE 109

)LJXUH ,PPXQRUHDFWLYH UHOD[LQ FRQWHQW WRWDO QJf LQ WKH JXLQHD SLJ XWHUXV GXULQJ SUHJQDQF\ DQG ODFWDWLRQ ; B 6(0f 'DWD H[SUHVVHG DV QJ SRUFLQH 1,+5;13 VWDQGDUG UHOD[LQ HTXLYDOHQWV GHWHUPLQHG E\ WKH KRPRORJRXV SRUFLQH UDGLRLPPXQRDVVD\ SUHVHQWHG LQ )LJXUH

PAGE 110

,008125($&7,9( 5(/$;,1 WRWDO QJ SHU XWHUXVf UR R R &' R &' R R R !LU LaLaU L L L U L L Lf§L L U77L L L L L UR 2 A 2 ‘L U U &' 2 L &' 2 U L L L U &2 + B &' RL P 2, R r Q r R A &' P R &' B ]r D = R R U R + + 7 R ‘r

PAGE 111

)LJXUH ,PPXQRUHDFWLYH UHOD[LQ FRQFHQWUDWLRQ QJJUDP ZHW ZHLJKWf GXULQJ SUHJQDQF\ DQG ODFWDWLRQ ; B6(0f 'DWD H[SUHVVHG DV QJ SRUFLQH 1,+5;13 VWDQGDUG UHOD[LQ HTXLYDOHQWV GHWHUPLQHG E\ WKH KRPRORJRXV SRUFLQH UDGLRLPPXQRDVVD\ SUHVHQWHG LQ )LJXUH

PAGE 112

&21& 00812 5 ( $ & 7, 9( 5(/$;,1 QJJURP ZHW ZHLJKWf

PAGE 113

, )LJXUH *HO ILOWUDWLRQ RI DQ 2'6 FUXGH XWHULQH H[WUDFW IURP ODWH SUHJQDQW JXLQHD SLJV PJf RQ D [ FP FROXPQ RI 6HSKDGH[ ILQHf 7KH FROXPQ ZDV HTXLOLEUDWHG ZLWK 0 DPPRQLXP DFHWDWH S+ 7KH IORZ UDWH ZDV PDLQWDLQHG DW POKU DQG IUDFWLRQV ZHUH FROOHFWHG HYHU\ PLQXWHV %LRORJLFDO DFWLYLW\ PRXVH XWHULQH PRWLOLW\ ELRDVVD\f LV LQGLFDWHG E\ WKH KDWFKHG DUHD 7KH ELRDFWLYH IUDFWLRQV ZHUH SRROHG WR IRUP WKH 6HSKDGH[ IUDFWLRQ

PAGE 114

)LJXUH ,RQ H[FKDQJH FKURPDWRJUDSK\ RI WKH 6HSKDGH[ ELRDFWLYH IUDFWLRQ PJf RQ D [ FP FROXPQ RI FDUER[\PHWK\OFHOOXORVH &0f 7KH FROXPQ ZDV HTXLOLEUDWHG DQG ULQVHG ZLWK 0 DPPRQLXP DFHWDWH EXIIHU S+ FRQGXFWLYLW\ P 0KRf XQWLO DOO XQDGVRUEHG PDWHULDO ZDV HOXWHG 7KH FROXPQ ZDV GHYHORSHG DW D UDWH RI POKU ZLWK D OLQHDU JUDGLHQW RI 0 DPPRQLXP DFHWDWH EXIIHU S+ f DQG LQFUHDVLQJ FRQFHQWUDWLRQV RI 1D&O 0 DPPRQLXP DFHWDWH ZLWK 0 1D&Of WR D ILQDO FRQGXFWLYLW\ RI P 0KR )UDFWLRQV POf ZHUH FROOHFWHG HYHU\ PLQXWHV

PAGE 115

$%625%$1&( QP )5$&7,21 12 ‘D 1 ( ? R! ] ; BO 8F X R } ] R ] R 2n

PAGE 116

)LJXUH $JDU GRXEOH LPPXQRGLIIXVLRQ SODWH 5 \O RI DQWLVHUXP SURGXFHG DJDLQVW SRUFLQH UHOD[LQ FRQFHQWUDWHG IRXU WLPHV E\ O\RSKLOL]DWLRQ 1,+ \O SRUFLQH 1,+5;13 UHOD[LQ \JPO /f *3 \O RI GD\ JXLQHD SLJ XWHUXV UHOD[LQ SUHSDUDWLRQ %LR*HO 3 PZ IUDFWLRQ PJPO Af

PAGE 118

r )LJXUH 7ZR GLPHQVLRQDO JHO HOHFWURSKRUHVLV 7KH ILUVW GLPHQVLRQ 1(3+*(f XWLOL]HG D JUDGLHQW RI S+ WR 7KH JHOV ZHUH VWDFNHG IRU PLQXWHV DW 9 DQG WKHQ UXQ IRU KU DW 9 7KH VHFRQG GLPHQVLRQ FRQVLVWHG RI D b 6'6 SRO\DFU\ODPLGH VODE JHO V\VWHP )LIWHHQ PDPSVVODE ZHUH XVHG DV WKH VWDFNLQJ FXUUHQW IRU KU 7KH FXUUHQW ZDV WXUQHG WR PDPSVVODE DQG WKH JHOV UXQ IRU DQ DGGLWLRQDO KU 7KH JHOV ZHUH IL[HG ZLWK b DFHWLF DFLG b HWK\O DOFRKRO DQG VWDLQHG ZLWK b FRRPDVLH EOXH 5 7KH JHOV ZHUH GHVWDLQHG ZLWK b DFHWLF DFLGb HWK\O DOFRKRO

PAGE 119

( ( [ R )LJXUH 0RXVH LQWHUSXELF OLJDPHQW ELRDVVD\ RI D 6HSKDGH[ PZ IUDFWLRQ RI JXLQHD SLJ XWHULQH H[WUDFW DQG D SRUFLQH UHOD[LQ VWDQGDUG 1,+5;13f 7ZHQW\ PLFH ZHUH XVHG DW HDFK GRVH OHYHO IRU WKH SRUFLQH VWDQGDUG DQG ILIWHHQ PLFH ZHUH XVHG DW HDFK GRVH OHYHO IRU WKH JXLQHD SLJ SUHSDUDWLRQ 7KH PHDQ LQWHUSXELF OLJDPHQW OHQJWK IRU WKH PLFH WUHDWHG ZLWK HVWUDGLRO DQG b EHQ]R SXUSXULQH % ZDV B ; B 6(0f 7KH EHVW ILW FXUYH IRU WKH 1,+5;13 SRUFLQH UHOD[LQ ZDV \ ORJ [f ZLWK D 6( DQG D ODPEGD YDOXH RI 7KH EHVW ILW FXUYH IRU WKH JXLQHD SLJ &0& SXULILHG UHOD[LQ ZDV \ ORJ [f ZLWK D 6( DQG D ODPEGD YDOXH RI 7KH PHDQ REVHUYHG SRWHQF\ RI JXLQHD SLJ XWHULQH UHOD[LQ ZDV 8PJ

PAGE 120

$33(1',; ,2',1$7,21 2) 68&&,1,0,'( 5(/$;,1 ,RGLQDWLRQ RI 1,+5;13 UHOD[LQ ZLWK WKH VXFFLQLPLGH PHWKRG $Q DOLTXRW RI HWK\O DFHWDWH ZDV GULHG ZLWK DQ H[FHVV RI DQK\GURXV VRGLXP VXOIDWH 7ZR PLOOLJUDPV RI 1VXFFLQLPLG\O K\GUR[\SKHQ\Of SURSLRQDWH ZHUH GLVVROYHG LQ PO RI WKH GULHG HWK\O DFHWDWH 7HQ PLFUROLWHUV RI WKH GLVVROYHG HVWHU 3Jf ZHUH WKHQ GULHG LQ D VPDOO YLDO XQGHU YDFXXP 7ZHQW\ILYH PLFURJUDPV RI UHOD[LQ LQ \O RI ERUDWH EXIIHU 0 S+ ZHUH DGGHG WR WKH GU\ HVWHU DQG DJLWDWHG DW r & IRU PLQ 7ZHQW\ PLFUROLWHUV \Jf RI WKH DF\ODWHG UHOD[LQ ZHUH DGGHG WR P&L SXUFKDVHG IURP $PHUVKDP &R $UOLQJWRQ +HLJKWV ,/ DQG PL[HG LQ D WXEH FRDWHG ZLWK LRGRJHQ \J GULHGf IRU PLQ ZLWK LQWHUPLWWHQW VKDNLQJ 7KH UHDFWLRQ PL[WXUH ZDV WKHQ OD\HUHG RQ D [ FP FROXPQ RI 6HSKDGH[ SUHHTXLOLEUDWHG ZLWK 0 VRGLXP SKRVSKDWH EXIIHU S+ ZLWK b JHODWLQ DQG HOXWHG ZLWK 0 VRGLXP SKRVSKDWH EXIIHU S+ 7HQ GURS DOLTXRWV ZHUH FROOHFWHG LQ [ PP GLVSRVDEOH JODVV WXEHV FRQWDLQLQJ PO 3%6b RYDOEXPLQ 7KH HOXWLRQ SDWWHUQ LV VKRZQ LQ )LJXUH 7KH SHDNV ZHUH WHVWHG IRU LPPXQRDFWLYLW\ ZLWK QHJDWLYH UHVXOWV ,OO

PAGE 121

)LJXUH 6HSDUDWLRQ RI VXFFLQLPLGH UHOD[LQ IURP XQERXQG FRPSRQHQWV XWLOL]LQJ D 6HSKDGH[ FKURPDWRJUDSK\ FROXPQ [ FPf 5DGLRDFWLYLW\ H[SUHVVHG DV &30nV SHU SL RI VROXWLRQ 1RQH RI WKH SHDNV ZHUH LPPXQRDFWLYH DV WHVWHG ZLWK DQ DQWLVHUXP WLWUDWLRQ WHVW

PAGE 122

& 30

PAGE 123

$33(1',; ,2',1$7,21 2) 5(/$;,1 :,7+ 7+( %2/721 $1' +817(5 5($*(17 ,RGLQDWLRQ RI 1,+5;13 UHOD[LQ ZLWK WKH %ROWRQ DQG +XQWHU UHDJHQW SK\GUR[\ SKHQ\O SURSLRQLF DFLG 1K\GUR[\VXFFLQLPLGH HVWHUf)LYH PLFURJUDPV UHOD[LQ GLVVROYHG LQ SL RI 0 VRGLXP ERUDWH EXIIHU S+ f ZHUH DGGHG WR WKH LRGLQDWHG %ROWRQ DQG +XQWHU UHDJHQW SXUFKDVHG IURP 1HZ (QJODQG 1XFOHDU &R 1RUWK %LOOHULFD 0$ DQG LQFXEDWHG LQ DQ LFH EDWK r &f IRU KU ZLWK LQWHUPLWWHQW VKDNLQJ $W WKH HQG RI WKH LQFXEDWLRQ SHULRG PO RI 0 JO\FLQH GLVVROYHG LQ 0 VRGLXP ERUDWH S+ f ZDV DGGHG DQG VWLUUHG IRU PLQ DW r & 7KH UHVXOWLQJ VROXWLRQ ZDV WKHQ OD\HUHG RQ D 6HSKDGH[ [ FPf FROXPQ HTXLOLEUDWHG ZLWK 0 VRGLXP SKRVSKDWH S+ f DQG b JHODWLQ 7HQ GURS DOLTXRWV ZHUH FROOHFWHG LQ [ PP GLVSRVDEOH JODVV FXOWXUH WXEHV FRQWDLQLQJ PO 3%6b RYDOEXPLQ 7KH HOXWLRQ SDWWHUQ LV VKRZQ LQ )LJXUH 7KH LRGLQDWHG UGD[LQ SHDN ZDV WHVWHG IRU LPPXQRDFWLYLW\ E\ HPSOR\LQJ DQ DQWLJHQDQWLERG\ WLWUDWLRQ )LJXUH f 5,$ 3URFHGXUH 'D\ f DGG SL RI 5 DQWLVHUXP GLOXWLRQ LQ 3%6 S+ f WR HDFK WXEH FRQWDLQLQJ WKH SL RI VWDQGDUG DQG XQNQRZQ VROXWLRQV DQG WR WKH FRXQW WXEHV f DGG SL RI 3%6 WR WKH EODQN WXEHV QRQVSHFLILF ELQGLQJf f PL[ WXEHV ZHOO DQG LQFXEDWH DW r & IRU KU 'D\ DGG SL RI AM UHOD[LQ &30 WR HDFK WXEH PL[ FRYHU DQG LQFXEDWH DW r & IRU KU

PAGE 124

'D\ DGG SL RI *$5 DW GLOXWLRQ DQG \O RI 156 WR DOO WKH WXEHV H[FHSW WKH WRWDO FRXQW WXEHV 0L[ FRYHU DQG LQFXEDWH DW r & IRU KU 'D\ FHQWULIXJH DW 530nV IRU PLQXWHV GHFDQW VXSHUQDWDQW DQG FRXQW SUHFLSLWDWH 7KLV SURFHGXUH ZDV QRW HPSOR\HG EHFDXVH WKH QRQVSHFLILF ELQGLQJ ZDV YHU\ KLJK LQ WKH ILUVW EDWFK RI %ROWRQ DQG +XQWHU UHDJHQW UHFHLYHG IURP 1HZ (QJODQG 1XFOHDU b RI WRWDO ELQGLQJf 7KH DGGLWLRQ RI PDOH UDEELW VHUXP GLOXWLRQf WR WKH ILUVW DQWLERG\ GHFUHDVHG WKH DPRXQW RI QRQVSHFLILF ELQGLQJ WR EHORZ b RI WRWDO ELQGLQJ EXW DOVR GHFUHDVHG VHQVLWLYLW\

PAGE 125

R F )LJXUH 6HSDUDWLRQ RI UHOD[LQ SUHSDUHG ZLWK WKH %ROWRQ DQG +XQWHU UHDJHQW IURP XQERXQG FRPSRQHQWV XWLOL]LQJ D 6HSKDGH[ FKURPn DWRJUDSK\ FROXPQ [ FPf 7XEHV ZHUH LPPXQRUHDFWLYH ZLWK 5 DQWLVHUXP 5DGLRDFWLYLW\ H[SUHVVHG DV &30nV SHU \O RI VROXWLRQ

PAGE 127

)LJXUH $QWLVHUXP WLWUDWLRQ FXUYH 3RROHG WXEHV ZHUH WHVWHG ZLWK GHFUHDVLQJ GLOXWLRQV RI 5 DQWLVHUXP DQG b DQWLJHQ ERXQG ZDV GHWHUPLQHG $ GLOXWLRQ RI ZDV HPSOR\HG LQ WKH 5,$ DSSUR[LPDWHO\ b ELQGLQJf b %RXQG DPRXQW RI AM UHOD[LQ SUHFLSLWDWHG E\ WKH 5 DQWLVHUXP

PAGE 128

R %281' ),1$/ $17,6(580 &21&

PAGE 129

%,2*5$3+,&$/ 6.(7&+ 5XEH -RVH 3DUGR ZDV ERUQ 2FWREHU LQ +DYDQD &XED +H UHFHLYHG KLV XQGHUJUDGXDWH HGXFDWLRQ DW WKH 8QLYHUVLW\ RI 0LDPL )ORULGD JUDGXDWLQJ ZLWK D %DFKHORU RI 6FLHQFH GHJUHH LQ ELRORJ\ LQ +H UHFHLYHG KLV 0DVWHU RI 6FLHQFH GHJUHH DW 0DUTXHWWH 8QLYHUVLW\ 0LOZDXNHH :LVFRQVLQ LQ +H WDXJKW ELRORJ\ DW 0DWWDWXFN &RPPXQLW\ &ROOHJH EHWZHHQ DQG +H KDV EHHQ HQUROOHG DW WKH 8QLYHUVLW\ RI )ORULGD VLQFH 6HSWHPEHU DQG LV QRZ D FDQGLGDWH IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\

PAGE 130

, FHUWLI\ WKDW FRQIRUPV WR DFFHSWDEOH DGHTXDWH LQ VFRSH DQG 'RFWRU RI 3KLORVRSK\ FHUWLI\ WKDW FRQIRUPV WR DFFHSWDEOH DGHTXDWH LQ VFRSH DQG 'RFWRU RI 3KLORVRSK\ FHUWLI\ WKDW FRQIRUPV WR DFFHSWDEOH DGHTXDWH LQ VFRSH DQG 'RFWRU RI 3KLORVRSK\ FHUWLI\ WKDW FRQIRUPV WR DFFHSWDEOH DGHTXDWH LQ VFRSH DQG 'RFWRU RI 3KLORVRSK\ KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI /\QQ + /DUNLQ &KDLUPDQ n3URIHVVRU RI $QDWRP\ ‘! KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI $VVRFLDWH 3URIHVVRU RI $QDWRP\ KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 7KRPDV +RLOLQJHU $VVRFLDWH 3URIHVVRU RI $QDWRP\ KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI f 0LFKDHO )LHOGV $VVRFLDWH 3URIHVVRU RI $QLPDO 6FLHQFH

PAGE 131

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

PAGE 132

81,9(56,7< 2) )/25,'$


PURIFICATION, CHARACTERIZATION AND LOCALIZATION
OF RELAXIN IN THE PREGNANT GUINEA PIG
BY
RUBE JOSE PARDO
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1982

I wish to dedicate this dissertation to my parents, Mr. and
Mrs. Rube Pardo, and my grandmother, Gilda de la Torriente. This
dissertation is also dedicated in the memory of my grandfather, Jose
Elias de la Torriente.

ACKNOWLEDGMENTS
I would like to express my appreciation to the members of my
supervisory committee for their help in the work presented in this
dissertation. I especially wish to thank Dr. Lynn Larkin, chairman of
my committee, for his help and financial backing. I also wish to
acknowledge the following individuals who have aided me in my doctoral
studies: Dr. Fuller Bazer, Dr. Don Cameron, Mr. Alberto de LaPaz,
Dr. Asgi Fazleabas, Dr. Michael Fields, Dr. Phillip Fields, Dr. Don
Hay, Dr. Thomas Hollinger, Dr. Satya Kalra, Dr. Ernst Kallenbach, Mr.
Denny Player, Mr. Lane Powell, Dr. Ray Roberts, Dr. Lynn Romrell, Mrs.
Pauletta Sanders, Mr. Will Sanders and Dr. Howard Suzuki. A special
thank you goes to my good friends and fellow graduate students, Phil
Ruiz, Wayne Barbee, and Pat Fitzgerald. Finally and most importantly,
I wish to express my deepest appreciation and love to my parents,
Mr. Rube Pardo and Mrs. Georgina T. Pardo; my grandmother, Gilda de la
Torriente; my sisters, Margarita and Georgina; my brother, Roberto;
and my brother-in-law, Bahram.
in

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF ABBREVIATIONS vi
ABSTRACT viii
INTRODUCTION 1
Relaxin Assays 2
Cellular Localization of Relaxin 8
Isolation and Characterization of Relaxin 13
Relaxin in the Guinea Pig 22
Statement of Problem 25
MATERIALS AND METHODS 26
General Procedures 26
Detection of Relaxin 29
Purification and Characterization of Guinea Pig Relaxin. ... 36
RESULTS 40
Detection of Guinea Pig Relaxin 40
Purification and Characterization of Guinea Pig Uterine
Relaxin 47
DISCUSSION 50
R19 Antiserum: Detection of Guinea Pig Relaxin 50
Detection of Guinea Pig Relaxin with the PAP Technique .... 51
Detection of Guinea Pig Relaxin with Radioimmunoassay 52
Endometrial Glands and Their Role in Relaxin Production. ... 55
Possible Actions of Uterine Relaxin in the Guinea Pig 58
Purification and Characterization of Guinea Pig Relaxin. ... 60
BIBLIOGRAPHY 63
APPENDIX 1 - TABLES 72
APPENDIX 2 - FIGURES 77

APPENDIX 3 - IODINATION OF SUCCINIMIDE RELAXIN Ill
APPENDIX 4 - IODINATION OF RELAXIN WITH THE BOLTON AND
HUNTER REAGENT 114
BIOGRAPHICAL SKETCH 120
v

LIST OF ABBREVIATIONS
Bo
CMC
CPM
DAB
EG
EGC
GAR
gww
H 5 E
L
lac
IP
mw
NEPHGE
NSB
NRS
ODS
PAP
PAGE
PBS
R19
RIA
RP
zero count tube
carboxymethyl cellulose
counts per minute
3,3' diaminobenzidine
endometrial gland(s)
endometrial gland cell(s)
goat anti-rabbit IgG
gram wet weight
hematoxylin and eosin
uterine lumen
lactating
late pregnant
molecular weight
non equilibrium polyacrylamide gel electrophoresis
nonspecific binding
normal rabbit serum
octadecylsilica
peroxidase antiperoxidase
polyacrylamide gel electrophoresis
phosphate buffered saline
antiserum made to purified porcine relaxin
radioimmunoassay
peroxidase reaction product
vx

RPM revolutions per minute
SC subcutaneous
SDS sodium dodecyl sulfate
SE surface epithelium of uterine lumen
T total count tube
TCA trichloroacetic acid
U unit(s) of relaxin activity
vi 1

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PURIFICATION, CHARACTERIZATION AND LOCALIZATION
OF RELAXIN IN THE PREGNANT GUINEA PIG
By
Rube Jose Pardo
May 1982
Chairman: Lynn H. Larkin
Major Department: Medical Sciences (Anatomy)
It has been shown using the peroxidase-antiperoxidase immunocyto-
chemical technique that the endometrial glands of the pregnant guinea
pig are the source of the hormone relaxin. The presence of relaxin
has been demonstrated in uteri from day 30, day 45, day 60 pregnant and
late pregnant animals (days 65-67). The density of reaction product
deposition increased as pregnancy proceeded, with high deposition occur¬
ring in days 45 and 60 of pregnancy and in late pregnant animals. Little
or no immunoperoxidase labeling was observed in tissues from day 15,
nonpregnant and lactating animals (3 days postpartum). Immunoperoxidase
labeling was not seen in nonendometrial gland components of the uterus.
High biological and immunological activities were found in extracts of
uteri taken on days 45 and 60 of pregnancy and in late pregnant animals.
When a crude extract of late pregnant uteri was chromatographed
in Sephadex G-50, a fraction containing relaxin activity eluted in the
6,000 molecular weight range. This fraction was active in the mouse
uterine motility bioassay (1.50 U/mg), and promoted lengthening of the
viii

interpubic ligament in estrogen primed female mice. The bioactive
Sephadex fraction was further purified in a carboxymethylcellulose
(CMC) ion exchange column. A single peak from the CMC column demon¬
strated relaxin bioactivity (3.87 U/mg) in the mouse uterine motility
bioassay. The CMC purified guinea pig relaxin was compared to CMC
purified porcine relaxin in a two dimensional gel electrophoresis system.
The two purified relaxins were of similar molecular weights, with the
porcine hormone being slightly more basic. The guinea pig relaxin
molecule appears to be similar to porcine relaxin according to the
following criteria: (1) A continuous line of identity was obtained when
a 6,000 molecular weight fraction of relaxin from uteri of day 60 preg¬
nant guinea pigs was tested with porcine relaxin and antirelaxin serum
in double immunodiffusion plate analyses. (2) Both relaxin molecules
were inactivated when reacted with antirelaxin serum in an antiserum
test employing the mouse uterine motility bioassay. (3) Both relaxin
molecules were inactivated by trypsin and dithiothrietol, but not by
moderate heat.
IX

INTRODUCTION
Many mammals that give birth to large mature young have mechanisms
to compensate for a narrow pelvic width. One of the most dramatic
examples of this is found in the guinea pig, which gives birth to
relatively large young. In the guinea pig, pubic separation is so
extreme that the two halves of the pelvis are freely movable during the
birth process. It was Hisaw's interest in this phenomenon which prompted
him to ask whether certain humoral factors were responsible for the
morphologic changes associated with this process. Hisaw (1926, 1927)
was the first to relate this pelvic separation to the presence of a
blood factor later called relaxin (Fevold, Hisaw and Meyer, 1930). Since
its discovery, relaxin has been recognized as a hormone of pregnancy and
has been detected in many species of animals.
The physiological effects of relaxin are mainly concerned with the
female reproductive tract of mammalian species. Three of these effects
have been extensively reviewed in the literature: (1) relaxation of the
ligaments which stabilize the pelvic bones, (2) inhibition of uterine
contractions, and (3) softening of the cervix at term (Hisaw and Zarrow,
1950; Hall, 1960; Schwabe et al., 1978; Porter, 1979). The relaxation
of pelvic ligaments and inhibition of uterine contractions are the basis
of two important bioassays which are used to detect relaxin.
The following portions of the introduction will concentrate on
four areas of study on relaxin: (1) detection of relaxin, (2) cellular
localization of relaxin, (3) isolation and characterization of relaxin
and (4) description of relaxin research in the guinea pig.
1

2
Relaxin Assays
Relaxation of Pelvic Ligaments
The first qualitative bioassay for relaxin was the guinea pig
pubic symphysis palpation assay developed by Fevold et al. (1930).
An attempt to quantitate this assay was made by Abramowitz et al. (1944).
A guinea pig unit (U) was defined by these investigators as the dose of
relaxin that in 6 hours caused relaxation of the pubic symphysis (deter¬
mined by palpation) in at least eight of twelve estrogen primed, cas¬
trated female guinea pigs. Two basic problems were associated with this
assay: (1) the degree of subjectivity was high and (2) repeated use of
the same guinea pigs at first sensitized them to relaxin but then made
the animals refractory to the hormone after several months of use (Noall
and Frieden, 1956). All studies before 1960 exclusively employed the
guinea pig pubic symphysis assay and can, therefore, be questioned for
the reasons explained above.
The mouse interpubic ligament assay was later developed by Steinetz
et al. (1960), and offered a more sensitive and objective method of
assaying relaxin. In this assay, groups of sexually immature female
mice (18-20 g) were primed with a single injection of 5 pg estradiol
and 7 days later received injections of relaxin standards or unknowns
(three dose levels) in 1% benzopurpurine-4B. Eighteen to twenty-four
hours later, the mice were killed and their pubes dissected free of con¬
nective tissue and fat. The interpubic distance was measured using a
dissecting microscope fitted with an ocular micrometer and a transillum-
inating source. With this assay, dose response curves could be compared
between two relaxin preparations to determine whether the relaxins

3
elicited similar (parallel dose response curves) or dissimilar responses
in the experimental animals.
Inhibition of Uterine Contractions
Krantz et al. (1950) were the first to describe the ability of
relaxin extracts to inhibit spontaneous contractions of rat, guinea pig
and mouse uteri maintained in vivo and in vitro. Kroc et al. (1959)
improved the uterine motility assay further by utilizing uteri from
sexually immature, estrogen primed mice in an in vitro system. This
bioassay is more economical because mice are less costly than the larger
rodents. Also, the mouse uterus requires less relaxin to reduce con¬
tractions, thereby conserving the hormone. The mouse uterine motility
assay has been recently modified by Larkin et al. (1981). In this
modified assay each uterine horn from sexually immature estrogen primed
mice is divided and suspended in an aerated organ bath of Locke's solu¬
tion. The uterine segment is attached to a heart lever against 1 g of
tension and contractions are monitored with an ink writing kymograph.
The relaxin standard or unknown is tested for the ability to inhibit
spontaneous uterine contractions. One section of the horn is treated
with the standard, and the other with the unknown. By doubling the con¬
centrations of standard and unknown in the organ bath at 4 min intervals,
the response of the two uterine segments may be compared and the potency
of the unknown determined.
The guinea pig pubic symphysis assay is the most subjective of the
assays mentioned but was the most widely used until 1960. The mouse
interpubic ligament assay offers the refinement of objectivity, since

4
quantitative comparisons of the slopes of the dose response curves between
unknowns and standards can be made. The mouse uterine motility assay
offers the quickest and most inexpensive method for assaying relaxin
bioactivity, but does not provide the dose response data which are
available with the mouse interpubic ligament assay. Thus a combination
of assays can be used to counteract the shortcomings of one single
assay. All relaxin bioassays are relatively insensitive when compared
with the levels of relaxin detected with radioimmunoassay (RIA).
Radioimmunoassay
In 1972, Bryant developed the first homologous RIA* for porcine
relaxin. In this assay, a relatively impure relaxin preparation (NIH-R-
Pl, 440 U/mg) was iodinated with the chloramine-T-method of Hunter and
Greenwood (1962). This impure preparation was also used for the produc¬
tion of antiserum and for the relaxin standards. This RIA was used by
Bryant and collaborators for several studies (Bryant, 1972; Bryant and
Stelmasiak, 1974; Bryant et al., 1975; Bryant and Chamley, 1976; Bryant
et al., 1976) before it was discovered by Sherwood and O'Byrne (1974)
that porcine relaxin contained no tyrosine residues and therefore could
not be iodinated by the chloramine-T method. It was likely that Bryant
either iodinated some peptide contaminants within the relaxin preparation
or perhaps labeled a prohormone which contained similar antigenic deter¬
minants to relaxin. This possibility has been explored by Bryant-
Greenwood and Greenwood (1979) in a recent publication in which they
*A homologous porcine RIA is an RIA where
1. the antirelaxin serum is produced against porcine relaxin
2. the iodinated hormone and the radioinert standards are
porcine relaxin.

5
compared the RIA utilizing NIH-R-P1 relaxin with an RIA using a highly
purified relaxin fraction (CM-a', 3,000 U/mg). The NIH-R-P1 relaxin
was iodinated by the chloramine-T method of Hunter and Greenwood (1962).
The CM-a' relaxin was reacted with a succinimide ester and iodinated by
the method of Bolton and Hunter (1973). It was found that antisera to
CM-a' relaxin crossreacted with NIH-R-P1 relaxin. Also highly purified
CM-a' relaxin crossreacted with antisera made to NIH-R-P1 relaxin.
However, when the two assays were used to detect relaxin in serum of
pregnant ewes, the assay based on the crude relaxin preparation (NIH-
R-Pl) read values of relaxin ten times greater than those read with the
assay utilizing the highly purified hormone (CM-a'). This was interpreted
to mean that the RIA utilizing NIH-R-P1 relaxin was reading a broad
spectrum of immunoactivity and could have been detecting tyrosine contain¬
ing contaminants or a relaxin prohormone that was not detected by the
RIA utilizing the highly purified hormone.
In 1975, Sherwood and his co-workers developed a homologous RIA
for porcine relaxin which took into account the hormone's total lack of
tyrosine residues (Sherwood et al., 1975). This RIA utilized a highly
purified relaxin preparation containing CM-a', CM-a and CM-B fractions,
which they called native relaxin. Initial efforts to iodinate native
relaxin with the chloramine-T method failed. Therefore, a novel approach
was employed to covalently bind tyrosine to relaxin through an amide
linkage using the agent N-carboxy-L-tyrosine anhydride. The resulting
molecule was named polytyrosyl relaxin, because it contained 1.67 moles
of tyrosine per mole of relaxin, and was used for the development of all
phases of the RIA. Sherwood et al. (1975) reported detecting levels of

6
porcine relaxin as low as 32 pg whereas previously used bioassays were
sensitive in the low microgram range. Utilizing this RIA the presence
of relaxin has been demonstrated in sera of pregnant pigs (Sherwood et
al., 1977a; 1977b). However, the assay did not detect relaxin in sera
from pregnant guinea pigs or pregnant cows (Sherwood et al., 1975).
This observation may have resulted because of a number of reasons, how¬
ever, two that should be considered are that the antirelaxin serum did
not crossreact with relaxin from these species and that serum levels of
the hormone were below the level of detection of the assay.
A RIA employing polytyrosyl relaxin also has been established in
the laboratory of Dr. B. G. Steinetz. A Sephadex G-50 relaxin fraction
containing 1,000 U/mg was used to develop the antiserum utilized in
Steinetz's RIA. The main difference in the RIA procedures of Steinetz
and of Sherwood was the employment of different antirelaxin sera. The
Steinetz assay system has been used to demonstrate the presence of
relaxin in sera from pregnant rats, mice, hamsters, guinea pigs, dogs,
monkeys and humans (O'Byme and Steintez, 1976; O'Byme et al., 1976;
O'Byrne et al., 1978).
The Bolton and Hunter (1973) method of iodination has been utilized
by several investigators in the development of a RIA for relaxin. In
this method, 3-(4-hydroxyphenyl)-propionic acid N-hydroxy-succinimide
ester is radioiodinated according to the method of Hunter and Greenwood
(1962). The ester is then reacted with relaxin and an iodinated phenyl
group is incorporated into the epsilon amino groups of lysine and N-
terminus of the relaxin molecule. Bryant-Greenwood and her co-workers

7
have used this preparation in the development of a homologous porcine
RIA (Bryant-Greenwood and Greenwood, 1979; Yamamoto et al., 1981). This
homologous porcine RIA has been used by Yamamoto et al. (1981) to
determine relaxin levels in the purification of relaxin from human
placental basal plates. Parallel displacement curves existed between
the porcine and human purified relaxins, although the RIA was less sensi¬
tive in detecting human relaxin. Parallel displacement curves indicate
similar antigenicity between molecules.
Using the Bolton and Hunter reagent to iodinate relaxin, Loumaye
et al. (1978) also employed a homologous porcine RIA. They were able
to detect relaxin in serum of pregnant women, in extracts of corpora
lútea of pregnancy and in corpora lútea cyst fluid of pregnant and non¬
pregnant women.
The only homologous nonporcine RIA system has been developed for
the detection of rat relaxin by Sherwood and Cmekovic (1979). Equal
quantities of two ion exchange chromatography fractions (CM-1 and CM-2)
of rat relaxin were pooled and iodinated by the method of Bolton and
Hunter (1973). Antisera were raised in rabbits against the CM-1 and
CM-2 rat relaxin fractions. These relaxin fractions were also employed
as the radioinert standard. The assay could measure in the range of
32-3000 pg of rat relaxin, using an antirelaxin serum dilution of
1:100,000.
The two most commonly used methods of labeling relaxin are the
method of Sherwood et al. (1975), which results in a polytyrosyl relaxin,
and the method of Bolton and Hunter, which employs succinimide relaxin.
Differences in the results of studies utilizing these RIA methods seem

8
to be related to the primary antisera employed in the assays rather than
the iodination procedure used for labeling the hormone.
While RIA and other immunologic techniques are being used increas¬
ingly to detect relaxin, the bioassay still remains the most widely
used technique for relaxin detection. Although the RIA has the advantage
of increased sensitivity, the bioassay detects the biologically active
hormone.
Cellular Localization of Relaxin
One of the key areas of study concerning relaxin's role in preg¬
nancy and parturition has been to determine the cellular location of the
hormone during these physiological states. Immunocytochemical tech¬
niques have been the most commonly employed methods used to detect the
cellular location of relaxin in tissues. These techniques have been
used successfully to detect cells containing relaxin in the pig, cow,
rat and human.
Pig
In the pig, there is good evidence that the corpus luteum of
pregnancy is the principle source of relaxin. Belt et al. (1971) were
the first investigators to correlate levels of relaxin with cytoplasmic
granules in porcine luteal tissue. The accumulation of dense cytoplasmic
granules in granulosa lutein cells of late pregnant pigs, and the
decline in the number of granules after gestation, closely paralleled
the rise and fall of bioassayable corpus luteum relaxin in the same
periods. Kendall et al. (1978) utilized the immunoperoxidase technique
to localize relaxin at the ultrastructural level in cytoplasmic granules
of porcine granulosa lutein cells. Larkin et al. (1977) used

9
immunofluorescent localization methods employing antiporcine relaxin
serum (R8) to localize relaxin in granulosa lutein cells or pregnant
pigs. Furtner studies with the porcine ovary by Arakari et al. (1980)
have shown that the antirelaxin serum employed is of utmost importance
in the localization of relaxin when using immunolabeling techniques. An
antiserum produced against a crude relaxin preparation (NIH-R-P1, 440
U/mg) gave a diffuse pattern of immunofluorescence in the corpus luteum
of pregnancy, with the fluorescence localized mainly in the connective
tissue elements. On the other hand, an antiserum produced against
purified relaxin (CM-a', 3,000 U/mg) gave a sharp and precise localiza¬
tion within the cytoplasm of the luteal cells. There have been no
reports of localization of relaxin in uterine or placental tissues in
the pig.
Cow
The ovary of the pregnant cow has been shown to be a source of
relaxin with bioassay techniques (Castro-Hemandez, 1976). Fields et al
(1980) detected relaxin with the immunoperoxidase technique in ovaries
taken from cows in the middle and late Stages of pregnancy. Relaxin was
localized in the cytoplasm of the granulosa lutein cells. Measurable
quantities of relaxin were not found with bioassay in bovine uterus or
placenta. The presence of relaxin in the bovine uterus and placenta
was not evaluated using immunocytochemical techniques.
Rat
The ovary of the pregnant rat contains large quantities of extract
able relaxin (Fields and Larkin, 1979; Sherwood and Crnekovic, 1979).
Relaxin has been detected in the rat ovary with bioassay, RIA and

10
inmunocytochemical techniques. Whereas it is well established that the
ovary is a source of relaxin in the pregnant rat, the metrial gland of
the uterus and the placenta have been implicated as tissues which may
also contain relaxin.
Dallenbach-Hellweg et al. (1965) reported immunofluorescent local¬
ization of relaxin in metrial gland cells of the pregnant rat uterus, but
not in the ovary or placenta. The antiserum utilized was made in rabbits
to porcine relaxin (1,000 U/mg). Results from this study should be viewed
with caution for two reasons. First, controls used in the study were not
stringent, since no attempt was made to absorb the antirelaxin serum
with purified porcine relaxin. Second, work of several laboratories
shows that the metrial gland of the rat does not contain relaxin. Larkin
(1974) tested tissue extracts from day 14 pregnant rats for relaxin bio¬
activity. Ovarian, but not metrial gland extracts contained bioassay-
able amounts of relaxin. Anderson et al. (1975) could not detect relaxin
in metrial glands of pregnant rats using immunofluorescence, but could
detect labeling in the ovary. The ovarian fluorescence was localized
in the cytoplasm of granulosa lutein cells. The antiserum employed by
Anderson et al. (1975) was raised against an even less pure porcine
relaxin preparation (NIH-R-P1, 440 U/mg), than that employed by Dallenbach-
Hellweg et al. (1965); however, controls were more complete. Other studies
(Anderson and Long, 1978) showed that ovarian extracts contained relaxin
activity, and metrial gland extracts did not. Zarrow and McClintock
(1966) injected I labeled antibody to porcine relaxin into pregnant
rats and discovered substantial accumulations of label in the ovary and

11
placenta. This study may be criticized on two points. First, whole
organs were counted for radioactivity and thus one cannot state with
certainty if cellular relaxin crossreacted with the labeled antibody or
if the antibody crossreacted with receptor bound relaxin present in the
tissue. Also organs with a high blood capacity like the placenta might
have sequestered blood bound labeled antibody. Second, the antibody
utilized in the study was produced against a very crude porcine relaxin
preparation (WL 1164 lot A, 150 U/mg powder).
Rabbit
The ovary, uterus and placenta of the pregnant rabbit have been
reported to contain relaxin. Zarrow and O'Connor (1966) found relaxin in
the rabbit gestational corpus luteum by employing an indirect immuno-
fluorescent labeling technique; however, it was difficult to determine
from the published photographs whether the label was located intra- or
extracellularly. The antibody utilized in the above study was produced
in rabbits to porcine relaxin (WL 1164, lot 8; 622 U/mg powder). No
fluorescence was found in uterine or placental tissue. Zarrow and
Rosenberg (1953) reported bioactive relaxin in the ovary, uterus and
maternal placenta of pregnant rabbits with the highest level appearing
in the maternal placenta. This study also showed that ovariectomy of
pregnant rabbits with subsequent progesterone replacement therapy did
not result in decreased blood levels of relaxin. Fields et al. (1981)
isolated relaxin from extracts of rabbit placentae. However, a cellular
source of the hormone in the placentae was not reported, leaving open
the possibility that the relaxin was blood borne. It appears that the
rabbit is a species which has extra-ovarian sources of relaxin, most
likely the uterus and/or placenta.

12
Human
The ovary and placenta appear to be sources of relaxin in the
pregnant human. Dallenbach and Dallenbach-Hellweg (1964) discovered the
presence of relaxin in basal plate cells of human placentae using an
indirect immunofluorescence technique. The antiserum employed was made
in rabbits to a porcine relaxin preparation (1,000 U/mg). This finding
has been substantiated by several recent studies. Fields and Larkin
(1981) also detected relaxin in basal plate cells of human term placentae
using the immunoperoxidase technique. An antiserum (R19) raised against
purified porcine relaxin was utilized in these studies. Fields and
Larkin (1981) also showed that placentae which gave a positive stain
for relaxin also contained bioassayable relaxin. Yamamoto et al. (1981)
have shown that basal plates of cesarean and vaginally delivered placentae
contain bioactive and immunoreactive relaxin. The decidua of the preg¬
nant human also has been shown to contain bioactive relaxin by Bigazzi
et al. (1980), but at this time a cellular source of relaxin has not
been found in this tissue.
The ovary has been established as a source of bioactive and immuno¬
reactive relaxin in the pregnant human (O'Byrne et al., 1978; Szalchter
et al., 1980; Weiss et al., 1976; Weiss et al., 1977). Relaxin also
has been shown to be present in the human gestational corpus luteum
using immunoperoxidase localization (Mathieu et al., 1981). The local¬
ization of relaxin appeared in the perinuclear area of the luteal cells.
It appears that animals which require the ovary for the maintenance
of pregnancy, i.e., the pig and the rat, also have the ovary as the
principal source of relaxin. On the other hand, animals which do not

13
require the ovary for the maintenance of pregnancy, like the rabbit and
the human, seem to have extraovarian sources of relaxin. The validity
of the above generalization will be tested as future studies encompass
a larger variety of species.
Isolation and Characterization of Relaxin
Relaxin has been isolated and characterized from the ovary of the
pregnant pig (Sherwood and O’Byme, 1974; Schwabe et al., 1976; 1977),
the ovary of the rat (Fields and Larkin, 1979; Sherwood, 1979; Walsh
and Niall, 1980), the placenta of the rabbit (Fields et al., 1981)
and the placenta and decidua of the human (Bigazzi et al., 1980; Fields
and Larkin, 1981; Yamamoto et al., 1981).
Pig
The ovary of the pig has been shown to be the most abundant source
of relaxin and the majority of the biochemical work has been accomplished
on relaxin extracted from this tissue (Schwabe et al., 1978).
Doczi (1963 U.S. patent 3,096,246) was the first investigator to
extract relaxin from porcine ovaries utilizing an acid-acetone extraction
solution. Griss et al. (1967) utilized a purification technique similar
to that used by Doczi to extract and partially purify relaxin from the
porcine ovary.
An initial extraction in a solution of hydrochloric acid, acetone
and water was conducted and then the relaxin containing extract was
fractionated with gel chromatography and anion exchange chromatography.
These separation techniques yielded a basic polypeptide with a molecular
weight (mw) of 5,000 to 10,000 that had both uterine relaxing activity
and the ability to cause lengthening of the interpubic ligament. Sherwood

14
and O'Byrne (1974) used an extraction procedure similar to that of
Doczi (1963) and Griss et al. (1967) and were the first to fully charact¬
erize the porcine relaxin molecule. Relaxin obtained by this procedure
could be separated by carboxymethyl cellulose (CMC) ion exchange chroma¬
tography into three fractions: CM-B, CM-a, and CM-a'. These fractions
had mw and isoelectric points of: 6340 and pH 10.55 (CM-B); 6370 and
pH 10.72 (CM-a) and 6180 and pH 10.77 (CM-a'). None of the fractions
contained amino acid residues of histidine, tyrosine or proline and all
had equal potency (2,000 to 3,000 U/mg), as determined by the mouse
interpubic ligament bioassay. Each fraction consisted of two subunits,
an alpha and a beta chain linked by disulfide bridges. Amino acid
analyses of the CM-a alpha subunit showed it to contain 22 amino acid
residues. The beta subunit contained some microheterogeneity with amino
acids ranging from 28 to 31 in number. Schwabe et al. (1976; 1977),
using the same purification scheme as Sherwood and O'Byme (1974),
sequenced the porcine relaxin molecule. They showed the alpha and beta
chains to contain 22 and 26 amino acid residues, respectively, and also
found that porcine relaxin lacked histidine, tyrosine or proline. James
et al. (1977) also published the primary structure for porcine relaxin.
These investigators used the same purification scheme as Sherwood and
O'Byme (1974), but obtained an amino acid sequence different from that
obtained by Schwabe et al. (1976; 1977). The difference in the alpha
chain was minor (glutamine instead of glutamic acid in the 10 position).
The beta chain was found to contain 29 amino acids, with amino acids
from the twenty-third position to the end terminus being of a different

15
sequence than those found by Schwabe et al. (1976; 1977). Walsh and
Niall (1980) utilized a novel approach in the purification of porcine
relaxin. Tissues were immediately frozen in liquid nitrogen upon
removal from the animals, and homogenized in a cold solution consisting
of trifluoroacetic acid, formic acid, hydrochloric acid and sodium
chloride. After centrifugation of the homogenate, the supernatant was
pumped through an octadecylsilica (ODS) column to which the relaxin and
other peptides adhered. The solution resulting from this procedure was
then chromatographed in gel and CMC ion exchange columns. The resulting
relaxin preparation consisted of one relaxin peak, which contained 31
amino acids in its beta chain, and eluted in the same position as CM-a
porcine relaxin (31 amino acids) in the CMC ion exchange chromatography
column run. The Walsh and Niall technique thus eliminated the molecular
microheterogeneity previously reported by other laboratories, and they
concluded that the microheterogeneity was due to degradation during
extraction.
Further characterization of the porcine relaxin molecule resulted
in the discovery that porcine relaxin and insulin were closely related
molecules. Although the amino acid sequences of the two hormones were
not the same, there was a striking similarity in tertiary structure,
including the presence of disulfide bridges at corresponding positions
in the molecules (Isaacs et al., 1978; Blundell, 1979). Clues that the
three-dimensional configuration of porcine relaxin is important to its
biological activity came from the work of Schwabe and Braddon (1976)
who showed that partial oxidation of the tryptophan at the 18 position

16
of the beta chain led to biological inactivation of the molecule. Reduc¬
tion of the disulfide bonds of the relaxin molecule with dithiothrietol
also eliminated its bioactivity (Schwabe et al., 1978).
Evidence for a prorelaxin compound has been accumulating from
several sources. James et al. (1977) suggest that relaxin might be
cleaved from a proinsulin like compound by proteolytic enzymes. Since
arginine is present at the N-terminus of the alpha chain as well as the
C terminus of the beta chain, they envision a prorelaxin precursor with
connections between the 30 position in the beta chain and the 1 position
in the alpha chain. The proteolytic cleavage would take place at this
position in the molecule. These investigators have identified forms of
relaxin in pig ovarian extracts which differ in net charge and amino
acid composition from the 6,000 mw relaxin molecule and feel that these
may perhaps be considered intermediates in the conversion of prohormone
to hormone.
Frieden and Yeh (1977) have acquired evidence for a prorelaxin
like compound in porcine ovarian extracts. When these investigators
chromatographed NIH relaxin (440 U/mg), they separated the material
into two protein peaks. Approximately 70% of the relaxin activity was
found in a peak eluting in the 6,000 mw range. However, approximately
10% of relaxin activity was concentrated in a 40,000 mw fraction. When
this higher mw fraction was exposed to trypsin, some of the high mw
material was converted to a 6,000 mw relaxin. This low mw relaxin was
indistinguishable from purified porcine relaxin in gel chromatography,
polyacrylamide gel electrophoresis (PAGE) and biological activity in the
guinea pig interpubic ligament bioassay. It appears from the above

17
studies that relaxin, like insulin, may be cleaved from a larger mw
precursor.
Cow
Bovine relaxin has been purified from ovaries of the late pregnant
cows (Fields et al., 1980). In this study, crude extracts were prepared
from corpora lútea by the technique of Griss et al. (1967). Chromatog¬
raphy of the crude extract on a Bio-Gel P-10 column demonstrated two
fractions having mw of 1,400 and 6,000. Both fractions were shown to
inhibit mouse uterine contractions in vitro and induce lengthening of
the mouse interpubic ligament. Immunodiffusion analyses showed a con¬
tinuous precipitin line between the two cow relaxin fractions, the NIH-
R-Pl porcine relaxin and an antiserum (R19), produced against purified
porcine relaxin. The 6,000 mw fraction gave 3 bands when electrofocused:
pH 8.8, pH 10.1 and pH 11.5. The pH 10.1 form of bovine relaxin had the
highest biological activity (250 U/mg) according to the mouse uterine
motility assay. The low mw relaxin lost activity in the presence of
dithiothrietol (Fields et al., 1980).
Rat
In 1979 Sherwood reported the purification and characterization of
rat relaxin. Ovaries were homogenized in a saline solution and two forms
of relaxin were obtained after fractionation of the crude ovarian extract
with Sephadex G-50 gel chromatography and CMC ion exchange chromatography.
The two forms were designated 01-1 and CM-2, and each contained comparable
specific activity when assayed with the mouse interpubic ligament bio¬
assay. CM-1 and CM-2 had isoelectric points of pH 7.6 and pH 9.4,

18
respectively, and both had raw of approximately 6,000. Unlike pig
relaxin, rat relaxin contained histidine, proline and tyrosine. Also,
although giving a linear log dose-response curve in the mouse interpubic
ligament bioassay, the slope of the line was not parallel to the assay
slope of the purified pig relaxin standard. These results supported
earlier findings by Larkin (1974), who used crude preparations from
rat and pig ovaries. Fields and Larkin (1979) also reported on the
isolation of rat ovarian relaxin. They isolated a fraction from a Bio-
Gel P-10 column which eluted in the mw range of porcine relaxin and
contained a potency of 60 U/mg in the mouse uterus bioassay. Electro-
focusing of the Sephadex fraction yielded 3 peaks with isoelectric
points of pH 9.0, 8.7 and 7.8. These peaks had activities of 325, 425
and 125 U/mg, respectively. Walsh and Niall (1980) isolated ovarian
relaxin from pregnant rats using the ODS technique previously mentioned
and obtained one major relaxin peak after preparation on a CMC ion
exchange chromatography column. No isofocusing data were presented for
the rat relaxin molecule in the Walsh and Niall (1980) study.
John et al. (1981) studied the sequence homologies between rat and
porcine relaxins. They isolated rat relaxin from late pregnant rat
ovaries (days 18-21 of pregnancy), according to the technique of Walsh
and Niall (1980). Amino acid sequencing studies showed the alpha chain
of rat relaxin to be 24 residues long and the beta chain to be 35 resi¬
dues long. The rat relaxin molecule contained tyrosine and histidine.
Only limited homology existed between rat and porcine relaxin, with
approximately 40% of the amino acid residues in corresponding positions
being identical. Antigenic dissimilarities between the porcine and rat

19
relaxins were also shown by the observation that only slight cross¬
reactivity existed between antisera produced against porcine relaxin and
rat relaxin (Larkin et al., 1979; Fields and Larkin, 1979; Sherwood and
Cmekovik, 1979) .
Rabbit
The placenta of the rabbit had been reported to contain relaxin by
Zarrow and Rosenberg in 1953. Of the tissues tested (ovary, uterus,
placenta), the maternal portion of the placenta seemed to contain the
highest levels of biologically active relaxin. Lower levels were seen
in the fetal placenta and uterus. Further proof that rabbit placentae
contained relaxin was demonstrated by Larkin et al. (1979) who showed
that antiserum produced against porcine relaxin inhibited the activity
of rabbit placental extracts in the mouse uterine motility assay. Also,
a reaction of identity was obtained when a Bio-Gel P-10 fraction of
rabbit placental extracts was compared with purified porcine relaxin
and an antiserum made against porcine relaxin in an agar double immuno¬
diffusion assay (Larkin et al., 1979).
This preliminary work led Fields et al. (1981) to purify relaxin
from the rabbit placenta. After extraction using a modified Griss
method (Griss et al., 1967), separation was achieved on a Bio-Gel P-30
column. The Bio-Gel P-30 fraction eluting at 6,000 daltons contained
low bioactivity in the mouse uterine motility bioassay (1.50 U/mg).
When this fraction was chromatographed in a CMC ion exchange column, a
single peak containing 15 U/mg was eluted. The mouse interpubic liga¬
ment assay was conducted on the CMC fraction. The dose response curve

20
for rabbit placental relaxin was parallel to the dose response curve of
the porcine standard. With this assay, the CMC fraction was calculated
to have a biological activity of 21.6 U/mg. Electrofocusing of the CMC
peak resulted in the separation of four distinct fractions.
Human
Whereas it has been established that the ovary is a source of
relaxin in the pregnant human (Weiss et al., 1976; Weiss et al, 1977;
O'Byrne et al., 1978; Szalchter et al., 1980), characterization of
relaxin from the ovary has not been accomplished. On the other hand,
recent reports of isolation of a placental relaxin have been published
(Fields and Larkin, 1981; Yamamoto et al., 1981). These recent reports
support the earlier work of Dallenbach and Dallenbach-Hellweg (1964) who
found immunoreactive relaxin in the basal plate of the human placenta
using indirect immunofluorescence. Bigazzi et al. (1980) have demon¬
strated relaxin production by the decidua capsularis in vitro and have
extracted relaxin from decidual tissue collected from term pregnancies.
Fields and Larkin (1981) first isolated and purified human placental
relaxin using the extraction technique of Griss et al. (1967). They
found human relaxin to be a peptide similar to porcine relaxin in molecu¬
lar weight. The biological activity of an isolated Bio-Gel P-30 fraction
(6,000 mw) as determined by the mouse uterine motility assay, was 11.9
U/mg. The same fraction produced a linear response in the mouse inter-
pubic ligament bioassay, which was parallel to the porcine standard.
Electrofocusing of the active fraction produced one peak having an
isoelectric point of pH 11.4 and a biological activity of 45 U/mg. The
electrofocused fraction exhibited a continuous line of identity with no

21
spurring in double immunodiffusion analyses when tested against purified
porcine relaxin. Incubation of the human relaxin with dithiothrietol
inactivated the hormone, indicating that disulfide bonds were necessary
for its biological activity.
Yamamoto et al. (1981) were also able to detect relaxin in extracts
of basal plates of human placentae. Placentae from cesarean deliveries
were found to contain five times higher relaxin levels than placentae
from normal deliveries. A single immunoreactive peak (as determined by
RIA) eluted in the 6,000 mw range from a Sephadex G-50 gel chromatography
column. The pooled active peak was applied to a CMC ion exchange column
and eluted with a salt gradient. Three relaxin fractions were obtained
from the CMC column and were called CMc-1, CMc-2, and CMc-3. CMc-1 and
CMc-2 eluted prior to the start of the salt gradient, while CMc-3
eluted at the start of the salt gradient (0.1 M NaCl). The three relaxin
peaks had parallel dilution curves when assayed in a homologous porcine
relaxin RIA. The CMc-2 fraction contained the only biological activity
as detected by the mouse uterine motility assay. Electrofocusing and
mouse interpubic ligament data were lacking.
Concurrent with the observations from Larkin's and Bryant-
Greenwood's laboratories were the observations of Bigazzi et al. (1980)
indicating that relaxin could be obtained by scraping the maternal
surface of fetal membranes gathered from normal deliveries. A homogenate
from the decidual tissue was obtained and fractionated. Only one fraction
from a Sephadex G-50 column contained relaxin biological activity as
shown by the rat uterine motility inhibition assay and the mouse inter¬
pubic ligament assay. This fraction eluted in the mw range of porcine

22
relaxin and had a tissue level of 15.0-33.5 U/mg of fresh tissue.
Further purification of the extract was not reported.
The relaxins studied to date appear similar in that they have
S-S linkages and an approximate mw of 6,000. Some differences, however,
exist among pig relaxin and relaxins purified from other species: (1)
the porcine relaxin has the highest specific activity in the bioassays,
(2) the relaxins from various species differ in isoelectric points, (3)
the two relaxins in which amino acid sequencing has been done appear
to be different, i.e., porcine relaxin contains no tyrosine or histidine
while rat relaxin does, and (4) a low mw form of bovine relaxin has
been reported. While not all relaxins have been studied with RIA, they
have all been characterized utilizing bioassay techniques.
Relaxin in the Guinea Pig
Early literature has suggested that nonovarian sources may be
very important in the production of relaxin in the guinea pig. Hisaw
(1926) was the first to discover that blood serum from pregnant guinea
pigs and rabbits, when injected subcutaneously (SC) into virgin guinea
pigs during early post-estrus, caused pubic symphysis relaxation six
hours later. Hisaw et al. (1944) further demonstrated that the pubic
ligaments of castrated guinea pigs pretreated with estradiol for 4 days
could respond to a single injection of progesterone and exhibit
increased pelvic mobility within 72 hours. Castrated, hysterectomized,
and estrogen-treated guinea pigs, on the other hand, did not respond to
progesterone treatment regardless of the progesterone dose. These same
animals could, however, respond to small quantities of relaxin within

23
six hours. These studies indicated that the estrogen primed uterus
could be induced to produce relaxin with progesterone treatment.
The status of relaxin in the guinea pig was equivocal because
some investigators obtained relaxation of the pelvis of the guinea pig
by estrogen therapy alone (Brouha, 1933) or with combinations of estrogen
and progesterone (Fugo, 1943). It should be pointed out that important
differences existed among Hisaw's observations and those of Brouha and
Fugo. The most obvious difference was that the time required to produce
a reaction in the relaxin treated, castrated animal pretreated with
estrogen was very short (6 hr). On the other hand, estrogen alone
(18-20 days) or combinations of estrogen and progesterone (2-4 days)
took much longer to elicit their effect.
Hisaw's early findings were confirmed by Zarrow (1947; 1948), who
found bioassayable relaxin in the blood of guinea pigs during middle and
late pregnancy, but not after parturition. He also noted relaxin activity
in extracts of the uterus and placentae on days 56 and 63 of pregnancy.
This added credence to Hisaw's theory that the uterus was responsible
for relaxin production in the pregnant guinea pig. Zarrow (1948)
further confirmed this by showing that progesterone could elicit forma¬
tion of relaxin in a castrated estrogen-primed guinea pig. Progesterone
did not cause production of relaxin in an estrogen-primed castrated and
hysterectomized animal regardless of the dose involved. This work,
although giving strong indication as to the tissue source of relaxin in
the guinea pig, relied exclusively on the cumbersome and subjective
guinea pig pubic symphysis assay. However, recent results have supported
the observations of Hisaw and Zarrow, rather than conclusions obtained by
Brouha and Fugo.

24
Recently, O'Byrne and Steinetz (1976) assayed sera from 4
pregnant guinea pigs at different stages of gestation with RIA. They
used a homologous RIA employing antibodies to porcine relaxin, which
was able to detect as little as 0.1 ng of the guinea pig relaxin. They
found that peripheral blood levels of relaxin gradually increased from
an average of less than 0.2 ng/ml in the 20 day pregnant guinea pigs
to just over 0.4 ng/ml in the 50 day pregnant animals. Postpartum
animals (24 hr after delivery) still contained high relaxin levels
(average 0.5 ng/ml). This study was only concerned with overall serum
levels of immunoreactive relaxin and did not look at individual tissue
levels. Bioassays were not conducted.
Boyd et al. (1981) used a homologous porcine RIA to assay plasma
relaxin immunoactivity in guinea pigs during the estrus cycle, throughout
mid to late pregnancy and parturition, and during lactation. Although
variability among animals was high, several major points could be drawn
from the study: (1) during the estrus cycle, relaxin levels were lowest
during estrus (2 ng/ml) and highest during portions of diestrus and
proestrus (5-6 ng/ml), (2) during the latter stages of pregnancy, relaxin
levels were higher (12-14 ng/ml), decreasing to basal levels after
parturition (2-4 ng/ml), and (3) during lactation, suckling did not
elevate relaxin levels in nursing dams, and in some instances, actually
decreased them.
In summary, work previous to 1950 indicated that relaxin was
present in the blood, uterus and placenta of pregnant guinea pigs. It
also showed that progesterone somehow stimulated production of relaxin

25
by the uterus in estrogen primed, castrated guinea pigs. Only recently
has RIA been employed to detect the presence of relaxin in serum of
pregnant and cycling guinea pigs.
In the past, the guinea pig was used extensively as an experimental
animal in relaxin work. This animal, however, has been neglected in
recent research due possibly to several reasons: (1) interest in guinea
pig relaxin decreased when newer, faster and less expensive bioassay
techniques using other animals became available, (2) corpora lútea of
the pig became established as the main source of relaxin, (3) cost of
keeping guinea pig colonies increased, compared to other laboratory
rodents, and (4) investigators focused on the ovary as being the only
source of relaxin in many mammals. There are, on the other hand, several
compelling reasons to study relaxin in the guinea pig. The guinea pig
is quite similar to the human in placentation, hormonal changes which
occur during gestation and the presence of an extra ovarian source of
relaxin (Zarrow, 1948; Pardo et al., 1980).
Statement of Problem
The primary goal of this research is to study relaxin in the
guinea pig. Studies proposed are designed to answer the following
questions: (1) Is relaxin produced by nonovarian sources in the guinea
pig? If so, what tissue and cell types produce the hormone? (2) What
are the tissue and serum levels of relaxin in the guinea pig throughout
pregnancy and lactation? (3) Can the rise and fall of serum and tissue
levels of relaxin be correlated with immunocytochemical studies? (4)
What are the physical and biochemical characteristics of the guiñe pig
relaxin molecule?

MATERIALS AND METHODS
General Procedures
Experimental Design, Treatment of Animals and
Collection of Specimens
Guinea pigs obtained from a local vendor were housed in the
University of Florida Health Center Animal Resources Department, and had
access to food and water ad libitum and a photoperiod of 12 hr light
and 12 hr dark. Adult females were housed with a male and pregnancy
was timed from the day on which sperm were found in a vaginal smear.
Animals used in ovariectomy studies were anesthetized with 0.88
ml/Kg Innovar-Vet purchased from Pitman-Moore, Inc., Washington Cross¬
ing, NJ, and bilaterally ovariectomized through two flank incisions.
Two weeks after the operation, the animals were started on a daily regi¬
men of hormone injections. Animals were given one of the following:
(1) estrogen alone (10 yg), (2) estrogen (10 yg) and progesterone (1 mg)
together, or (3) no injections. The hormones were mixed in sesame seed
oil and injected SC at the back of the neck. Estradiol dipropionate
was obtained from Ciba Pharmaceutical Products, Inc., Summit, NJ. Pro¬
gesterone was obtained from Eli Lilly and Co., Indianapolis, IN. Injec¬
tions were given daily at approximately 11:00 AM for 15 days (time needed
for estrogen-progesterone treated animals to undergo relaxation of the
pelvic ligaments (Zarrow, 1948)). Two animals were used for each of the
three treatments and were monitored daily for pelvic flexibility by
manual palpation.
26

27
All animals were killed at the same time of the day (11:00 am
+_ 1 hr). The animals were anesthetized with pentobarbitol (2.5 mg/100 g
body weight) and exsanguinated via cardiac puncture. The reproductive
tract was removed immediately and portions of the uterus were fixed in
Bouin's solution for histologic study. This tissue was processed for
paraffin embedding. The remainder of the uterus was frozen at -20° C
and later used in the extraction procedure.
Antirelaxin Sera
Antisera against highly purified porcine relaxin was produced in
New Zealand white rabbits as described by Larkin et al. (1977). In this
technique, 2 mg of a pig relaxin preparation (WL 150, 150 U/mg) obtained
from Warner Lambert, Inc., Morris Plains, NJ, were run on PAGE. The
bands were localized by fixing them in trichloroacetic acid (TCA) (15%) ,
and staining in 0.6% Coomassie blue in 15% TCA. Three bands were present
and were named Cl, C2, and C3; Cl being the closest to the anode. The
C2 bands were then cut out of the gels and homogenized in an equal volume
of Freund's complete adjuvant and injected into New Zealand white rabbits.
Subsequent injections were given with the gels homogenized in Freund's
incomplete adjuvant. The injection schedule was as follows: Rabbit 19
was given one SC injection per week for six weeks. The injections con¬
taining six-C2 bands were given dorsally between the scapulae. Booster
injections consisting of six-C2 bands were given approximately every
two months.
R19 antiserum has been shown to inhibit the biological activity
of porcine, cow and rabbit relaxins in vitro (Larkin et al., 1979).

28
Also it has been used to detect relaxin immunocytochemically in cells of
the cow ovary (Fields et al., 1980], and human placenta (Fields and
Larkin, 1981).
Tissue Extraction
Preparation of crude uterine extracts was accomplished by utilizing
one of two methods. Initially, tissues were extracted with the acid-
acetone procedure of Griss et al. (1967). This procedure was employed
for the extraction of uteri taken from individual animals and the extract
was used for bioassay and RIA experiments. Recently, a new extraction
procedure for relaxin was reported by Walsh and Niall (1980). This
newer technique was employed to extract relaxin from uteri and the
resulting preparations were used in purification and characterization
studies. A more detailed account of these techniques is given below.
Griss procedure.—The extraction procedure of Griss et al. (1967),
was used for extraction of uteri utilized in bioassay and RIA experi¬
ments. Cold extraction solution (acetone:water:hydrochloric acid, 5.0:
2.83:0.17 ratio) was added to minced frozen tissues at a ratio of 5:1
(ml/g), and homogenized in a Sorvall Omni-mixer at 4° C. The extract
was incubated for 24 hr at 4° C and then centrifuged at 3000 RPM's
(4° C) for 30 min in a Beckman J-21c centrifuge equipped with a JA-14
rotor. Five volumes of acetone were added to the supernatant and the mix¬
ture was stored at -20° C for 24 hr. The majority of the supernatant
was decanted and the precipitate pelleted by centrifugation at 3000 RPM's
for 10 min in a JA-14 rotor and air dried. The dried powder was weighed
and stored in a sealed container at room temperature.

29
Walsh and Niall procedure.—The extraction procedure of Walsh
and Niall (1980) was utilized in the purification and characterization
stages of the research because it had been reported to yield more
relaxin with less proteolysis. Uteri were removed from late pregnant
guinea pigs (65-67 days), immediately frozen in liquid nitrogen and
stored at -80° C in a Reveo freezer until extracted. Twenty gram aliquots
of minced frozen uterus were placed in 200 ml of a cold solution of
15% trifluoroacetic acid, 5% formic acid, 1% NaCl and 1 M HC1. The
tissue was homogenized for 2 min in a Sorvall Omni mixer. The homogen¬
ate was centrifuged (4° C) for 30 min at 10,000 RPM's in the JA-14
rotor. The resulting supernatant was filtered through Whatman filter
paper (No. 541) and a 0.45 ym pore millipore filter. Octadecylsilica
columns, purchased from Waters Associates, Millford, MA, were preequili¬
brated by passing 30 ml of an 80% acetonitrile, 0.1% trifluoroacetic
acid solution followed by a 30 ml wash of distilled water. The relaxin
containing supernatant was pumped through three ODS columns twice and
the columns were washed with 30 ml of a 10% acetonitrile, 0.1% tri¬
fluoroacetic acid solution. The eluate was evaporated to near dryness
at 38° C and resuspended in a known volume of 0.01 M ammonium acetate
buffer pH 5.
Detection of Relaxin
Immunocytochemical Localization of Relaxin
Immunoperoxidase staining was conducted as described by Stemberger
(1979) according to the following protocol. All dilutions of antisera
were carried out with phosphate buffered saline (PBS) pH 7.4, and the
incubations were conducted at room temperature. Paraffin sections

30
(6 pm in thickness) were deparaffinized immediately prior to use. Normal
goat serum (1/30 dilution) was applied to the sections for 30 min. The
slides were drained but not rinsed and 4 drops of either R19 antiserum
or control solutions of varying concentrations were applied to the
sections for 30 min. The slides were rinsed with a stream of PBS and placed
in Copeland jars containing PBS for three, three min rinses. The slides
were drained of excess PBS, and blotted to absorb excess PBS from around
the sections. Four drops of goat antirabbit IgG (GAR) (1/20 dilution),
purchased from Polysciences Inc., Warrington, PA, were applied to the
sections for 30 min. The sections were rinsed, drained and blotted as
described previously. Four drops of peroxidase-antiperoxidase (PAP)
(1/80 dilution with 0.05 M tris saline pH 7.6), purchased from Stem-
berger-Meyer Immunocytochemicals, Jarretsville, MD, were applied to the
sections for 30 min. The sections were rinsed, drained and blotted as
described previously. The PAP was visualized by incubating the slides
in a 5 mg% DAB solution (3,3' diaminobenzidine) type II, purchased from
Sigma, St. Louis, MO, with 0.01% ^2^2 ^or min. The slides were then
washed in distilled water for 5 min, briefly treated with 1% 0s0^,
rinsed in distilled water, dehydrated through alcohols and xylene, and
covers lips were applied. The following controls were carried out: (1)
substitution of the R19 antiserum with serum from a male rabbit that had
not been immunized against relaxin (NRS), (2) omission of the R19 anti¬
serum and replacement with PBS (pH 7.4), (3) absorption of the R19 anti¬
serum with porcine relaxin standard (NIH-RXN-P1), and (4) successive
dilutions of the R19 antiserum.

31
Bioassay of Relaxin Containing Extracts
All mice used in the bioassays were females of the ICR strain
which were initially obtained from Flow Laboratories (Dublin, VA).
Mouse uterine motility.--The in vitro mouse uterus bioassay as
described by Kroc et al. (1959) and modified by Larkin et al. (1981) was
employed to detect relaxin in tissue extracts. Female mice (16-18 g)
were primed with 0.1 ml of estradiol dipropionate (50 yg/ml). The mice
were killed 7 or 8 days later and their uteri removed. Each horn of the
uterus was divided in to two portions and each portion was suspended in
a test tube containing 20 ml of Locke's solution at 37° C. The uterine
segments were attached to a heart lever against 1 g tension and con¬
tractions were recorded on an ink writing kymograph. Specific volumes
of either NIH-RXN-P1 standard relaxin preparation or unknown solutions
of known concentrations were added to the tubes so that the bath concen¬
trations were doubled every 4 min. Specific activities were calculated
using the following equation:
Specific Activity of Unknown = x — x ^pAS
VU CU
where VS is the volume (in yl) of standard relaxin preparation needed to
reduce the uterine contractions by half, VU is the volume (in yl) of
unknown relaxin preparation needed to reduce the uterine contractions by
half, CS is the concentration of the standard relaxin preparation (in
yg/ml), CU is the concentration of the unknown relaxin preparation (in
yg/ml), and SpAS is the specific activity of the NIH-RXN-P1 relaxin stand¬
ard (U/mg protein).
Mouse interpubic ligament.--The in vivo assay for relaxin activity
was employed according to the technique of Steinetz et al. (1960). The

32
length of the interpubic ligament was determined in a three point
parallel line assay employing 20 mice at each dose level of the relaxin
standard and 15 mice at each dose level of the unknown. At day 0,
virgin prepuberal female mice (18-20 g weight) were primed with an SC
injection of 5 yg estradiol cypionate purchased from the Upjohn Co.,
Kalamazoo, MI, in 0.1 ml of sesame seed oil.
On day 7, the relaxin standard (NIH-RXN-P1) and unknowns of com¬
parable levels of activity (as determined by the mouse uterine motility
bioassay) were injected SC in 0.2 ml of a 1% solution of benzopurpurine-
4B. Control mice received 0.1 ml of estradiol cypionate and 0.2 ml 1%
benzopurpurine-4B. The dose levels for the NIH-RXN-P1 standard were
0.5 yg, 0.25 yg and 0.125 yg per mouse. The dose levels of the guinea
pig Sephadex G-50 fraction were 1 mg, 0.5 mg and 0.25 mg. Eighteen to
twenty-four hours later the mice were killed in a CC^ chamber, the abdom¬
inal cavities opened and the uteri examined for evidence of estrogen prim¬
ing. No mice exhibited "threadlike" uteri due to lack of priming. The
anal and vulval areas and upper half of the trunk were dissected away
with scissors, thereby removing the skin and all pelvic organs surround¬
ing the pubic symphysis. The pelvis was positioned under a light source
allowing a beam of light to pass through the pubic ligament. The short¬
est distance between the edges of the pubic bones was measured with a
dissecting microscope fitted with an occular micrometer. Results were
evaluated by the method of least-squares of variance; the computer
program was PROC CLM of Statistical Analysis System (Barr and Goodnight,
1976). The mathematical model was preparation (NIH versus guinea pig)

33
and dose (3 levels). Dose effects were examined further by polynomial
regression. Differences in dose/trends between preparations (NIH versus
guinea pig) were examined by tests of heterogeneity of regression. A
valid assay is one in which there is a significant (P>0.01) linear
regression of response to log dose, no divergence from parallelism to
the NIH-RXN-P1 standard, no quadratic regression components, and a
lambda value of less than 0.4. The results were expressed as U/mg
relative to the NIH-RXN-P1 porcine relaxin standard.
Radioimmunoassay
Three different iodination methods were attempted in the devel¬
opment of the homologous porcine RIA for guinea pig relaxin. Since
sufficient amounts of purified guinea pig relaxin were not available
for the RIA experiments, it became necessary to employ porcine relaxin
for both the immunization procedure and for iodination. The first two
procedures involved the use of the Bolton and Hunter reagent for the
iodination of porcine relaxin (NIH-RXN-P1) (Bolton and Hunter, 1973).
These two procedures were not utilized in the research reported and
specific information about these assays will be found in Appendices
3 and 4. In the third procedure, polytyrosyl relaxin was iodinated by
the method of O'Byrne and Steinetz (1976).
Iodination of polytyrosyl relaxin.--The iodination was conducted
according to the technique of O'Byrne and Steinetz (1976) with some
modifications. Polytyrosyl relaxin (5 yg), donated by Dr. B. G. Steinetz
of the Ciba Geigy Corp., Ards ley, NY, was dissolved in 50 yl of 0.1 M
sodium phosphate buffer pH 7.5. The polytyrosyl solution and 1 m Ci
12 S
I purchased from the Amersham Corp. were added to a 10 x 75 mm test

34
tube coated with 100 yg dried iodogen. Iodination was achieved util¬
izing the technique of Markwell and Fox (1978). Iodogen (1,3,4,6-
tetrachloro-3,6-diphenylglycouril) was purchased from the Pierce
Chemical Corp., Rockford, IL. The reaction solution was mixed at room
temperature for 15 min with intermittent shaking and then transferred
to a 1 x 18 cm column of Sephadex G-25 preequilibrated with 0.5 M
sodium phosphate buffer pH 7.0. Fractions (20 drops/tube) from the
gravity fed column were collected in 10 x 75 mm tubes containing 0.5 ml
of PBS 1% ovalbumin pH 7.0. Ten microliters of the pooled assay tubes
of the polytyrosyl relaxin peak contained 160,000 cpm of radioactivity.
Approximately 33% of the -^I-labelled polytyrosyl relaxin was precipi-
1 25
table in antibody excess. The I-labelled polytyrosyl relaxin was
used for four weeks after iodination before a noticeable drop in sensi¬
tivity was noticed in the RIA.
nr
Development of RIA utilizing I-labelled polytyrosyl relaxin.--
Fractions containing the ■’■‘^I-labelled relaxin were pooled and employed
in the development of the RIA used to detect guinea pig relaxin. For
the detection of relaxin in crude uterine extracts, 20 mg of the acid-
acetone extracted powder was suspended in 1 ml of PBS-1% ovalbumin,
pH 7.0 and the resulting suspension centrifuged to remove nonsolubilized
material. The supernatant was then diluted 1:1 with PBS-1% ovalbumin,
and tested in the RIA.
Double antibody RIAs were conducted in 10 x 75 mm disposable glass
culture tubes. Quantities of relaxin standard solutions (NIH-RXN-P1)
containing 3.25-2000 pg of relaxin in PBS-1% ovalbumin or volumes of
uterine extract supernatants were added to the culture tubes. Sufficient

35
quantities of PBS-1% ovalbumin were added to each tube to bring the
volume to 500 yl. One hundred microliters of R19 antiserum (1:25,000
final dilution) in 0.05 M ethylene diamine tetraacetic acid-PBS con¬
taining 6% male rabbit serum were added to each tube. The tubes were
vortexed and then incubated at 4° C for 24 hr. One hundred microliters
1
of I-labelled relaxin (10,000-15,000 CPM) in PBS-1% ovalbumin were
added to each tube, the tubes were vortexed and then incubated for 24 hr
at 4° C. The tubes were then centrifuged at 3000 RPM's for 30 min,
drained of supernatant and the pellets counted in a Searle analytic
gamma counter. A standard curve employing NIH-RXN-P1 relaxin was run
concurrent with every assay. Radioactivity expressed as % bound was
plotted in a % bound versus log dilution curve and unknown guinea pig
values were read off the standard curves and expressed as porcine relaxin
equivalents. The following controls were employed: (1) Total count tube
i or
(T): 100 yl of i'i0I-labelled polytyrosyl relaxin gives the total
amount of isotope added to each tube. (2) Nonspecific binding (NSB) :
primary antiserum (R19) was omitted to determine whether there was
any nonspecific binding of the I-labelled relaxin to other assay
components. (3) "Zero" count tube (Bo): radioinert relaxin was not
added to determine the maximum amount of possible binding of the 1 JI-
labelled relaxin to the antirelaxin serum. Percent binding (% B) was
determined by dividing the radioactivity of the standard or unknown tubes
(bound) by the "zero" count tube (Bo). Nonspecific radioactive binding
was subtracted from all values before calculations were made.
o. D bound-NSB
'O D —
Bo-NSB

36
RIA characterization.--The specificity of the homologous porcine
RIA used to detect guinea pig relaxin was tested in two experiments.
First, dilution curves obtained with crude and semi-purified preparations
of guinea pig relaxin were compared to the dilution curves obtained
using purified NIH-RXN-P1 porcine relaxin. Secondly, the levels of
relaxin crossreactivity were determined in preparations of crude
extracts taken from uteri in varying stages of pregnancy. Interassay
reproducibility was determined by measuring the variability in a 125
pg sample of porcine NIH-RXN-P1 relaxin between 4 different standard
dilution curves. Intraassay reproducibility was determined by measur¬
ing a 125 pg sample of porcine NIH-RXN-P1 relaxin in the same assay 6
different times.
Purification and Characterization of Guinea
Pig Relaxin
Purification
Gel filtration.—The Walsh and Niall (1980) procedure was utilized
to extract 118.50 g of uterine tissue from 5 late pregnant guinea pigs
and the crude extract from the ODS extraction (188.8 mg protein) was
suspended in 10 ml ammonium acetate buffer, pH 5.0. Protein was
determined by the method of Lowry et al. (1951). The protein solution
was layered on a Sephadex G-50 (fine) column (2 x 100 cm), purchased
from Pharmacia Fine Chemicals, Uppsala, Sweden, and equilibrated with
the same buffer. The column was developed at room temperature at a rate
of 7.5 ml/hr. Material eluting from the columns was monitored at 280 mm
wavelength with a Beckman Acta III Spectrophotometer. Fractions were
collected every 24 min. Fractions containing the protein peaks were

37
pooled, lyophilized, and assayed using the mouse uterine motility
bioassay. Columns were calibrated by using a series of low mw markers
and by using a porcine relaxin standard. Buffer containing sodium
azide (0.05%) was pumped through the column between experiments to
eliminate bacterial growth.
Bio-Gel P-30, purchased from Bio-Rad Laboratories, Richmond, CA,
was utilized as the gel chromatrography resin to prepare the relaxin
fraction used in the double immunodiffusion experiments. This gel was
equilibrated and run in exactly the same manner as the Sephadex G-50
resin.
Ion exchange chromatography.--The active fraction from the Sephadex
column (35.5 mg) was applied to a 0.8 x 5 cm CMC column (CM-52) purchased
from Whatman Ltd., Springfield, England, and then equilibrated with
0.01 M ammonium acetate buffer, pH 5.0 until all unadsorbed material was
removed. The column was developed at a rate of 9 ml/hr with a linear
NaCl gradient (0.1 M to 0.3 M) in 0.01 M ammonium acetate buffer pH 5.0
to a final conductivity of 20 m Mho. Fractions (1.5 ml) were collected
every 10 min.
Characterization
Double immunodiffusion studies.--Double immunodiffusion plates
were employed as described by Clausen (1969) for the microtechnique.
Petri dishes of 12 x 60 mm (1.5% agar in 0.85% saline with 0.1% sodium
azide) were used. A center well was filled with 5 yl of R19 antiserum
and peripheral wells were filled with 5 pi of a Bio-Gel P-30 6,000 mw
fraction from guinea pig uterus (6 mg/ml), and NIH-RXN-P1 porcine
relaxin. The substances were allowed to diffuse at room temperature for
24 hr.

38
Two dimensional gel electrophoresis.--Two dimensional gel electro¬
phoresis was conducted using a modification of the Horst et al. (1980)
technique. The first dimension employed a NEPHGE (nonequilibrium pH
gradient electrophoresis) system using tubes 14-15 cm long with an inner
diameter of 2.5 mm. The NEPHGE system was modified to accommodate more
basic polypeptides as described by Sanders et al. (1980), resulting
in an effective pH gradient of 5.4 to 9.8. The lower chamber of the
electrophoresis apparatus was filled with 0.04 M NaOH and the upper
chamber with 0.04 H^SO^. One hundred micrograms of either guinea pig
CMC purified relaxin or NIH-RXN-P1 relaxin were layered on the gels.
The gels were allowed to stack at 75 V for 15 min and then run for 2.5
hr at 400 V. The proteins were separated in the second dimension on
15% sodium dodecyl sulfate (SDS) polyacrylamide slab gels. Electrophor¬
esis buffer (3 g tris, 14.4 g glycine and 1.0 g per liter SDS) was placed
in the reservoirs and the gels were run toward the anode. Fifteen mamp/
slab were used as the stacking current for 2 hr. The current was turned
up to 30 mamp/slab and the gels were run for 4-5 hr. The gels were fixed
with 7% acetic acid:40% ethyl alcohol, and stained with 0.125% Coomassie
blue R250. The gels were destained in 7% acetic acid:10% ethyl alcohol.
Reduction with dithiothrietol.--Five hundred microliters of an ODS
crude* relaxin fraction (30 mg/ml H2O) from late pregnant guinea pig
*0DS crude relaxin is a partially purified uterine extract taken after the
initial purification step in the ODS procedure. This extract was tested in
the mouse uterine motility assay, without being altered (control), and after
the addition of different agents (experimental). A ratio was determined by
dividing the final volume of the experimental by the final volume of the
control. The assays were run twice and an average value was computed. The
greater the experimental to control ratio, the greater the ability of the
agent to inhibit the action of relaxin.

39
uterus was reduced by the addition of dithiothrietol (final concentra¬
tion, 0.1 M). The solution was incubated for 1 hr at room temperature.
A nontreated sample from the same fraction was tested as a control, and
potencies were compared using the mouse uterine motility bioassay.
Heating.--One hundred microliters of an ODS crude relaxin fraction
(30 mg/ml H?0) from late pregnant guinea pig uterus was heated at 70° C
for 1 hr. A nontreated sample from the same fraction was tested as a
control, and potencies were compared using the mouse uterine motility
bioassay.
Trypsin digestion.--Seven hundred microliters of an ODS crude
relaxin fraction (30 mg/ml H^O) from late pregnant guinea pig uterus
was incubated with the trypsin at a final concentration of 0.1 mg
trypsin/mg protein. The solution was incubated for 1.5 hr at room
temperature (pH 7.0). A nontreated sample from the same fraction was
tested as a control, and potencies were compared using the mouse uterine
motility bioassay.
In vitro assay of antisera (Larkin et al., 1979).--Fifty micro¬
liters of R19 antiserum were added to one of the tubes in the mouse
uterine motility bioassay, while the other tube received 50 pi of NRS
as a control. An ODS relaxin preparation of late pregnant guinea pig
uterus (30 mg/ml H^0) was then added to each tube in equal concentrations,
and the volumes required to inhibit the uterine contractions were com¬
pared.

RESULTS
Detection of Guinea Pig Relaxin
Immunocytochemical Localization of Uterine Relaxin
The PAP immunocytochemical technique was used to examine the
ovary, placenta, uterus, spleen and liver of guinea pigs. The endo¬
metrial gland cells (EGC) of the uterus was the only cell type that
showed heavy deposition of peroxidase reaction product (RP), indicating
the presence of relaxin. The ovary demonstrated weak staining, while
the liver, spleen and placentae did not stain. Therefore, subsequent
studies employed the uterus. Tissue samples were taken from guinea
pigs at different stages of pregnancy, during lactation and in ovari-
ectomized animals undergoing estrogen-progesterone treatments to deter¬
mine periods of relaxin production.
Relaxin was not detected in sections of uteri taken from nonpreg¬
nant ovariectomized control (no hormone treatment or estrogen treated
animals) or day 15 pregnant animals. Day 30 was the earliest stage of
pregnancy studied that showed accumulation of relaxin in the EGC. At
this stage, only a few glands contained relaxin (Figure 1). Control
(NRS treated) sections did not exhibit RP (Figure 2).
Hematoxylin and eosin (H 5 E) stained sections viewed at higher
magnification revealed that the EGC were low columnar cells with basally
located nuclei, and could be easily distinguished from uterine surface
epithelium (SE) (Figure 3). A section adjacent to that shown in
Figure 3 stained with R19 antiserum and the PAP technique demonstrated
40
Á

41
even deposition of RP in the cytoplasm of EGC with no nuclear staining
(Figure 4). Not all EGC within a single gland exhibited RP (Figure 4).
Sections of uteri taken on day 45 of pregnancy showed that a higher
percentage of endometrial glands (EG) contained relaxin than did day 30
tissue (Figure 5). Control (NRS treated) sections did not exhibit RP
(Figure 6). No remarkable features were noted at a higher magnification
in H § E stained sections beyond those mentioned for the day 30 EGC
(Figure 7). A section adjacent to that shown in Figure 7 treated with
R19 antiserum and the PAP technique showed RP in most, but not all of
the EGC (Figure 8). The EGC that were stained had RP evenly distributed
throughout the cytoplasm (Figure 8).
All EG in sections of the day 60 uteri demonstrated dense accumu¬
lations of RP (Figure 9). Control (NRS treated) sections did not have
RP (Figure 10). At higher magnifications, H 5 E stained EGC appeared to
contain granular accumulations of acidophilic material in the luminal
portions of the cytoplasm (Figure 11). Individual granules could not
be clearly resolved in these cells although under higher magnification
structures resembling granules could be detected in the luminal portion
of the cells. Electron micrographs of EGC from day 60 pregnant animals
showed dense accumulations of granules located in the apical areas of
the cells (data not shown). A section adjacent to that shown in Figure 11
treated with R19 antiserum and the PAP technique, showed that every EGC
in every gland exhibited RP (Figure 12). While the pattern of staining
varied somewhat between animals, the most characteristic feature was a
dense accumulation of RP in the apical portion of the cells, with little
or no stain observed in the basal cytoplasmic regions.

42
As in day 60 tissue, sections of uteri taken from the late preg¬
nant group of animals revealed that all glands gave a positive reaction
for relaxin (Figure 13). Control (NRS treated) sections did not exhibit
RP localization (Figure 14). High magnification of H § E stained tissue
showed little differences between individual EGC (Figure 15). A section
adjacent to that shown in Figure 15 stained with R19 antiserum and the
PAP technique showed a striking pattern of RP deposition in some EGC
which differed markedly from the day 60 tissue (Figure 16). Some cells
demonstrated RP throughout the cytoplasm. Other cells had a dense
accumulation of labeling localized in a juxtanuclear region in the apical
portion of the EGC, with a conspicuous absence of stain from the other
areas of the cell cytoplasm (Figure 16).
Sections of uteri from the lactating group of guinea pigs demon¬
strated a low percentage of glands that gave a positive reaction for
relaxin (Figure 17). Control (NRS treated) sections did not exhibit
deposition of RP (Figure 18). High magnification of H 8 E treated
sections showed the EGC to be tall columnar type cells with a large
number of mitotic figures (Figure 19). Few endometrial glands showed
deposition of RP, and those that did had RP evenly distributed throughout
the cytoplasm of the cells (Figure 20).
The group of animals ovariectomized and treated with estradiol
and progesterone produced preparations that most resembled tissue taken
from animals on day 45 of pregnancy. When a section of uterus from this
group of animals was treated with R19 antiserum and the PAP technique,
the majority of the EG gave a positive reaction for relaxin (Figure 21).

43
Control (NRS treated) tissue did not show deposition of RP (Figure 22).
High magnification of H § E treated sections revealed the EGC to be
cuboidal cells with few distinguishing features (Figure 23). A section
adjacent to that shown in Figure 23, treated with R19 serum and the PAP
technique, showed that the deposition of RP in this uterus was less
dense than in glands of uteri taken from animals in the latter stages
of pregnancy (Figure 24). In all stages studied, RP was found only in
EGC, that is, no RP was noted in the uterine stroma, luminal epithelium
or uterine cervical glands. The following represent results of control
studies utilized in the PAP procedure: (1) immunoperoxidase labeling
was abolished when antiserum R19 was absorbed with purified porcine
(NIH-RXN-P1) relaxin prior to incubation with tissue sections, (2) suc¬
cessive dilutions (1/10-1/100,000) of antiserum R19 eventually abolished
tissue immunolabeling, and (3) staining was eliminated when NRS was sub¬
stituted for R19, GAR or PAP in the immunolabeling procedure.
Overall, these results support evidence obtained by others that
the uterus is a source of relaxin in the guinea pig.
Biologically Active Uterine Relaxin During
Pregnancy and Lactation
The preceding cytological evidence is extended by bioassays which
show that biologically active relaxin is only detected in the uterus, and
that the activity peaks in the later stages of pregnancy (Table 1; Fig¬
ures 25 and 26). Uteri from day 30 pregnant animals contained low bio¬
logical activity (0.15 +^0.09 units per gram wet weight (U/gww); 0.63 +_
0.40 U total). At day 45 of pregnancy, a significant increase in uterine
relaxin biological activity was noted (2.19 _+ 0.51 U/gww; 13.71 ^2.52
U total). Uterine relaxin levels further increased at day 60 of pregnancy

44
(2.62 ;+ 0.20 U/gww; 46.80 _+ 2.85 U total). Uteri from late pregnant
animals showed the highest biological activity levels (3.76 + 0.76
U/gww; 57.75 _+ 7.35 U total). Relaxin levels dropped in lactating
animals (0.23 +_0.14 U/gww; 1.50 +_ 1.17 U total).
Statistical Analyses of Bioassay Data
Total activity.—A regression analysis of the bioassay data
(Table 1; Figure 25) showed that total biological activity of the
uterine extracts was different over time of pregnancy with a high level
of significance (p<0.0001), and a quadratic component with a high cor-
2
relation coefficient (r = 0.907). A Duncan's multiple range test
for total biological activity values showed the following results (alpha
level = 0.05).
Ip 60_ 45 lac 30
Stages interconnected by bars are statistically indistinguishable from
each other according to the Duncan's multiple range test.
Specific activity.—A regression analyses of bioassay data (Table 1;
Figure 26) showed that specific activity (U/gww) of the uterine extracts
was different over time of pregnancy with a high level of significance
(p<0.0001) and a quadratic component (r = 0.739). A Duncan's multiple
range test for specific biological activity values showed the following
results (alpha level = 0.05)
lp 6£ 45 lac 30
Crude extracts of liver and placenta of late pregnant guinea pigs
showed no relaxin activity in the mouse uterine motility bioassay. In
an attempt to find some relaxin activity, the placental extract was
further purified by passing it through a Bio-Gel P-30 column. The

45
fraction eluting in the 6,000 mw range contained very low activity
(0.27 U/mg) (data not shown). The liver extract was not purified or
tested further. It should be emphasized that neither of these two
tissues exhibited any demonstrable immunolabeling when tested with R19
antiserum and the PAP immunocytochemical technique. Therefore, the
relaxin activity in the placental extract may have been the result of
blood borne relaxin.
Radioimmunoassay of Uterine Relaxin During
Pregnancy and Lactation
RIA characterization.--The iodination curve (Figure 27), antiserum
titration curve (Figure 28), and dilution curves (Figure 29) obtained
with NIH-RXN-P1 relaxin support the validity of the RIA employed in the
present study. The dilution curves of the NIH-RXN-P1 relaxin and the two
guinea pig relaxin preparations were of similar slopes (Figure 29).
Therefore, it was deemed feasible to utilize NIH-RXN-P1 for the develop¬
ment of standard curves. The RIA did not detect relaxin in serum of
pregnant guinea pigs. The interassay coefficient of variation for the
RIA (4 assays) was 15.4% at 125 pg and the intra-assay coefficient of
variation (6 samples) at 125 pg was 4.2%. The RIA was capable of detect¬
ing levels of relaxin that ranged from 32 pg to 1,000 pg.
RIA of uterine extracts.—
(1) Total amount of immunoreactive relaxin per uterus.--The RIA
of crude extracts showed that uteri from day 15 pregnant guinea pigs
(Table 2; Figure 30) contained the least amount of relaxin (0.40 +_0.14
ng). Data are expressed as nanogram (ng) porcine relaxin equivalents.
Amounts of relaxin increased in uterine extracts from day 30 pregnant
animals (11.44 +_ 4.51 ng). At day 45 of pregnancy, uterine relaxin

46
levels increased to 148.47 +_ 21.52 ng and were highest by day 60 of
pregnancy (172.67 +_ 19.17 ng). By late pregnancy, total uterine relaxin
levels had decreased to 101.90 +_ 25.78 ng and a wide variability existed
between animals. In the lactating animals the uterine relaxin levels had
decreased to 4.78 +^0.61 ng. A regression analysis of the RIA data
(Table 2; Figure 30) showed that total amounts of immunoreactive relaxin
were different (p<0.0001) over time of pregnancy, with a quadratic com-
ponent (r^ = 0.831). A Duncan's multiple range test for total relaxin
immunoactivity showed the following results (alpha level = 0.05).
60 45 lp 30 lac 15
(2) Concentration of immunoreactive relaxin.--Uterine extracts
from day 15 pregnant animals (Figure 31) contained low concentrations of
relaxin (0.25 0.05 ng/gww) which increased at day 30 of pregnancy
(2.06 ^0.44 ng/gww). The highest specific activity was found in the
uteri of day 45 pregnant animals (24.72 +_ 6.31 ng/gww). Uteri of day
60 and late pregnant animals had lower relaxin concentrations (9.83 +_
0.48 ng/gww and 6.45 +_ 1.74 ng/gww, respectively). While uteri from
lactating animals again contained very low concentrations of relaxin
(1.01 +_ 0.29 ng/gww).
A regression analysis of the RIA data (Table 2 and Figure 31)
showed that specific relaxin immunoactivity (ng/gww) of the crude uterine
extracts was different (p<0.0001) over time of pregnancy, with a quad-
ratic component (r = 0.754). A Duncan's multiple range test for specific
immunoactivity values showed the following results (alpha level = 0.05).
45_ 60 l£ 30 lac 15

47
Purification and Characterization of Guinea Pig
Uterine Relaxin
Purification: OPS Procedure
Uteri of the late pregnant guinea pigs were purified with the
extraction procedure of Walsh and Niall (1980). Five guinea pigs in the
late stages of pregnancy (days 65-67) were killed and 118.5 g wet weight
of uteri were utilized (Table 3). The ODS extracted material (188.8 mg)
contained low but detectable activity in the mouse uterine motility
assay (0.32 U/mg). The ODS material was fractionated in a Sephadex
column (Figure 32) and the 6,000 mw fraction had an activity of 1.50
U/mg. This active fraction from the Sephadex G-50 column (35.5 mg) was
further chromatographed in a CMC ion exchange column. The most active
fractions from the CMC column (tubes 55-90) contained 1.7 mg protein,
had an activity of 3.87 U/mg, and the peak protein fraction (tube 70)
eluted in the 7 m Mho conductance range (Figure 33). Similarly run
NIH-RXN-P1 porcine relaxin eluted in the 10 m Mho conductance range (data
not shown). RIA of every tenth tube of the CMC column run showed that
immunoreactive relaxin was present in the eluate. The regions containing
the highest immunoreactive relaxin (^300 ng/ml/OD; tubes 80-90) also
exhibited bioactivity (Figure 33).
Characterization
Double immunodiffusion studies.--Analyses of a Bio-Gel P-30
relaxin fraction (6 mg/ml) from the guinea pig uterus (day 60 of
pregnancy) tested against R19 antiserum to porcine relaxin and NIH-
RXN-P1 porcine relaxin showed a single precipitin line with no spurring
(Figure 34).

48
Two dimensional gel electrophoresis.--Carboxymethyl cellulose
purified guinea pig relaxin tested in a two dimensional gel electro¬
phoresis system showed that guinea pig relaxin migrated in the same mw
range as did NIH-RXN-P1 relaxin, which is known to have a mw of 6,000
(Figure 35). An equilibrium isoelectric point of a molecule cannot be
resolved with NEPHGE. Nevertheless, it was apparent that the guinea
pig relaxin molecule did not migrate as far in the first dimension
(pH 6.9) as did NIH-RXN-P1 porcine relaxin (pH 7.2), indicating that the
guinea pig relaxin molecule had a lower pH than the porcine molecule
(Figure 35). This observation supported data from the CMC column run,
which showed guinea pig relaxin eluting earlier (7 m Mho range) than
NIH-RXN-P1 porcine relaxin (10 m Mho range (Figure 33)). Figure 35 is
a representation of a slab gel, made up of a composite of two separately
run gels.
Mouse interpubic ligament assay.--A Sephadex G-50 6,000 mw fraction
from guinea pig uterine extracts was tested in the mouse interpubic
ligament assay (Figure 36). A positive response was obtained as noted
by linear response to log-dose of the Sephadex fraction. The data
indicate a valid assay according to the following criteria: (1) parallel¬
ism existed between the guinea pig relaxin fraction and the NIH-RXN-P1
porcine relaxin standard, and (2) a lambda value (standard error/slope)
of less than 0.4 was obtained. The specific activity of the Sephadex
fraction was calculated to be 0.8 U/mg protein. The mouse uterine motil¬
ity assay of the same fraction gave a biological activity of 1.5 U/mg.
The best fit curve for the NIH-RXN-P1 porcine relaxin was y = 1.2 (log x)
+ 0.83, with a standard error (SE) = 0.086 and a lambda value of 0.08.

49
The guinea pig CMC purified relaxin had a best fit curve of y = 0.87
(log x) + 0.64 with an SE = 0.097 and a lambda value of 0.12. Control
(estrogen treated) animals had a mean interpubic ligament length of
0.89 + 0.75 (X + SEM) .
Physiochemical characteristics.—Treatment of the ODS crude
extract with dithiothrietol, trypsin and R19 reduced its ability to
inhibit uterine contractions (Table 4). These results indicate that
the guinea pig relaxin molecule depends on intact disulfide linkages
and structural integrity for its biological activity. Also, blocking
the immunologically active sites of the relaxin molecule with anti-
relaxin antibodies inhibits the hormone's biological activity. Heat
(70° C) did not adversely affect the guinea pig relaxin molecule.
Dithiothrietol and trypsin tested by themselves did not alter the ampli¬
tude or frequency of the contractions.

DISCUSSION
R19 Antiserum: Detection of Guinea Pig Relaxin
A major portion of this dissertation utilized techniques which
employed antirelaxin serum (R19). R19 was produced in rabbits against
highly purified porcine relaxin (Larkin et al., 1977). This antiserum
has been shown to crossreact with relaxin from different species; i.e.,
cow (Fields et al., 1980), human (Fields and Larkin, 1981), and rabbit
(Fields et al., 1981). R19 also has the ability to detect guinea pig
relaxin, as demonstrated by the following observations from the current
study: (1) Double immunodiffusion agar plate assays showed a reaction
of identity when partially purified uterine relaxin from day 60 preg¬
nant guinea pigs was tested against highly purified pig relaxin and
R19. (2) R19 was shown to be effective in inhibiting the action
of guinea pig relaxin using the in vitro mouse uterine motility anti¬
serum assay. (3) The immunoperoxidase labeling data from different
stages of gestation correlated well with bioassay and radioimmunoassay
data of crude uterine extracts from these stages; that is, the stages
demonstrating the greatest degree of labeling were also the stages
with the highest relaxin levels (days 45, 60 of pregnancy and late
pregnant animals). (4) All the controls in the immunolocalization
experiments were negative, including the observation that immunoperoxi¬
dase labeling was eliminated when the R19 antiserum was absorbed with
purified porcine relaxin standard (NIH-RXN-P1) prior to tissue incubation.
50

51
Detection of Guinea Pig Relaxin with the
PAP Technique
The R19 antiserum discussed in the previous section was employed
in immunocytochemical studies on sections of uteri from pregnant and
lactating guinea pigs, as well as in uteri of guinea pigs induced to
undergo pelvic relaxation with estrogen and progesterone treatments.
The immunocytochemical studies show an increase during pregnancy in
RP localized in the EG. This suggests that there is an increase in the
accumulation of relaxin in these cells, supporting the contention that
the EG are the sites for relaxin production. Of the intervals evalu¬
ated, day 30 was the first stage of pregnancy where a small amount of
immunoperoxidase labeling (relaxin) was noted in some EG. At this
stage, very few glands were labeled and in those glands that did label,
the reaction production was detected in only a few of the gland cells.
In uterine tissue from day 48 animals almost all of the EG appeared to
contain relaxin, but even at this stage, not all of the EGC of a given
gland were labeled. Thus it appears that there exists a gradual build up
of relaxin by the EGC. This is most clearly seen in day 60 and late
pregnant stages where dense deposits of RP were localized in the apical
cytoplasm of the EGC. In uteri from lactating animals (3 days post¬
partum), relaxin was absent from most of the EG, and a high incidence
of mitotic figures were noticed in the glandular epithelium.
Results from experiments involving nonpregnant, ovariectomized-
hormone treated animals provide strong evidence for a non-ovarian source
of relaxin. When ovariectomized animals were treated with estrogen,
no RP was seen localized over the EGC. When ovariectomized animals
were treated with estrogen and progesterone, RP was seen localized over

52
the EGC. These results agree with and extend the results of Zarrow
(1948), who noted that hysterectomized and ovariectomized guinea pigs
did not undergo pelvic relaxation or contain serum relaxin when treated
with a similar course of steroid injections. It is not known how
estrogen and progesterone induce the accumulation or synthesis of
relaxin in the EGC.
Detection of Guinea Pig Relaxin with
Radioimmunoassay
There have been no publications to date that report on the produc¬
tion of antisera to purified guinea pig relaxin. All studies dealing
with radioimmunological detection of relaxin in the guinea pig have
employed antiporcine relaxin sera (Sherwood et al., 1975; O'Byrne and
Steinetz, 1976; Bryant-Greenwood and Greenwood, 1979; Boyd et al., 1981;
Nagao and Bryant-Greenwood, 1981). It seems logical that differences
in the specificity and sensitivity of the various antisera could be
responsible for the wide variations in relaxin levels reported between
different laboratories which have studied relaxin in the guinea pig.
For example, the present RIA (employing R19 antiserum) detected porcine
relaxin as well as relaxin from guinea pig uterine extracts, but not
guinea pig serum relaxin. This antiserum was produced in rabbits to
highly purified porcine realxin. Sherwood et al. (1975) also reported
that an antiserum produced in rabbits to highly purified porcine relaxin
detected only porcine relaxin. These investigators were not able to
detect relaxin in sera of pregnant guinea pigs. This is in agreement
with the present study. However, Sherwood et al. (1975) did not report
attempts to detect relaxin in extracts of relaxin containing tissues.

53
O'Byrne and Steinetz (1976) on the other hand demonstrated that ani-
serum R6 crossreacted with relaxin in serum from a variety of pregnant
animals: humans, baboons, rhesus monkeys, dogs, cats, guinea pigs, rats
and mice. The R6 antiserum was produced in rabbits to a relaxin
fraction (Sephadex G-50: 1,000 U/mg) . O'Byme and Steinetz (1976) did
not publish bioassay results. The RIA employed in the present studies,
as well as in the experiments of Sherwood et al. (1975) and O'Byme and
Steinetz (1976), used polytyrosyl relaxin. It seems that the difference
in immunological specificity encountered by the three laboratories
stemmed not from the polytyrosyl relaxin, but from the use of different
antisera or possibly from the technique employed. Nagao and Bryant-
Greenwood (1981) found that an antiserum produced to a relatively impure
porcine relaxin fraction (Sephadex G-50 column cut) appeared capable of
detecting a greater range of "relaxin" immunoactivity than an antiserum
produced against a purified relaxin fraction (porcine CM-a'). Perhaps
this extended range is due to the ability of the more "impure" antisera
to crossreact with metabolites of relaxin or with nonrelaxin components
of serum. This is not unreasonable to propose since it has been shown
by investigators working in Dr. Bryant-Greenwood's laboratory (Arakari
et al., 1980) that the antiserum to impure relaxin recognizes connective
tissue elements in immunofluorescence studies involving the pregnant
sow ovary. Nagao and Bryant-Greenwood (1981) utilized the antiserum
raised against CM-a' to assay uterine extracts taken from guinea pigs in
different stages of gestation. The highest levels encountered by these
investigators were of an order of magnitude 10 to 100 times greater than

54
the levels encountered in the present study. If the bioassay levels of
the present study are converted to ng porcine relaxin equivalents,*
they fall in the range of relaxin levels found by Nagao and Bryant-
Greenwood (1981) utilizing RIA. It appears that the antiserum used by
Bryant-Greenwood detected greater amounts of relaxin than the R19
antiserum used in the present study. It seems obvious from these com¬
parisons, that a direct correlation cannot be made between RIA data
obtained from different laboratories if different labeling techniques
and antisera were used. It is reasonable, however, to compare relative
data from the same system if the appropriate controls are carried out.
An important aspect of the present investigation was the correla¬
tion of RIA studies with bioassay and immunological localization studies.
Stages of pregnancy which demonstrated high levels of biologically and
immunologically active relaxin in uterine extracts, were also the stages
which displayed increased immunoperoxidase labeling in the uterine tissue
sections. A comparison of relaxin levels detected with bioassay and
radioimmunoassay at the different stages of pregnancy raises an interest¬
ing point. Bioassay of crude uterine extracts detected the highest con¬
centration of relaxin in late pregnant animals. On the other hand,
concentrations of immunoreactive relaxin were highest in the day 45
pregnant animals, but decreased by day 60 of pregnancy and continued to
decline during late pregnancy. It seems that the bioassay detected levels
*This comparison is made by using the following conversion.
ng equivalent relaxin =
biological activity (Units) x 1,000 ng of porcine relaxin
3 Units

55
of relaxir! that were not detected by the RIA. One may speculate on
the possibility that accumulations of relaxin (prorelaxin) that are
biologically active, but not immunoreactive, are present in those latter
stages of pregnancy. The present study did not report on serum relaxin
levels. Serum studies undertaken in other laboratories, however, showed
that biologically active (Zarrow, 1948), and immunoreactive (O'Byrne
and Steinetz, 1976; Boyd et al., 1981) serum relaxin levels increased
as pregnancy proceeded in guinea pigs, dropping just prior to parturition.
This trend seen in serum relaxin levels approximates activity detected
in the tissue extracts and tissue sections with techniques employed in
the present investigation, supporting the hypothesis that uterine relaxin
may play an influential role in pregnancy and parturition in the guinea
pig-
Endometrial Glands and Their Role in Relaxin Production
EG classically have been assumed to play a role during pregnancy
where they serve a nutritive and/or supportive role to the preimplanta¬
tion embryo (Finn, 1977). In the mouse, the end result of glandular
differentiation is the secretion of periodic acid Schiff positive
material from the EG into the uterine lumen, and it has been shown that
estrogen and progesterone together can induce uterine glandular secre¬
tions (Finn, 1971; Finn and Martin, 1971). In the pig, Bazer (1975)
has shown that a uterine specific purple protein is secreted by the
glandular endometrial epithelium throughout pregnancy. Most animals
studied have been shown to produce uterine specific proteins, especially
during the early stages of pregnancy (Aitken, 1979). Animals with an

56
epitheliochorial or syndesmochorial type of placentation may provide a
source of nutrition to the developing embryo through the production of
uterine specific proteins which diffuse through the placenta. Animals
with a hemochorial type of placentation, like the human and the guinea
pig, derive most of their embryonic nutrition from the maternal blood¬
stream. It has, nevertheless, been shown that amniotic fluid from
humans in the second trimester of pregnancy contains uterine specific
proteins (Sutcliffe et al., 1978). The EG and/or the SE are most
likely active during pregnancy and produce proteins which possibly come
into contact with the embryo and fetus.
Direct and indirect evidence from other laboratories has been
accumulated which implicates the uterus as an important source of
relaxin in the guinea pig: (1) Frieden and Adams (1977) have shown
that softening of the pelvic ligaments can be detected by palpation as
early as mid-pregnancy in the guinea pig, which is the approximate time
(day 30) when accumulation of relaxin was initially detected in the EGC
with immunoperoxidase labeling, RIA, and bioassay. (2) Porter (1972)
has shown that a uterine quieting substance, most likely relaxin, is
present in the blood of pregnant guinea pigs. (3) Zarrow (1948) and
Nagao and Bryant-Greenwood (1981) have detected the presence of relaxin
in sera of ovariectomized estrogen-progesterone treated guinea pigs.
(4) Catchpole (1969) has shown that the guinea pig is able to proceed
through a normal pregnancy and parturition after ovariectomy as early
as day 38 of pregnancy, thus strongly indicating a nonovarian source of
relaxin for this species.

57
The uterus of the guinea pig has been known to be a source of
relaxin for some time (Zarrow, 1948). Day 30 of pregnancy seems to be
the approximate time when measurable levels of relaxin first appear
in the uterus and blood of guinea pigs (O'Byme and Steinetz, 1976;
Boyd et al., 1981; Nagao and Bryant-Greenwood, 1981). This is also
approximately 10-15 days after the first detectable rise in serum
estrogen and progesterone (Challis et al., 1971). As shown by Zarrow
(1948) 10-15 days is the time required for injections of estrogen and
progesterone to evoke the synthesis of relaxin by the uterus of non¬
pregnant ovariectomized guinea pigs.
This latency period appears to be shorter than 15 days, since by
this time, treatment with estrogen and progesterone had evoked a change
in the interpubic ligament length of the guinea pigs. Nagao and Bryant-
Greenwood (1981) detected a rise in uterine relaxin 11 days after ovari¬
ectomized guinea pigs were primed with estrogen and progesterone. However,
no one has reported a systematic study to determine when relaxin is first
produced by the uterus following estrogen and progesterone stimulation.
Autoradiographic evidence indicates that estrogen primed, ovari¬
ectomized guinea pigs contain progesterone receptors in the endometrial
7
glands, as evidenced by accumulation of H progesterone in the cytoplasm
and nuclei of the EGC (Stumpf, 1968; Sar and Stumpf, 1974; Warembourg,
1974). This is consistent with the contention that the EG are a target
tissue for the steroid hormones,and all evidence indicates that estrogen
and progesterone are necessary for relaxin synthesis by the EG.

58
Possible Actions of Uterine Relaxin in the Guinea Pig
As evidence accumulates that the corpus luteum of the pregnant
female is not the only source of relaxin, it becomes apparent that
relaxin may be produced by other tissues and act locally as well as
systemically. Perhaps this is best illustrated by the guinea pig,
which may have an ovarian source of relaxin (Nagao and Bryant-Greenwood,
1981), but also contains a uterine source as well. Relaxin produced by
the uterus of this animal may have systemic as well as local effects.
Relaxin has been shown to be present in the systemic blood of pregnant
guinea pigs with bioassay (Zarrow, 1948) and RIA (O'Byrne and Steinetz,
1976; Boyd et al., 1981). Also Zarrow (1948) has shown that relaxin is
present in systemic blood of ovariectomized-estrogen and progesterone
treated guinea pigs. Effects such as changes which occur in the inter-
pubic ligament prior to parturition, in mammary gland development, as
well as in the maintenance of uterine quiescence could be proposed as
possible systemic effects of relaxin.
An increasing body of evidence on the other hand, indicates that
relaxin may act as a local hormone in the guinea pig. Immunolocalization
studies from this laboratory illustrate a distinct staining pattern in
some of the EGC in uterine tissue taken from guinea pigs in the latter
stages of pregnancy. In day 60 and late pregnant animals, RP was
localized apically in some EGC, i.e. immunolabeling was not seen in the
basal areas of the cytoplasm in these cells. Also electron micrographs
of EG taken from the same stages showed a large number of apical gran¬
ules. These observations may suggest that relaxin is being released
into the uterine lumen of the endometrial glands. Relaxin synthesized

59
and released from the EG into the uterine lumen could: (1) Have free
access to fetal-maternal tissues during pregnancy. Harkness and Hark-
ness (1956; 1957) have shown that the tensile strength of rat fetal
membranes is greatly reduced during the birth process. Relaxin may
be responsible for this phenomenom. (2) Maintain uterine quiescence
during pregnancy. Although there is no direct evidence for this assump¬
tion, the proximity of the uterine endometrium to the myometrium could
possibly allow for local diffusion of relaxin to occur. Porter (1972)
has shown that relaxin is most likely the hormone responsible for keeping
the uterus quiescent in the guinea pig during pregnancy. (3) Effect
cervical softening at term. MacLennan et al. (1980), have found that
topical application of porcine relaxin to the posterior fornix of the
vagina resulted in softening of the cervix in a significant number of
women.
One of the most interesting problems remaining to be investigated
is how relaxin enters the systemic circulation from the EG and what
course it follows to reach the target tissues. Studies to identify
target organs for relaxin become very important when one considers the
possibilities of local effects of relaxin.
Relaxin may be released into the uterine lumen, and make its way
back through the endometrial stroma into the bloodstream. While the
findings in the present study did not indicate direct secretion of
relaxin into the uterine stromal compartment, this cannot be ruled out.
Work in other systems by Bazer and Thatcher (1977) suggested an
endocrine-exocrine mechanism for the release of prostaglandin F 2-alpha

60
(PGF 2-alpha) by the uterine endometrial glands of the pig. It was
postulated by these investigators that in the nonpregnant state, PGF
2-alpha is produced in an endocrine fashion by the EG under the control
of progesterone. On the other hand, in a pregnant animal, the estrogen
released by the conceptus changes the pattern of release to an exocrine
fashion, and the hormone is released into the uterine lumen.
Purification and Characterization of Guinea Pig Relaxin
The guinea pig relaxin molecule demonstrated characteristics similar
to relaxins isolated from other species (Sherwood and O'Byrne, 1974;
Fields and Larkin, 1979; Sherwood, 1979; Fields and Larkin, 1981; Fields
et al., 1980; 1981; Reinig et al., 1981) according to the following
criteria: (1) mw of approximately 6,000, (2) basic isoelectric point,
(3) ability to inhibit uterine contractions (mouse uterine motility
bioassay), (4) ability to induce interpubic ligament formation in estrogen
primed mice (mouse interpubic ligament assay), (5) susceptibility to
enzyme digestion with trypsin and to the strong reducing agent dithio-
thrietol, indicating its proteinaceous nature, and its reliance on
disulfide bonds for its biological activity, and (6) resistance to
moderate heat. Guinea pig relaxin was antigenically similar to porcine
relaxin in that: (1) a reaction of identity was obtained when an extract
of guinea pig uterus from a day 60 pregnant animal was reacted against
antiserum to purified porcine relaxin and porcine relaxin (NIH-RXN-P1),
(2) an antiserum produced against porcine relaxin inhibited the ability
of guinea pig extracts to retard spontaneous uterine contractions,
(3) parallel dilution curves were obtained between relaxin containing
uterine extracts of late pregnant guinea pigs and porcine relaxin

61
(NIH-RXN-P1) in a homologous porcine relaxin RIA, and (4) the biologic¬
ally active guinea pig relaxin CMC peak (Figure 33) was also immuno¬
logical ly active in the homologous porcine RIA.
Guinea pig relaxin displayed a very low specific biological
activity when compared to porcine relaxin. The present studies confirmed
preliminary work done by Pardo et al., 1980, utilizing a different extrac¬
tion procedure, who showed that crude uterine extracts of day 60 preg¬
nant guinea pigs contained low biological activity (0.14 U/mg) when
tested with the mouse uterine motility bioassay. Higher activity levels
were reported for relaxin separated on a column of Bio-Gel (36 U/mg)
(Pardo et al., 1980). This is in contrast to levels reported in the
present study. The significance of this discrepancy is unclear and may
relate to different separation techniques, as well as variability of the
bioassays. The low biological activity of guinea pig relaxin is not
unreasonable to expect since relaxins isolated from all species except
the pig had low biological activity. This discrepancy in activity levels
between the pig and other species perhaps reflects differences in
specificity in the different bioassays. A good example of this phenome-
nom was found in relaxin isolated from the shark (Reinig et al., 1981).
Shark relaxin has been found to be ineffective in the mouse bioassays
(uterine motility and interpubic ligament formation). However, shark
relaxin is active when guinea pigs were employed for the uterine motility
and interpubic ligament assays. It is clear, however, more than one
bioassay should be employed when considering whether a preparation
contains relaxin. Guinea pig relaxin was effective in both the mouse

62
uterine motility bioassay and in the mouse interpubic ligament bioassay,
and thus at least in these aspects it remains similar to relaxin from
other mammalian species.
Future studies can be proposed to answer many of the questions as
yet unexplained. First, what, if any, is the contribution of the ovary
to the synthesis of relaxin? Second, how do the steroid hormones
initiate the synthesis of relaxin by the EGC, and why a synchronization
of secretion is not apparent under a uniform hormonal milieu? Third,
what is the possible mechanism of relaxin release by the EGC? Fourth,
how does uterine relaxin reach its target organs with respect to the
events of pregnancy and parturition; e.g., maintaining the uterus
quiescent during pregnancy and preparing the cervix and pelvic ligaments
for parturition?
It is hoped that the research presented in this dissertation will
amplify the knowledge of relaxin physiology in the guinea pig, and
advance an understanding of the parturition process in this animal as
well as in other species.

BIBLIOGRAPHY
Abramowitz, A. A., W. L. Money, M. X. Zarrow, R. V. N. Talmage, L. H.
Kleinholz, and F. L. Hisaw. (1944) Preparation, biological assay
and properties of relaxin. Endocrinology, 34: 103-114.
Abramson, D., E. Hurwitt, and G. Lesnik. (1937) Relaxin in human
serum as a test of pregnancy. Surg. Gynecol. Obstet., 65: 335-339.
Aitken, R. J. (1979) Uterine proteins. In: Oxford Reviews of
Reproductive Biology. (A. A. Finn, ed.) Clarendon Press, Oxford,
England.
Anderson, M. L., and J. A. Long. (1978) Localization of relaxin in the
pregnant rat. Bioassay of tissue extracts and cell fractionation
studies. Biol. Reprod., 18: 110-117.
Anderson, M. L., J. A. Long, and T. Hayashida. (1975) Immunofluorescence
studies on the localization of relaxin in the corpus luteum of
the pregnant rat. Biol. Reprod., 13: 499-504.
Arakari, R. F., R. G. Kleinfeld, and G. D. Bryant-Greenwood. (1980)
Immunofluorescence studies using antisera to crude and to purified
porcine relaxin. Biol. Reprod., 23: 153-159.
Barr, A. J., and J. H. A. Goodnight. (1976) Users Guide to the Statisti¬
cal Analysis System. North Carolina State University, Raleigh,
N. C.
Bazer, F. W. (1975) Uterine protein secretions: Relationship to
development of the conceptus. J. Anim. Sci., 41: 1376-1382.
Bazer, F. W., and W. W. Thatcher. (1977) Theory of maternal recognition
of pregnancy in swine based on estrogen controlled endocrine
versus exocrine secretion of Prostaglandin F2-alpha by the uterine
endometrium. Prostaglandins, 14: 397-401.
Belt, W. D., L. L. Anderson, L. F. Cavazos, and R. M. Melampy. (1971)
Cytoplasmic granules and relaxin levels in porcine corpora lútea.
Endocrinology, 89: 1-10.
Bigazzi, M., E. Nardi, P. Bruni, and F. Petrucci. (1980) Relaxin in
human decidua. J. Clin. Endocrinol. Metab., 51: 939-941.
Blundell, T. (1979) Conformation and molecular biology of polypeptide
hormones. I. Insulin, insulin-like growth factor and relaxin.
TIBS. March, 1979: 51-54.
63

64
Bolton, A. E., and W. M. Hunter. (1973) The labelling of proteins to
high specific radioactivities by conjugation to a 12Si_containing
acylating agent. Biochem. J., 133: 529-539.
Boyd, S., J. Z. Kendall, N. Mentó, and G. D. Bryant-Greenwood. (1981)
Relaxin immunoactivity in plasma during the reproductive cycle of
the female guinea pig. Biol. Reprod., 24: 405-414.
Brouha, L. (1933) Recherches sur la mobilisation de la symphyse
pubienne chez le cobaye impubere. Compte Rendu Soc. Biol., 113:
406-408.
Bryant, G. D. (1972) The detection of relaxin in porcine, ovine and
human plasma by radioimmunoassay. Endocrinology, 91: 1113-1117.
Bryant, G. D., and W. A. Chamley. (1976) Changes in relaxin and pro¬
lactin immunoactivities in ovine plasma following suckling. J.
Reprod. Fértil., 46: 457-459.
Bryant, G. D., M. E. A. Panter, and T. Stelmasiak. (1975) Immunore-
active relaxin in human serum during the menstrual cycle. J.
Clin. Endocrinol. Metab., 41: 1065-1069.
Bryant, G. D., J. F. Sassin, E. D. Weitzman, S. Kapen, and A. Frantz.
(1976) Relaxin immunoactivity in human plasma during a 24-hour
period. J. Reprod. Fértil., 48: 389-392.
Bryant, G. D., and T. Stelmasiak. (1974) The specificity of radio¬
immunoassay for relaxin. Endo. Res. Commun., 1: 415-433.
Bryant-Greenwood, G. D., and F. C. Greenwood. (1979) Specificity of
radioimmunoassays for relaxin. J. Endocrinol., 81: 239-247.
Castro-Hernandez, A. (1976) Isolation and purification of bovine
luteal polypeptides with relaxin hormone activity (Doctoral
Dissertation, University of Florida).
Catchpole, H. R. (1969) Hormonal mechanisms during pregnancy and
parturition. In: Reproduction in Domestic Animals. (H. H. Cole
and P. T. Cupps, eds.) Academic Press, New York.
Challis, J. R. G., R. B. Heap, and D. V. Illingsworth. (1971) Concentra¬
tions of oestrogen and progesterone in the plasma of non-pregnant,
pregnant and lactating guinea pigs. J. Endocrinol., 51: 333-345.
Clausen, J. (1969) Immunochemical Techniques for the Identification and
Estimation of Macromolecules. (T. S. Work, E. Work, eds.)
American Elsevier, New York, p. 521.

65
Dallenbach, F. D., and G. Dallenbach-Hellweg. (1964) Immunohistologische
untersuchungen zur lokalisierung des relaxins in menschlicher
plazenta and decidua. Virch. Arch. Path. Anat., 337: 301-316.
Dallenbach-Hellweg, G., J. V. Battista, and F. D. Dallenbach. (1965)
Immunohistological and histochemical localization of relaxin in
the metrial gland of the pregnant rat. Am. J. Anat., 117:
435-450.
Fevold, H., F. L. Hisaw, and R. K. Meyer. (1930) The relaxative hor¬
mone of the corpus luteum, its purification and concentration.
J. Am. Chem. Soc., 52: 3340-3348.
Fields, M. J., P. A. Fields, A. Castro-Hernandez, and L. H. Larkin.
(1980) Evidence for relaxin in corpora lútea of late pregnant
cows. Endocrinology, 107: 869-875.
Fields, P. A., and L. H. Larkin. (1979) Isolation of rat relaxin.
Anat. Rec., 193: 537.
Fields, P. A., and L. H. Larkin. (1981) Purification and immunohisto-
chemical localization of relaxin in the human term placenta. J.
Clin. Endocrinol. Metab., 52: 79-85.
Fields, P. A., L. H. Larkin, and R. J. Pardo. (1981) Purification of
relaxin from the placenta of the rabbit. Ann. N. Y. Acad. Sci.
380: 76-86.
Finn, C. A. (1977) The implantation reaction. In: Biology of the
Uterus (R. M. Wynn, ed.) Plenum Press, New York.
Finn, C. A., and L. Martin. (1971) Endocrine control of the prolifer¬
ation and secretion of uterine glands in the mouse. Acta Endocrinol.
Suppl., 155: 139.
Frieden, E. H., and W. C. Adams. (1977) The response to endogenous
relaxin of guinea pigs refractory to porcine relaxin. Proc. Soc.
Exp. Biol. Med., 155: 558-561.
Frieden, E. H., and F. L. Hisaw. (1953) The biochemistry of relaxin.
Rec. Prog. Horm. Res., 8: 333-372.
Frieden, E. H., and L. Yeh. (1977) Evidence for a "pro-relaxin" in
porcine relaxin concentrates. Proc. Soc. Exp. Biol. Med., 154:
407-411.
Fugo, N. W. (1943) Relaxation of the pelvic ligaments of castrate
hysterectomized guinea pigs induced by progesterone. Proc. Soc.
Exp. Biol. Med., 54: 200-201.

66
Griss, G., J. Keck, R. Engelhom, and H. Tuppy. (1967) The isolation
and purification of an ovarian polypeptide of uterine-relaxing
activity. Biochim. Biophys. Acta, 140: 45-54.
Hall, K. (1960) Relaxin. J. Reprod. Fértil., 1: 368-384.
Harkness, M. L. R., and R. D. Harkness. (1956) Changes in the foetal
membrane during pregnancy in the rat. J. Physiol., 129: 788.
Harkness, M. L. R., and R. D. Harkness. (1957) Changes in the physical
properties of the uterine cervix of the rat during pregnancy.
J. Physiol., 148: 524-547.
Hisaw, F. L. (1926) Experimental relaxation of the pubic ligament of
the guinea pig. Proc. Soc. Exp. Biol. Med., 23: 661-663.
Hisaw, F. L. (1927) Experimental relaxation of the symphysis pubis of
the guinea pig. Anat. Rec., 37: 126.
Hisaw, F. L., and M. X. Zarrow. (1950) The physiology of relaxin.
Vit. and Horm., 8: 151-178.
Hisaw, F. L., M. X. Zarrow, W. L. Money, R. V. N. Talmage, and A. A.
Abramowitz. (1944) Importance of the female reproductive tract
in the formation of relaxin. Endocrinology, 34: 122-134.
Horst, M. N., S. M. M. Basha, G. A. Baumbach, E. H. Mansfield, and R. M.
Roberts. (1980) Alkaline urea solubilization, two-dimensional
electrophoresis and lectin staining of mammalian cell plasma
membrane and plant seed proteins. Anal. Biochem., 102: 399-408.
Hunter, W. M., and F. C. Greenwood. (1962) Preparation of iodine-131
labelled human growth hormone of high specific activity. Nature,
194: 495-496.
Isaacs, N., R. James, H. Niall, G. Bryant-Greenwood, G. Dodson, A. Evans,
and A. C. T. North. (1978) Relaxin and its structural relation¬
ship to insulin. Nature, 271: 278-281.
James, R., H. Niall, S. Kwok, and G. Bryant-Greenwood. (1977) Primary
structure of porcine relaxin: Homology with insulin and related
growth factors. Nature, 267: 544-546.
John, M. J., B. W. Borjesson, J. R. Walsh, and H. Niall. (1981)
Limited sequence homology between porcine and rat relaxins: Impli¬
cations for physiological studies. Endocrinology, 108: 726-729.
Kendall, J. Z., C. G. Plopper, and G. D. Bryant-Greenwood. (1978)
Ultrastructural immunoperoxidase demonstration of relaxin in
corpora lútea from a pregnant sow. Biol. Reprod., 18: 94-98.

67
Krantz, J. C., H. H. Bryant, and C. J. Carr. (1950) The action of
aqueous corpus luteum extract upon uterine activity. Surg.
Gynecol. Obstet., 90: 372-375.
Kroc, R. L., B. G. Steinetz, and V. L. Beach. (1959) The effects of
estrogens, progestagens, and relaxin in pregnant and non-pregnant
laboratory rodents. Ann. N. Y. Acad. Sci., 75: 942-980.
Larkin, L. H. (1974) Bioassay of rat metrial gland extracts for
relaxin using the mouse interpubic ligament technique. Endocrin¬
ology, 94: 567-570.
Larkin, L. H., P. A. Fields, and R. M. Oliver. (1977) Production of
antisera against electrophoretically separated relaxin and immuno-
fluorescent localization of relaxin in the porcine corpus luteum.
Endocrinology, 101: 679-683.
Larkin, L. H., P. A. Fields, and R. J. Pardo. (1981) Mouse uterus bio¬
assay for relaxin. In: Relaxin. (G. D. Bryant-Greenwood, H. D.
Niall and F. C. Greenwood, eds.) Elsevier, North Holland.
Larkin, L. H., C. A. Suarez-Quian, and P. A. Fields. (1979) In vitro
analyses of antisera to relaxin. Acta Endocrinol., 92: 568-576.
Loumaye, E., B. Teuwissen, and K. Thomas. (1978) Characterization of
relaxin radioimmunoassay using Bolton-Hunter reagent. Gynecol.
Obstet. Invest., 9: 262-267.
Lowry, 0. H., N. J. Roseborough, A. L. Farr, and R. J. Randall. (1951)
Protein measurement with the folin phenol reagent. J. Biol. Chem.,
193: 265-275.
MacLennan, A. H., R. C. Green, G. D. Bryant-Greenwood, F. C. Greenwood,
and R. F. Seamark. (1980) Ripening of the human cervix and
induction of labor with purified porcine relaxin. Lancet, Feb. 2,
1980, pp. 220-223.
Marcus, G. J. (1974) Mitosis in the rat uterus during the estrus cycle,
early pregnancy, and early pseudopregnancy. Biol. Reprod. 10:
447-452.
Markwell, M. A. K., and C. F. Fox. (1978) Surface-specific iodination
of membrane proteins of viruses and eukaryotic cells using 1, 3,
4, 6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. Biochem.,
17: 4807-4817.
Mathieu, P. H., J. Rathier, and K. Thomas. (1981) Localization of
relaxin in human gestational corpus luteum. Cell Tiss. Res., 219:
213-216.

68
Nagao, R., and G. D. Bryant-Greenwood. (1981) Evidence for a uterine
relaxin in the guinea pig. In: Relaxin. (G. D. Bryant-Greenwood,
H. D. Niall and F. C. Greenwood, eds.) Elsevier, North
Holland.
Noall, M. W., and E. H. Frieden. (1956) Variations of sensitivity of
ovariectomized guinea pigs to relaxin. Endocrinology, 5: 659-664.
O'Byrne, E. M., F. F. Flitcraft, W. K. Sawyer, J. Hochman, G. Weiss, and
B. G. Steinetz. (1978) Relaxin bioactivity and immunoactivity
in human corpora lútea. Endocrinology, 102: 1641-1644.
O'Byrne, E. M., W. K. Sawyer, M. C. Butler, and B. G. Steinetz. (1976)
Serum immunoreactive relaxin and softening of the uterine cervix
in pregnant hamsters. Endocrinology, 99: 1333-1335.
O'Byrne, E. M., and B. G. Steinetz. (1976) Radioimmunoassay (RIA) of
relaxin in sera of various species using an antiserum to porcine
relaxin. Proc. Soc. Exp. Biol. Med., 152: 272-276.
Pardo, R., L. H. Larkin, and P. A. Fields. (1980) Immunocytochemical
localization of relaxin in endometrial glands of the pregnant guinea
pig. Endocrinology, 107: 2110-2112.
Porter, D. G. (1972) Myometrium of the pregnant guinea pig: The
probable importance of relaxin. Biol. Reprod., 7: 458-464.
Porter, D. G. (1979) Relaxin: Old Hormone, new prospect. In: Oxford
Reviews of Reproductive Biology, Vol. 1 (C. A. Finn, ed.) Clarendon
Press, Oxford, England.
Reinig, J. W., D. N. Lambert, C. Schwabe, L. K. Gowan, B. G. Steinetz,
and E. M. O'Byrne. (1981) Isolation and characterization of
relaxin from the sand tiger shark (odontaspis taurus). Endocrin¬
ology, 109: 537-543.
Sanders, M. M., V. E. Groppi, Jr., and E. T. Browning. (1980) Resolu¬
tion of basic cellular proteins including histone variants by
two-dimensional gel electrophoresis: Evaluation of lysine to
arginine ratios and phosporylation. Anal. Biochem., 103: 157-
165.
Sar, M., and W. E. Stumpf. (1974) Cellular and subcellular localization
of ^H-progesterone or its metabolites in the oviduct, uterus,
vagina and liver of the guinea pig. Endocrinology, 94: 1116-1125.
Schwabe, C., and S. A. Braddon. (1976) Evidence for one essential
tryptophan residue at the active site of relaxin. Biochem.
Biophys. Res. Commun., 68: 1126-1132.

69
Schwabe, C., J. K. McDonald, and B. G. Steinetz. (1976) Primary
structure of the A-chain of porcine relaxin. Biochem. Biophys.
Res. Commun., 70: 397-405.
Schwabe, C., J. K. McDonald, and B. G. Steinetz. (1977) Primary
structure of the B-chain of porcine relaxin. Biochem. Biophys.
Res. Commun., 75: 503-510.
Schwabe, C., B. G. Steinetz, G. Weiss, A. Segaloff, J. K. McDonald,
E. O'Byrne, J. Hochman, B. Carriere, and L. Goldsmith. (1978)
Relaxin. Rec. Prog. Horm. Res., 34: 123-211.
Sherwood, 0. D. (1979) Purification and characterization of rat
relaxin. Endocrinology, 104: 886-892.
Sherwood, 0. D., and V. E. Crnekovic. (1979) Development of a homo¬
logous radioimmunoassay for rat relaxin. Endocrinology, 104:
893-897.
Sherwood, 0. D., P. A. Martin, C. C. Chang, and P. J. Dziuk. (1977a)
Plasma relaxin levels in pigs with corpora lútea induced during
late pregnancy. Biol. Reprod., 17: 97-100.
Sherwood, 0. D., P. A. Martin, C. C. Chang, and P. J. Dziuk. (1977b)
Plasma relaxin levels during late pregnancy and at parturition in
pigs with altered utero-ovarian connections. Biol. Reprod.,
17: 101-103.
Sherwood, 0. D., and E. M. O'Byrne. (1974) Purification and character¬
ization of porcine relaxin. Arch. Biochm. Biophys., 160: 185-196.
Sherwood, 0. D., K. R. Rosentreter, and M. L. Birkhimer. (1975) Develop¬
ment of a radioimmunoassay for porcine relaxin using bI labelled
polytyrosyl-relaxin. Endocrinology, 96: 1106-1112.
Steinetz, B. G., V. L. Beach, R. L. Kroc, N. R. Stasilli, R. E. Nussbaum,
P. J. Nemith, and R. K. Dun. (1960) Bioassay of relaxin using
a reference standard: A simple and reliable method utilizing
direct measurement of interpubic ligament formation in mice.
Endocrinology, 67: 102-115.
Sternberger, L. A. (1979) Immunocytochemistry. John Wiley and Sons,
New York.
3
Stumpf, W. E. (1968) Subcellular distribution of H-estradiol m rat
uterus by quantitative autoradiography--a comparison between ^H-
estradiol and %-norethynodrel. Endocrinology, 83: 777-782.

70
Sutcliffe, R. G., D. J. H. Brock, L. B. V. Nicholson, and E. Dunn.
(1978) Fetal- and uterine-specific antigens in human amniotic
fluid. J. Reprod. Fértil., 54: 85-90.
Szalchter, N., E. O'Byrne, L. Goldsmith, B. G. Steinetz, and G. Weiss.
(1980) Myometrial inhibiting activity of relaxin-containing
extracts of human corpora lútea. Am. J. Obstet. Cynecol., 136:
584-586.
Walsh, J. R., and H. D. Niall. (1980) Use of an octadecylsilica
purification method minimizes proteolysis during isolation of
porcine and rat relaxins. Endocrinology, 107: 1258-1260.
Warembourg, M. (1974) Radiographic study of the guinea pig uterus
after injection and incubation with "ll-progesterone. Endocrinology,
94: 665-670.
Weiss, G., E. M. O'Byrne, J. A. Hochman, L. T. Goldsmith, I. Rifkin, and
B. G. Steinetz. (1977) Secretion of progesterone and relaxin by
the human corpus luteum at midpregnancy and at term. Obstet.
Gynecol., 50: 679-681.
Weiss, G., E. M. O'Byrne, J. A. Hochman, B. G. Steinetz, L. Goldsmith,
and J. G. Flitcraft. (1978) Distribution of relaxin in women
during pregnancy. Obstet. Gynecol., 52: 568-570.
Weiss, G., E. M. O'Byme, and B. G. Steinetz. (1976) Relaxin: A
product of the corpus luteum of pregnancy. Science, 194: 948-949.
Yamamoto, S., S. C. M. Kwok, F. C. Greenwood, and G. D. Bryant-Greenwood.
(1981) Relaxin purification from human placental basal plates.
J. Clin. Endocrinol. Metab., 52: 601-607.
Zarrow, M. X. (1947) Relaxin content of blood, urine and other tissues
of pregnant and postpartum guinea pigs. Proc. Soc. Exp. Biol.
Med., 66: 488-491.
Zarrow, M. X. (1948) The role of the steroid hormones in the relaxation
of the symphysis pubis of the guinea pig. Endocrinology, 42: 129-
140.
Zarrow, M. X., E. G. Holmstrom, and H. A. Salhanick. (1955) The con¬
centration of relaxin in the blood serum and other tissues of
women during pregnancy. J. Clin. Endocrinol. Metab., 15: 22-27.
Zarrow, M. X., and J. A. McClintock. (1966) Localization of I
labelled antibody to relaxin. J. Endocrinol., 36: 377-387.

71
Zarrow, M. X., and W. B. O'Connor. (1966) Localization of relaxin in
the corpus luteum of the rabbit. Proc. Soc. Exp. Biol. Med.,
121: 612-614.
Zarrow, M. X., and B. Rosenberg. (1953) Sources of relaxin in the
rabbit. Endocrinology, 53: 593-598.

APPENDIX 1
TABLES

Table 1. Bioactlve relaxin content of uteri from guinea pigs in different stages of pregnancy and lactation
STAGE OF ANIMAL
GESTATION NO.
(g) (mg)
WET WT. DRY WT.
TOTAL UNITS OF
RELAXIN PER UTERUS
UNITS OF RELAXIN TER
GRAM WET WEICIIT
DAY 30
4
6.1
61.0
(2)
1.65
0.25
5
2.6
23.0
(2)
0.87
(0.63 + 0.40)**
0.33
(0.15 + 0.09)
6
7.2
106.0
(1)
0
0
7
4.0
38.0
(1)
0
0
DAY 45
8
8.0
443.5
(2)
20.84
2.61
9
3.2
195.0
(2)
9.17
(13.71 + 2.52)
2.86
(2.19 + 0.51)
10
5.0
280.0
(1)
13.16
2.63
11
17.5
248.0
(2)
11.66
0.66
DAY 60
12
17.0
273.4
(3)
42.83
2.19
13
18.0
346.5
(3)
43.43
(46.80 + 2.85)
2.41
(2.62 + 0.20)
14
20.0
391.0
(2)
55.13
2.76
15
14.7
243.7
(2)
45.82
3.12
Late
16
13.0
321.0
(2)
50.08
3.85
Pregnant
17
16.0
268.2
(2)
41.84
2.62
18
26.0
495.6
(2)
77.31
(57.75 + 7.35)
2.97
(3.76 + 0.76)
19
11.0
234.9
(2)
73.52
6.68
20
17.2
194.3
(4)
46.00
2.68
Lactating
21
6.3
53.9
(1)
0
0
22
4.0
50.0
(1)
0
(1.50 + 1.17)
0
(0.23 + 0.14)
23
3.0
35.6
(1)
1.07
0.36
24
9.2
105.0
(1)
4.94
0.54
* Twenty milligrams
(20 mg) of
Acid Acetone
ext racted
powder was homogenized in
1 ml of distilled water.
** Units of
activity
are expressed as porcine NIH-RXN-
PI standard relaxin equivalents (X + SEM) determined
by the mouse uterine motility bioassay.
( )
denotes
the number of bioassays
conducted.

Table 2
Immunoreactive relaxin content of uteri taken from guinea pigs in different stages of pregnancy and lactation.
(ng)
( ng)
STAGE OF
ANIMAL
(«>
fag)
TOTAL G.P. RELAXIN
AMT.
RELAXIN
PREGNANCY
NO.
WET WT.
DRY WT.
PER UTERUS PER GRAM
WET WEIGHT
DAY 15
1
2.0
13.0
0.65
0.33
2
1.5
10.0
0.40 (0.40 + 0.14)**
0.27
(0.25 + O.i
3
1.0
9.0
0.16
0.16
DAY 30
4
6.1
61.0
12.20
2.00
5
2.6
23.0
1.46
6
7.2
106.0
23 85 ni.44 + 4.51)
3.31
(2.06 + 0.<
7
4.0
38.0
5.89
1.47
DAY 45
8
8.0
443.5
199.58
24.95
9
3.2
195.0
33.52
10
5.0
280.0
168.00 (148.47 + 21.52)
33.60
(24.72 + 6
11
17.5
248.0
119.04
6.80
DAY 60
12
17.0
273.4
158.57
9.33
13
18.0
346.5
173 25
9.63
14
20.0
391.0
224Í83 <172-67 ± 19.17)
11.24
(9.83 + 0.'
15
14.7
243.7
134.04
9.12
LATE
16
13.0
321.0
72.23
5.56
PREGNANT
17
16.0
268.2
64.37
4.02
18
26.0
495.6
183.37 (101.90 + 25.78)
7.05
(6.45 + l.;
19
11.0
234.9
140.94
12.81
20
17.2
194.3
48.58
2.82
1.AC TAT I NG
21
6.3
53.9
5.66
0.90
22
A .0
50.0
3.00 ,, „„ „
0.75
23
3.0
35.6
5 52 (4-78 ± °-6D
1.84
(1.01 + o.:
24
9.2
105.0
4.94
0.54
‘Twenty milligrams
of Acid
Acetone extracted
powder was
homogenized in 1 ml of distilled
water.
**Activity expressed as ng porcine NIII-RXN-P1 standard relaxin equivalents (X + SFM) as determined by a
homologous porcine radioimmunoassay. All assays were conducted in duplicate
'-J
4^

Table 3. Protein yield and potencies of late pregnant guinea pig uterus throughout the ODS purification procedure.
Fraction
Kecovery (mg)
Protein Yielda
(mg/g Fresh Tissue)
Total Units
Spec 1fic Activity
(U/mr.)
ODS extracted guinea pig
uterus
188.80
1.59
60.00
(2) 0.32
Bioactlve Sepliadex C-50
f raction^
35.50
0.30
53.19
(2) 1.50
Carboxymethylcellulose ion
exchange chromatography (CHC)c
1.70
0.01
6.39
(2) 3.87
Yields represent the values calculated from 5 uteri. The mouse uterine molility bioassay was utilized to determine
the potency of the preparations. ( ) denotes the number of bioassays conducted.
a Total wet weight of the ute-ri was 118.5 grams.
Bioassays were conducted on the pooled relaxln containing fractions
C Bioassays were conducted on the pooled relaxln containing fractions
from the chromatography run.
from the CMC run.

76
Table 4. Physiochemical characteristics of guinea pig
uterine relaxin. The source of relaxin was an
ODS* purified uterine preparation.
Reduced Activity
No Change
Dithiothrietol
24x
Heating at 70° C
2x
Trypsin
24x
R19 antiserum
1 lx
*ODS crude relaxin is a partially purified uterine extract
taken after the initial purification step in the ODS pro¬
cedure. This extract was tested in the mouse uterine motility
assay, without being altered (control), and after experimental
treatments. A ratio was determined by dividing the final
volume of the experimental by the final volume of the control.
The assays were run twice and an average value was computed.
The greater the experimental to control ratio, the greater
the ability of the agent to inhibit the action of relaxin.

APPENDIX 2
FIGURES

Figures 1-4 represent sections of uteri taken from guinea pigs
on day 30 of pregnancy.
1.Transverse section of uterus stained using the PAP tech¬
nique with R19 antiserum (1:500 dilution). Arrow - endometrial
glands exhibiting RP; arrowhead - endometrial glands lacking RP.
X 40.
2. Section adjacent to that shown in Figure 1 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo¬
metrial glands (arrows). X 40.
3. Hematoxylin and eosin stained section. X 500.
4. Section stained using the PAP technique with R19 anti¬
serum (1:500 dilution). Note that not all endometrial gland cells
demonstrate RP. Clear areas in basal regions of the endometrial
gland cells represent unstained nuclear profiles. X 500.

79
*ir

Figures 5-8 represent sections of uteri taken from guinea pigs
on day 45 of pregnancy.
5. Transverse section of the guinea pig uterus taken on
day 45 of pregnancy and stained using the PAP technique with R19
antiserum (1:500 dilution). Arrow - endometrial glands exhibiting
RP; arrowhead - endometrial glands lacking RP. A higher percentage
of glands are labeled than in day 30 tissue, however, not all glands
have RP at this stage. X 40.
6. Section adjacent to that shown in Figure 5 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over
endometrial glands (arrows). X 40.
7. Hematoxylin and eosin stained section. X 500.
8. Section stained using the PAP technique with R19 anti¬
serum (1:500 dilution). While not all cells in each gland show
the presence of RP, note that the majority of cells in each gland
show a heavy accumulation of RP. X 500.

81

Figures 9-12 represent sections of uteri taken from guinea
pigs on day 60 of pregnancy.
9.Transverse section of the guinea pig uterus taken on
day 60 of pregnancy stained using the PAP technique with R19 anti¬
serum (1:500 dilution). Note that all of the endometrial glands
exhibit RP. X 40.
10. Section adjacent to that shown in Figure 9 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo¬
metrial glands. X 40.
11. Hematoxylin and eosin stained section. Note what appear
to be dense aggregates of material near the luminal surface of the
endometrial gland cells. X 500.
12. Section stained using the PAP technique with R19 anti¬
serum (1:500 dilution). Dense aggregates of RP similar to that
demonstrated in Figure 9 are shown in the luminal surfaces of the
EGC. Clear areas in base of EGC represent unstained nuclear
profiles. X 500.

83

Figures 13-16 represent sections of uteri taken from late
pregnant guinea pigs.
13. Transverse section of the guinea pig uterus from late
pregnant animals (day 65 or 66 of pregnancy) and stained using the
PAP technique with R19 antiserum (1:500 dilution). Note all of the
endometrial glands exhibit RP. X 40.
14. Section adjacent to that shown in Figure 13 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo¬
metrial glands. X 40.
15. Hematoxylin and eosin stained section. Continuity
between the lumen of an endometrial gland and the lumen of the
uterus can be seen in the lowest of the three gland profiles. Note
the difference in cytoplasmic and nuclear staining densities between
the EGC and cells of the uterine lumen epithelium. X 500.
16. Section adjacent to that shown in Figure 15 stained
using the PAP technique with R19 antiserum (1:500 dilution). Note
that some of the endometrial gland cells have RP distributed through¬
out the cytoplasm, some have no RP and in some cells (lower gland)
the RP is localized in a specific supranuclear region. Note also
that the extent of the gland can be determined by the region where
deposition of RP ceases in cells that are continuous with the
uterine lumen epithelium. This pattern of deposition of RP
corresponds with differences in staining noted in H § E stained
tissue (Figure 15). X 500.

85
16

Figures 17-20 represent sections of uteri taken from lactating
guinea pigs.
17.Transverse section of guinea pig uterus taken from lac¬
tating animals (3 days postpartum) and stained using the PAP
technique with R19 antiserum (1:500 dilution). Arrow - endometrial
glands exhibiting RP; arrowhead - endometrial glands lacking RP.
X 40.
18. Section adjacent to that shown in Figure 17 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo¬
metrial glands (arrows). X 40.
19. Hematoxylin and eosin stained section. Note the large
number of mitoses in the glands (arrows). X 500.
20. Section stained using the PAP technique with R19 anti¬
serum (1:500 dilution). Note that RP is located in only a few
cells of the gland and that the pattern of deposition of RP is
variable from cell to cell. X 500.

87

Figures 21-24 represent sections of uteri taken from ovari-
ectomized hormone treated animals.
21. Transverse section of guinea pig uterus taken from
ovariectomized animals treated with estrogen (10 yg) and progester¬
one (2 mg) daily for 15 days stained using the PAP technique with
R19 antiserum (1:500 dilution). Arrow - endometrial glands exhibit¬
ing RP. X 40.
22. Section adjacent to that shown in Figure 21 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo¬
metrial glands (arrows). X 40.
23. Hematoxylin and eosin stained section. Glandular cells
are cuboidal with densely staining nuclei. X 500.
24. Section stained using the PAP technique with R19 anti¬
serum (1:500 dilution). Only a few endometrial gland cells do not
show the presence of relaxin. Location of RP varies from cell to
cell, however, the majority of cells appear to have RP distributed
throughout the cytoplasm. X 500.

89

Figure 25. Biologically active relaxin content o_f uteri
taken from guinea pigs during pregnancy and lactation (X +_SEM) .
Data expressed as total units per uterus as determined by the mouse
uterine motility bioassay of uterine extracts.

STAGE OF PREGNANCY/ LACTATION
UNITS OF RELAXIN ACTIVITY (total units per uterus)
_ tv> oi & ai
o o o o o o
i i i i ti i" r i r i i i i i i i i i i i i vt 'i"ii i i i i
to
8 0

Figure 26. Biologically active relaxin content in the guinea
pig uterus during pregnancy and lactation (X +_ SEM). Data expressed
as units per gram wet weight of uterus as determined by the mouse
uterine motility bioassay of uterine extracts.

STAGE OF PREGNANCY/ LACTATION
UNITS OF RELAXIN ACTIVITY (units per gram wet weight)
to

125
Figure 27. Separation of I polytyrosyl relaxin from unbound components
utilizing a Sephadex G-25 chromatography column (1 x 18 cm). Tubes 10-15 were
immunoreactive with antiserum R19.

C PM
VO
cn

Figure 28. Antiserum titration curve. Pooled tubes 10-13 were tested
with decreasing dilutions of R19 antiserum, and % antigen bound was determined.
A dilution of 1:28,800 was employed in the RIA (approximately 25-30% binding).
% Bound = amount of 125j relaxin precipitated by the R19 antiserum.

% BOUND
80
70
60
50
40
1/1,800 1/7,200 I/28JBOO 1/115,200
FINAL ANTISERUM CONC.
1/230,400
10

Figure 29. Inhibition by late pregnant guinea pig acid-
acetone crude extracts and ODS crude extracts in a RIA using ^^1
polytyrosyl porcine relaxin. R19 antiporcine relaxin serum (1:27,000
dilution) and NIH-RXN-P1 porcine relaxin standard. Standard porcine
relaxin (NIH-RXN-P1) (•), ODS crude extracts (*) and acid acet>ne
crude extracts (â– ). % Bound = Bound-NSB
Bo-NSB

I o
1 1 1 I 1 1
N|H ,, ,
31.25 62.5
125
250
500
1000
pg
RXN-PI
1
1
1
GP ...
3.5
7
14
28
56
112 pg
ODSCRUDE
1
GP
62.5
125
250
500
1000
2000 jjg
ACID ACETONE
CRUDE
AMOUNT OF MATERIAL

Figure 30. Immunoreactive relaxin content (total ng) in the
guinea pig uterus during pregnancy and lactation (X +_ SEM). Data
expressed as ng porcine NIH-RXN-P1 standard relaxin equivalents,
determined by the homologous porcine radioimmunoassay presented
in Figure 29.

IMMUNOREACTIVE RELAXIN
(total ng per uterus)
cn
to -t* O o O o c
>ll| -| "7 I I I I I I" I" I I I I I I I
ro cn
o o o
t 11 "i" r r i i i -i ii
H
> _
CD (ji
m
oj
o
*
CJl
o
n
“0
Z) &
m
CD
z-0
> ó
o
t
J
CD
o
r i i i â– â–  r
>
O
>
H
O
o
200r

Figure 31. Immunoreactive relaxin concentration (ng/gram
wet weight) during pregnancy and lactation (X +_ SEM). Data expressed
as ng porcine NIH-RXN-P1 standard relaxin equivalents, determined
by the homologous porcine radioimmunoassay presented in Figure 29.

CONC. I MMUNO R E A C TI VE RELAXIN (ng/grom wet weight)
103

I
Figure 32. Gel filtration of an ODS crude uterine extract from late pregnant guinea pigs
(188.8 mg) on a 2 x 100 cm column of Sephadex G-50 (fine). The column was equilibrated with
0.01 M ammonium acetate pH 5.0. The flow rate was maintained at 7.5 ml/hr and fractions were
collected every 24 minutes. Biological activity (mouse uterine motility bioassay) is indicated
by the hatched area. The bioactive fractions were pooled to form the Sephadex G-50 fraction.
104

Figure 33. Ion exchange chromatography of the Sephadex G-50 bioactive fraction
(35.5 mg) on a 0.8 x 5 cm column of carboxymethylcellulose (CM52). The column was
equilibrated and rinsed with 0.01 M ammonium acetate buffer, pH 5.0 (conductivity, 0.9
m Mho), until all unadsorbed material was eluted. The column was developed at a rate of
9 ml/hr with a linear gradient of 0.01 M ammonium acetate buffer (pH 5.0) and increasing
concentrations of NaCl (0.1 M ammonium acetate with 0.3 M NaCl), to a final conductivity
of 20 m Mho. Fractions (1.5 ml) were collected every 10 minutes.

ABSORBANCE 280nm
FRACTION NO.
â– a
ó
N.
E
\
o>
z
X
<
UJ
a:
u.
o
»-
z
=3
O
2
<
o
O'

Figure 34. Agar double immunodiffusion plate. R19-5 yl of
antiserum produced against porcine relaxin concentrated four times
by lyophilization; NIH-5 yl porcine NIH-RXN-P1 relaxin (100 yg/ml
1L0). G.P.-5 yl of day 60 guinea pig uterus relaxin preparation
(Bio-Gel P-30 6,000 mw fraction, 6 mg/ml 1^0).

108

109
*
. Figure 35. Two dimensional gel electrophoresis. The first
dimension (NEPHGE) utilized a gradient of pH 5.4 to 9.8. The gels
were stacked for 15 minutes at 75 V and then run for 2.5 hr at
400 V. The second dimension consisted of a 15% SDS polyacrylamide
slab gel system. Fifteen mamps/slab were used as the stacking
current for 2 hr. The current was turned to 30 mamps/slab and the
gels run for an additional 4-5 hr. The gels were fixed with 7%
acetic acid: 40% ethyl alcohol and stained with 0.125% coomasie
blue R250. The gels were destained with 7% acetic acid:10% ethyl
alcohol.

110
E
E
x
o
ÃœJ
Figure 36. Mouse interpubic ligament bioassay of a Sephadex
G-50 6,000 mw fraction of guinea pig uterine extract, and a porcine
relaxin standard (NIH-RXN-P1). Twenty mice were used at each dose
level for the porcine standard, and fifteen mice were used at each
dose level for the guinea pig preparation. The mean interpubic
ligament length for the mice treated with estradiol and 1% benzo-
purpurine 4 B was 0.89 +_ 0.075 (X +_ SEM) . The best fit curve for
the NIH-RXN-P1 porcine relaxin was y = 1.2 (log x) + 0.83, with
a SE = 0.086 and a lambda value of 0.08. The best fit curve for
the guinea pig CMC purified relaxin was y = 0.87 (log x) + 0.64
with a SE = 0.097 and a lambda value of 0.12. The mean observed
potency of guinea pig uterine relaxin was 0.8 U/mg.

APPENDIX 3
IODINATION OF SUCCINIMIDE RELAXIN
Iodination of NIH-RXN-P1 relaxin with the succinimide method.--
An aliquot of ethyl acetate was dried with an excess of anhydrous sodium
sulfate. Two milligrams of N-succinimidyl 3(4-hydroxyphenyl) propionate
were dissolved in 10 ml of the dried ethyl acetate. Ten microliters of
the dissolved ester (2 Pg) were then dried in a small vial under vacuum.
Twenty-five micrograms of relaxin in 50 yl of borate buffer, 0.1 M pH 8.5,
were added to the dry ester and agitated at 0° C for 15 min. Twenty
microliters (10 yg) of the acylated relaxin were added to 0.5 mCi
125
I purchased from Amersham Co., Arlington Heights, IL., and mixed in
a tube coated with iodogen (100 yg dried), for 15 min with intermittent
shaking. The reaction mixture was then layered on a 1 x 18 cm column of
Sephadex G-25 preequilibrated with 0.05 M sodium phosphate buffer pH
7.0 with 0.025% gelatin, and eluted with 0.05 M sodium phosphate buffer
pH 7.0. Ten drop aliquots were collected in 10 x 75 mm disposable glass
tubes containing 0.5 ml PBS-5% ovalbumin. The elution pattern is shown
in Figure 1-1. The peaks were tested for immunoactivity with negative
results.
Ill

125
Figure 1-1. Separation of I succinimide relaxin from unbound components
utilizing a Sephadex G-25 chromatography column (1 x 18 cm). Radioactivity expressed
as CPM's per 10 pi of solution. None of the peaks were immunoactive as tested with
an antiserum titration test.

C PM
113

APPENDIX 4
IODINATION OF RELAXIN WITH THE BOLTON AND HUNTER REAGENT
Iodination of NIH-RXN-P1 relaxin with the Bolton and Hunter reagent
(p-hydroxy phenyl propionic acid, N-hydroxysuccinimide ester).—Five
micrograms relaxin dissolved in 10 pi of 0.1 M sodium borate buffer
(pH 8.5) were added to the iodinated Bolton and Hunter reagent purchased
from New England Nuclear Co., North Billerica, MA, and incubated in an
ice bath (0° C) for 1.5 hr with intermittent shaking. At the end of
the incubation period, 0.5 ml of 0.2 M glycine dissolved in 0.1 M
sodium borate (pH 8.5) was added and stirred for 15 min at 0° C. The
resulting solution was then layered on a Sephadex G-25 (1 x 18 cm) column
equilibrated with 0.05 M sodium phosphate (pH 7.5) and 0.025% gelatin.
Ten drop aliquots were collected in 10 x 75 mm disposable glass culture
tubes containing 0.5 ml PBS-5% ovalbumin. The elution pattern is shown
in Figure 2-1. The iodinated relaxin peak was tested for immunoactivity
by employing an antigen-antibody titration (Figure 2-2).
RIA Procedure
Day 1: (1) add 50 pi of R19 antiserum (1:3000 dilution in PBS
pH 7.0) to each tube containing the 200 pi of standard
and unknown solutions, and to the "0" count tubes.
(2) add 50 pi of PBS to the blank tubes (nonspecific
binding).
(3) mix tubes well and incubate at 4° C for 24 hr.
Day 2: add 50 pi of ^5j relaxin (50,000 CPM to each tube, mix,
cover and incubate at 4° C for 24 hr.
114

115
Day 3: add 100 pi of GAR at 1/29 dilution and 50 yl of NRS to
all the tubes except the total count tubes. Mix, cover,
and incubate at 4° C for 48 hr.
Day 5: centrifuge at 3000 RPM's for 30 minutes, decant supernatant
and count precipitate.
This procedure was not employed because the nonspecific binding
was very high in the first batch of Bolton and Hunter reagent received
from New England Nuclear (15-20% of total binding). The addition of
male rabbit serum (1/60 dilution) to the first antibody decreased the
amount of nonspecific binding to below 5% of total binding, but also
decreased sensitivity.

1 o c
Figure 2-1. Separation of I relaxin prepared with the Bolton and
Hunter reagent, from unbound components, utilizing a Sephadex G-25 chrom¬
atography column (1 x 18 cm). Tubes 17-25 were immunoreactive with R19
antiserum. Radioactivity expressed as CPM's per 10 yl of solution.


Figure 2-2. Antiserum titration curve. Pooled tubes 17-25 were
tested with decreasing dilutions of R19 antiserum and % antigen bound was
determined. A dilution of 1:18,000 was employed in the RIA (approximately
50% binding). % Bound = amount of 125j relaxin precipitated by the R19
antiserum.

5/o BOUND
FINAL ANTISERUM CONC.
119

BIOGRAPHICAL SKETCH
Rube Jose Pardo was born October 22, 1950, in Havana, Cuba. He
received his undergraduate education at the University of Miami, Florida,
graduating with a Bachelor of Science degree in biology in 1972. He
received his Master of Science degree at Marquette University, Milwaukee,
Wisconsin, in 1974. He taught biology at Mattatuck Community College
between 1974 and 1977. He has been enrolled at the University of
Florida since September, 1977, and is now a candidate for the degree
of Doctor of Philosophy.
120

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
/
Vi
/â– >
Lynn H. Larkin, Chairman
'Professor of Anatomy
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Associate Professor of Anatomy
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Thomas G. Hoilinger
Associate Professor of Anatomy
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Michael J. Fields
Associate Professor of Animal Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Satya ^P. Kalra
Associate Professor of Obstetrics
and Gynecology
This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
May 1982
Dean for Graduate Studies and Research

UNIVERSITY OF FLORIDA
3 1262 08554 3493