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PURIFICATION, CHARACTERIZATION AND LOCALIZATION
OF RELAXIN IN THE PREGNANT GUINEA PIG
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
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.
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.
TABLE OF CONTENTS
ACKNOWLEDGMENTS . . .....
LIST OF ABBREVIATIONS . . .
ABSTRACT . . . ...
INTRODUCTION . . . .
Relaxin Assays . .
Cellular Localization of Relaxin .. ...
Isolation and Characterization of Relaxin.
Relaxin in the Guinea Pig .
Statement of Problem . .
MATERIALS AND METHODS . .
General Procedures . .
Detection of Relaxin . .
Purification and Characterization of Guinea Pig Relaxin.
RESULTS . . . .
Detection of Guinea Pig Relaxin . .
Purification and Characterization of Guinea Pig Uterine
Relaxin . . .......
DISCUSSION . . . .
R19 Antiserum: Detection of Guinea Pig Relaxin .
Detection of Guinea Pig Relaxin with the PAP Technique .
Detection of Guinea Pig Relaxin with Radioimmunoassay .
Endometrial Glands and Their Role in Relaxin Production. .
Possible Actions of Uterine Relaxin in the Guinea Pig .
Purification and Characterization of Guinea Pig Relaxin .
BIBLIOGRAPHY . . . .
APPENDIX 1 TABLES . . .
APPENDIX 2 FIGURES . . .
: : : : ::::
APPENDIX 3 IODINATION OF SUCCINIMIDE RELAXIN. ... 111
APPENDIX 4 IODINATION OF RELAXIN WITH THE BOLTON AND
HUNTER REAGENT ................. 114
BIOGRAPHICAL SKETCH ....................... .. 120
LIST OF ABBREVIATIONS
Bo zero count tube
CMC carboxymethyl cellulose
CPM counts per minute
DAB 3,3' diaminobenzidine
EG endometrial gland(s)
EGC endometrial gland cell(s)
GAR goat anti-rabbit IgG
gww gram wet weight
H & E hematoxylin and eosin
L uterine lumen
lp late pregnant
mw molecular weight
NEPHGE non equilibrium polyacrylamide gel electrophoresis
NSB nonspecific binding
NRS normal rabbit serum
PAP peroxidase antiperoxidase
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
R19 antiserum made to purified porcine relaxin
RP peroxidase reaction product
RPM revolutions per minute
SDS sodium dodecyl sulfate
SE surface epithelium of uterine lumen
T total count tube
TCA trichloroacetic acid
U unit(s) of relaxin activity
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
Rube Jose Pardo
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
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
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.
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
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
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).
In 1972, Bryant developed the first homologous RIA* for porcine
relaxin. In this assay, a relatively impure relaxin preparation (NIH-R-
P1, 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
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-P1) 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
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'Byrne and Steintez, 1976; O'Byrne 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
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 relaxirn 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
lutea of pregnancy and in corpora lutea cyst fluid of pregnant and non-
The only homologous nonporcine RIA system has been developed for
the detection of rat relaxin by Sherwood and Crnekovic (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
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
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
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.
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
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 ovary of the pregnant cow has been shown to be a source of
relaxin with bioassay techniques (Castro-Hernandez, 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.
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
immunocytochemical 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-PI, 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 131I labeled antibody to porcine relaxin into pregnant
rats and discovered substantial accumulations of label in the ovary and
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).
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.
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
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'Byrne, 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).
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
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
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'Byrne (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'Byrne (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
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
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
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
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
studies that relaxin, like insulin, may be cleaved from a larger mw
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 lutea 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-P1 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).
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 CM-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,
respectively, and both had mw 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
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
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
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.
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
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 NaC1). 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
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
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 ruch 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.
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
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
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 lutea 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 guine pig
MATERIALS AND METHODS
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 libitwn 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 pg), (2) estrogen (10 jg) 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
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 -200 C
and later used in the extraction procedure.
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; C1 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
R19 antiserum has been shown to inhibit the biological activity
of porcine, cow and rabbit relaxins in vitro (Larkin et al., 1979).
Also it has been used to detect relaxin immunocytochemically in cells of
the cow ovary (Fields et al., 1980), and human placenta (Fields and
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 40 C. The extract
was incubated for 24 hr at 40 C and then centrifuged at 3000 RPM's
(40 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 -200 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.
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 -800 C in a Revco 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% NaCI and 1 M HC1. The
tissue was homogenized for 2 min in a Sorvall Omni mixer. The homogen-
ate was centrifuged (40 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 pm 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 380 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 Sternberger
(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
(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 Stern-
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% H202 for 5-8 min. The slides were then
washed in distilled water for 5 min, briefly treated with 1% 0 04'
rinsed in distilled water, dehydrated through alcohols and xylene, and
coverslips 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.
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 pg/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 370 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 = VS x CS x SpAS
where VS is the volume (in ul) of standard relaxin preparation needed to
reduce the uterine contractions by half, VU is the volume (in pl) of
unknown relaxin preparation needed to reduce the uterine contractions by
half, CS is the concentration of the standard relaxin preparation (in
ug/ml), CU is the concentration of the unknown relaxin preparation (in
ug/ml), and SpAS is the specific activity of the NIH-RXN-PI 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
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 ug 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 pg, 0.25 ug and 0.125 ug 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 CO2 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)
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.
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-Pl) (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 ig), donated by Dr. B. G. Steinetz
of the Ciba Geigy Corp., Ardsley, NY, was dissolved in 50 pl of 0.1 M
sodium phosphate buffer pH 7.5. The polytyrosyl solution and 1 m Ci
1251 purchased from the Amersham Corp. were added to a 10 x 75 mm test
tube coated with 100 pg dried iodogen. lodination 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 125I-labelled polytyrosyl relaxin was precipi-
table in antibody excess. The 125I-labelled polytyrosyl relaxin was
used for four weeks after iodination before a noticeable drop in sensi-
tivity was noticed in the RIA.
Development of RIA utilizing 12SI-labelled polytyrosyl relaxin.--
Fractions containing the 125I-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
quantities of PBS-1% ovalbumin were added to each tube to bring the
volume to 500 il. 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 40 C for 24 hr. One hundred microliters
of 125I-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 40 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
(T): 100 pi of 125I-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 125I-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 1251
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.
% B bound-NSB
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-PI 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
Purification and Characterization of Guinea
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 methodof 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 7.5ml/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
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
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.
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 vl of R19 antiserum
and peripheral wells were filled with 5 ul of a Bio-Gel P-30 6,000 mw
fraction from guinea pig uterus (6 mg/ml), and NIH-RXN-PI porcine
relaxin. The substances were allowed to diffuse at room temperature for
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 H2SO4. 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 destined in 7% acetic acid:10% ethyl alcohol.
Reduction with dithiothrietol.--Five hundred microliters of an ODS
crude* relaxin fraction (30 mg/ml H20) from late pregnant guinea pig
*ODS 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.
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 H20) from late pregnant guinea pig uterus was heated at 700 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
Trypsin digestion.--Seven hundred microliters of an ODS crude
relaxin fraction (30 mg/ml H20) 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
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 il of NRS
as a control. An ODS relaxin preparation of late pregnant guinea pig
uterus (30 mg/ml H20) was then added to each tube in equal concentrations,
and the volumes required to inhibit the uterine contractions were com-
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 & 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
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 & 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.
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).
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
(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-
relation coefficient (r2 = 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 (r2 = 0.739). A Duncan's multiple
range test for specific biological activity values showed the following
results (alpha level = 0.05)
Ip 60 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
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
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 (r2 = 0.831). A Duncan's multiple range test for total relaxin
immunoactivity showed the following results (alpha level = 0.05).
60 45 Ip 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 (r2 = 0.754). A Duncan's multiple range test for specific
immunoactivity values showed the following results (alpha level = 0.05).
45 60 Ip 30 lac 15
Purification and Characterization of Guinea Pig
Purification: ODS 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).
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
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
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.
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
(700 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.
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.
Detection of Guinea Pig Relaxin with the
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
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
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.
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'Byrne 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'Byrne 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
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
of relaxing 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
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
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.
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'Byrne 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
glands, as evidenced by accumulation of 3H 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.
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
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 phenomenon. (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
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
(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 concepts 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
(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-
logically 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
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
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
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(1953) Sources of relaxin in the
N N N
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0 1 0 1 1 0 I 0
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I t I I I
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II I I II I -0 0 e l
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I 7 I 0 I 0
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Table 4. Physiochemical characteristics of guinea pig
uterine relaxin. The source of relaxin was an
ODS* purified uterine preparation.
Heating at 700 C 2x
R19 antiserum llx
*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.
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.
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.
.- r ":,'. a
r. I .
P'& ^H-^^^^^^^" ^IBJ^R^ W.
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.
S-- 'r' .- -
t .- Ia*.!*-
-. ...* .
. ac t ...
4p ,.:^ -
5 1+-. .-
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.
. lrr .
^.* \ "V
i' ^ ^
*r I t-
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.
I ", 16 .
I'. .. ,15
Figures 17-20 represent sections of uteri taken from lactating
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.
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.
;! ~ ~ 4'. 4k~ .
*3 At ar
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 ug) 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.
-' + (
9 a 21
n'- ~` ,e
Figure 25. Biologically active relaxin content of 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.
30 45 60 Ip lac
STAGE OF PREGNANCY/ LACTATION
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