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

MISSING IMAGE

Material Information

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

Subjects

Subjects / Keywords:
Guinea Pigs -- physiology   ( mesh )
Relaxin -- physiology   ( mesh )
Anatomical Sciences thesis Ph.D   ( mesh )
Dissertations, Academic -- Anatomical Sciences -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000342609
oclc - 08368430
notis - ABX8758
System ID:
AA00009113:00001


This item is only available as the following downloads:


Full Text
















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











BY

RUBE JOSE PARDO


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


UNIVERSITY OF FLORIDA


1982















I wish to dedicate this dissertation to my parents, Mr. and

Mrs. Rube Pardo, and my grandmother, Gilda de la Torriente. This

dissertation is also dedicated in the memory of my grandfather, Jose

Elias de la Torriente.














ACKNOWLEDGMENTS

I would like to express my appreciation to the members of my

supervisory committee for their help in the work presented in this

dissertation. I especially wish to thank Dr. Lynn Larkin, chairman of

my committee, for his help and financial backing. I also wish to

acknowledge the following individuals who have aided me in my doctoral

studies: Dr. Fuller Bazer, Dr. Don Cameron, Mr. Alberto de LaPaz,

Dr. Asgi Fazleabas, Dr. Michael Fields, Dr. Phillip Fields, Dr. Don

Hay, Dr. Thomas Hollinger, Dr. Satya Kalra, Dr. Ernst Kallenbach, Mr.

Denny Player, Mr. Lane Powell, Dr. Ray Roberts, Dr. Lynn Romrell, Mrs.

Pauletta Sanders, Mr. Will Sanders and Dr. Howard Suzuki. A special

thank you goes to my good friends and fellow graduate students, Phil

Ruiz, Wayne Barbee, and Pat Fitzgerald. Finally and most importantly,

I wish to express my deepest appreciation and love to my parents,

Mr. Rube Pardo and Mrs. Georgina T. Pardo; my grandmother, Gilda de la

Torriente; my sisters, Margarita and Georgina; my brother, Roberto;

and my brother-in-law, Bahram.














TABLE OF CONTENTS


Page


ACKNOWLEDGMENTS . . .....

LIST OF ABBREVIATIONS . . .


. iii

. vi


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 . . .


viii


1


. 26


111 -


: : : : ::::


.












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

lac lactating

lp late pregnant

mw molecular weight

NEPHGE non equilibrium polyacrylamide gel electrophoresis

NSB nonspecific binding

NRS normal rabbit serum

ODS octadecylsilica

PAP peroxidase antiperoxidase

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

R19 antiserum made to purified porcine relaxin

RIA radioimmunoassay

RP peroxidase reaction product












RPM revolutions per minute

SC subcutaneous

SDS sodium dodecyl sulfate

SE surface epithelium of uterine lumen

T total count tube

TCA trichloroacetic acid

U unit(s) of relaxin activity














Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


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

By

Rube Jose Pardo

May 1982

Chairman: Lynn H. Larkin
Major Department: Medical Sciences (Anatomy)

It has been shown using the peroxidase-antiperoxidase immunocyto-

chemical technique that the endometrial glands of the pregnant guinea

pig are the source of the hormone relaxin. The presence of relaxin

has been demonstrated in uteri from day 30, day 45, day 60 pregnant and

late pregnant animals (days 65-67). The density of reaction product

deposition increased as pregnancy proceeded, with high deposition occur-

ring in days 45 and 60 of pregnancy and in late pregnant animals. Little

or no immunoperoxidase labeling was observed in tissues from day 15,

nonpregnant and lactating animals (3 days postpartum). Immunoperoxidase

labeling was not seen in nonendometrial gland components of the uterus.

High biological and immunological activities were found in extracts of

uteri taken on days 45 and 60 of pregnancy and in late pregnant animals.

When a crude extract of late pregnant uteri was chromatographed

in Sephadex G-50, a fraction containing relaxin activity eluted in the

6,000 molecular weight range. This fraction was active in the mouse

uterine motility bioassay (1.50 U/mg), and promoted lengthening of the


viii











interpubic ligament in estrogen primed female mice. The bioactive

Sephadex fraction was further purified in a carboxymethylcellulose

(CMC) ion exchange column. A single peak from the CMC column demon-

strated relaxin bioactivity (3.87 U/mg) in the mouse uterine motility

bioassay. The CMC purified guinea pig relaxin was compared to CMC

purified porcine relaxin in a two dimensional gel electrophoresis system.

The two purified relaxins were of similar molecular weights, with the

porcine hormone being slightly more basic. The guinea pig relaxin

molecule appears to be similar to porcine relaxin according to the

following criteria: (1) A continuous line of identity was obtained when

a 6,000 molecular weight fraction of relaxin from uteri of day 60 preg-

nant guinea pigs was tested with porcine relaxin and antirelaxin serum

in double immunodiffusion plate analyses. (2) Both relaxin molecules

were inactivated when reacted with antirelaxin serum in an antiserum

test employing the mouse uterine motility bioassay. (3) Both relaxin

molecules were inactivated by trypsin and dithiothrietol, but not by

moderate heat.














INTRODUCTION

Many mammals that give birth to large mature young have mechanisms

to compensate for a narrow pelvic width. One of the most dramatic

examples of this is found in the guinea pig, which gives birth to

relatively large young. In the guinea pig, pubic separation is so

extreme that the two halves of the pelvis are freely movable during the

birth process. It was Hisaw's interest in this phenomenon which prompted

him to ask whether certain humoral factors were responsible for the

morphologic changes associated with this process. Hisaw (1926, 1927)

was the first to relate this pelvic separation to the presence of a

blood factor later called relaxin (Fevold, Hisaw and Meyer, 1930). Since

its discovery, relaxin has been recognized as a hormone of pregnancy and

has been detected in many species of animals.

The physiological effects of relaxin are mainly concerned with the

female reproductive tract of mammalian species. Three of these effects

have been extensively reviewed in the literature: (1) relaxation of the

ligaments which stabilize the pelvic bones, (2) inhibition of uterine

contractions, and (3) softening of the cervix at term (Hisaw and Zarrow,

1950; Hall, 1960; Schwabe et al., 1978; Porter, 1979). The relaxation

of pelvic ligaments and inhibition of uterine contractions are the basis

of two important bioassays which are used to detect relaxin.

The following portions of the introduction will concentrate on

four areas of study on relaxin: (1) detection of relaxin, (2) cellular

localization of relaxin, (3) isolation and characterization of relaxin

and (4) description of relaxin research in the guinea pig.











Relaxin Assays

Relaxation of Pelvic Ligaments

The first qualitative bioassay for relaxin was the guinea pig

pubic symphysis palpation assay developed by Fevold et al. (1930).

An attempt to quantitate this assay was made by Abramowitz et al. (1944).

A guinea pig unit (U) was defined by these investigators as the dose of

relaxin that in 6 hours caused relaxation of the pubic symphysis (deter-

mined by palpation) in at least eight of twelve estrogen primed, cas-

trated female guinea pigs. Two basic problems were associated with this

assay: (1) the degree of subjectivity was high and (2) repeated use of

the same guinea pigs at first sensitized them to relaxin but then made

the animals refractory to the hormone after several months of use (Noall

and Frieden, 1956). All studies before 1960 exclusively employed the

guinea pig pubic symphysis assay and can, therefore, be questioned for

the reasons explained above.

The mouse interpubic ligament assay was later developed by Steinetz

et al. (1960), and offered a more sensitive and objective method of

assaying relaxin. In this assay, groups of sexually immature female

mice (18-20 g) were primed with a single injection of 5 pg estradiol

and 7 days later received injections of relaxin standards or unknowns

(three dose levels) in 1% benzopurpurine-4B. Eighteen to twenty-four

hours later, the mice were killed and their pubes dissected free of con-

nective tissue and fat. The interpubic distance was measured using a

dissecting microscope fitted with an ocular micrometer and a transillum-

inating source. With this assay, dose response curves could be compared

between two relaxin preparations to determine whether the relaxins












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).

Radioimmunoassay

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
porcine relaxin.








5


compared the RIA utilizing NIH-R-P1 relaxin with an RIA using a highly

purified relaxin fraction (CM-a', 3,000 U/mg). The NIH-R-P1 relaxin

was iodinated by the chloramine-T method of Hunter and Greenwood (1962).

The CM-a' relaxin was reacted with a succinimide ester and iodinated by

the method of Bolton and Hunter (1973). It was found that antisera to

CM-a' relaxin crossreacted with NIH-R-P1 relaxin. Also highly purified

CM-a' relaxin crossreacted with antisera made to NIH-R-P1 relaxin.

However, when the two assays were used to detect relaxin in serum of

pregnant ewes, the assay based on the crude relaxin preparation (NIH-

R-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-

pregnant women.

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

1:100,000.

The two most commonly used methods of labeling relaxin are the

method of Sherwood et al. (1975), which results in a polytyrosyl relaxin,

and the method of Bolton and Hunter, which employs succinimide relaxin.

Differences in the results of studies utilizing these RIA methods seem











to be related to the primary antisera employed in the assays rather than

the iodination procedure used for labeling the hormone.

While RIA and other immunologic techniques are being used increas-

ingly to detect relaxin, the bioassay still remains the most widely

used technique for relaxin detection. Although the RIA has the advantage

of increased sensitivity, the bioassay detects the biologically active

hormone.

Cellular Localization of Relaxin

One of the key areas of study concerning relaxin's role in preg-

nancy and parturition has been to determine the cellular location of the

hormone during these physiological states. Immunocytochemical tech-

niques have been the most commonly employed methods used to detect the

cellular location of relaxin in tissues. These techniques have been

used successfully to detect cells containing relaxin in the pig, cow,

rat and human.

Pig

In the pig, there is good evidence that the corpus luteum of

pregnancy is the principle source of relaxin. Belt et al. (1971) were

the first investigators to correlate levels of relaxin with cytoplasmic

granules in porcine luteal tissue. The accumulation of dense cytoplasmic

granules in granulosa lutein cells of late pregnant pigs, and the

decline in the number of granules after gestation, closely paralleled

the rise and fall of bioassayable corpus luteum relaxin in the same

periods. Kendall et al. (1978) utilized the immunoperoxidase technique

to localize relaxin at the ultrastructural level in cytoplasmic granules

of porcine granulosa lutein cells. Larkin et al. (1977) used











immunofluorescent localization methods employing antiporcine relaxin

serum (R8) to localize relaxin in granulosa lutein cells or pregnant

pigs. Furtner studies with the porcine ovary by Arakari et al. (1980)

have shown that the antirelaxin serum employed is of utmost importance

in the localization of relaxin when using immunolabeling techniques. An

antiserum produced against a crude relaxin preparation (NIH-R-P1, 440

U/mg) gave a diffuse pattern of immunofluorescence in the corpus luteum

of pregnancy, with the fluorescence localized mainly in the connective

tissue elements. On the other hand, an antiserum produced against

purified relaxin (CM-a', 3,000 U/mg) gave a sharp and precise localiza-

tion within the cytoplasm of the luteal cells. There have been no

reports of localization of relaxin in uterine or placental tissues in

the pig.

Cow

The ovary of the pregnant cow has been shown to be a source of

relaxin with bioassay techniques (Castro-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.

Rat

The ovary of the pregnant rat contains large quantities of extract-

able relaxin (Fields and Larkin, 1979; Sherwood and Crnekovic, 1979).

Relaxin has been detected in the rat ovary with bioassay, RIA and











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).

Rabbit

The ovary, uterus and placenta of the pregnant rabbit have been

reported to contain relaxin. Zarrow and O'Connor (1966) found relaxin in

the rabbit gestational corpus luteum by employing an indirect immuno-

fluorescent labeling technique; however, it was difficult to determine

from the published photographs whether the label was located intra- or

extracellularly. The antibody utilized in the above study was produced

in rabbits to porcine relaxin (WL 1164, lot 8; 622 U/mg powder). No

fluorescence was found in uterine or placental tissue. Zarrow and

Rosenberg (1953) reported bioactive relaxin in the ovary, uterus and

maternal placenta of pregnant rabbits with the highest level appearing

in the maternal placenta. This study also showed that ovariectomy of

pregnant rabbits with subsequent progesterone replacement therapy did

not result in decreased blood levels of relaxin. Fields et al. (1981)

isolated relaxin from extracts of rabbit placentae. However, a cellular

source of the hormone in the placentae was not reported, leaving open

the possibility that the relaxin was blood borne. It appears that the

rabbit is a species which has extra-ovarian sources of relaxin, most

likely the uterus and/or placenta.











Human

The ovary and placenta appear to be sources of relaxin in the

pregnant human. Dallenbach and Dallenbach-Hellweg (1964) discovered the

presence of relaxin in basal plate cells of human placentae using an

indirect immunofluorescence technique. The antiserum employed was made

in rabbits to a porcine relaxin preparation (1,000 U/mg). This finding

has been substantiated by several recent studies. Fields and Larkin

(1981) also detected relaxin in basal plate cells of human term placentae

using the immunoperoxidase technique. An antiserum (R19) raised against

purified porcine relaxin was utilized in these studies. Fields and

Larkin (1981) also showed that placentae which gave a positive stain

for relaxin also contained bioassayable relaxin. Yamamoto et al. (1981)

have shown that basal plates of cesarean and vaginally delivered placentae

contain bioactive and immunoreactive relaxin. The decidua of the preg-

nant human also has been shown to contain bioactive relaxin by Bigazzi

et al. (1980), but at this time a cellular source of relaxin has not

been found in this tissue.

The ovary has been established as a source of bioactive and immuno-

reactive relaxin in the pregnant human (O'Byrne et al., 1978; Szalchter

et al., 1980; Weiss et al., 1976; Weiss et al., 1977). Relaxin also

has been shown to be present in the human gestational corpus luteum

using immunoperoxidase localization (Mathieu et al., 1981). The local-

ization of relaxin appeared in the perinuclear area of the luteal cells.

It appears that animals which require the ovary for the maintenance

of pregnancy, i.e., the pig and the rat, also have the ovary as the

principal source of relaxin. On the other hand, animals which do not











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).

Pig

The ovary of the pig has been shown to be the most abundant source

of relaxin and the majority of the biochemical work has been accomplished

on relaxin extracted from this tissue (Schwabe et al., 1978).

Doczi (1963 U.S. patent 3,096,246) was the first investigator to

extract relaxin from porcine ovaries utilizing an acid-acetone extraction

solution. Griss et al. (1967) utilized a purification technique similar

to that used by Doczi to extract and partially purify relaxin from the

porcine ovary.

An initial extraction in a solution of hydrochloric acid, acetone

and water was conducted and then the relaxin containing extract was

fractionated with gel chromatography and anion exchange chromatography.

These separation techniques yielded a basic polypeptide with a molecular

weight (mw) of 5,000 to 10,000 that had both uterine relaxing activity

and the ability to cause lengthening of the interpubic ligament. Sherwood











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

extraction.

Further characterization of the porcine relaxin molecule resulted

in the discovery that porcine relaxin and insulin were closely related

molecules. Although the amino acid sequences of the two hormones were

not the same, there was a striking similarity in tertiary structure,

including the presence of disulfide bridges at corresponding positions

in the molecules (Isaacs et al., 1978; Blundell, 1979). Clues that the

three-dimensional configuration of porcine relaxin is important to its

biological activity came from the work of Schwabe and Braddon (1976)

who showed that partial oxidation of the tryptophan at the 18 position











of the beta chain led to biological inactivation of the molecule. Reduc-

tion of the disulfide bonds of the relaxin molecule with dithiothrietol

also eliminated its bioactivity (Schwabe et al., 1978).

Evidence for a prorelaxin compound has been accumulating from

several sources. James et al. (1977) suggest that relaxin might be

cleaved from a proinsulin like compound by proteolytic enzymes. Since

arginine is present at the N-terminus of the alpha chain as well as the

C terminus of the beta chain, they envision a prorelaxin precursor with

connections between the 30 position in the beta chain and the 1 position

in the alpha chain. The proteolytic cleavage would take place at this

position in the molecule. These investigators have identified forms of

relaxin in pig ovarian extracts which differ in net charge and amino

acid composition from the 6,000 mw relaxin molecule and feel that these

may perhaps be considered intermediates in the conversion of prohormone

to hormone.

Frieden and Yeh (1977) have acquired evidence for a prorelaxin

like compound in porcine ovarian extracts. When these investigators

chromatographed NIH relaxin (440 U/mg), they separated the material

into two protein peaks. Approximately 70% of the relaxin activity was

found in a peak eluting in the 6,000 mw range. However, approximately

10% of relaxin activity was concentrated in a 40,000 mw fraction. When

this higher mw fraction was exposed to trypsin, some of the high mw

material was converted to a 6,000 mw relaxin. This low mw relaxin was

indistinguishable from purified porcine relaxin in gel chromatography,

polyacrylamide gel electrophoresis (PAGE) and biological activity in the

guinea pig interpubic ligament bioassay. It appears from the above












studies that relaxin, like insulin, may be cleaved from a larger mw

precursor.

Cow

Bovine relaxin has been purified from ovaries of the late pregnant

cows (Fields et al., 1980). In this study, crude extracts were prepared

from corpora 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).

Rat

In 1979 Sherwood reported the purification and characterization of

rat relaxin. Ovaries were homogenized in a saline solution and two forms

of relaxin were obtained after fractionation of the crude ovarian extract

with Sephadex G-50 gel chromatography and CMC ion exchange chromatography.

The two forms were designated 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

Crnekovik, 1979).

Rabbit

The placenta of the rabbit had been reported to contain relaxin by

Zarrow and Rosenberg in 1953. Of the tissues tested (ovary, uterus,

placenta), the maternal portion of the placenta seemed to contain the

highest levels of biologically active relaxin. Lower levels were seen

in the fetal placenta and uterus. Further proof that rabbit placentae

contained relaxin was demonstrated by Larkin et al. (1979) who showed

that antiserum produced against porcine relaxin inhibited the activity

of rabbit placental extracts in the mouse uterine motility assay. Also,

a reaction of identity was obtained when a Bio-Gel P-10 fraction of

rabbit placental extracts was compared with purified porcine relaxin

and an antiserum made against porcine relaxin in an agar double immuno-

diffusion assay (Larkin et al., 1979).

This preliminary work led Fields et al. (1981) to purify relaxin

from the rabbit placenta. After extraction using a modified Griss

method (Griss et al., 1967), separation was achieved on a Bio-Gel P-30

column. The Bio-Gel P-30 fraction eluting at 6,000 daltons contained

low bioactivity in the mouse uterine motility bioassay (1.50 U/mg).

When this fraction was chromatographed in a CMC ion exchange column, a

single peak containing 15 U/mg was eluted. The mouse interpubic liga-

ment assay was conducted on the CMC fraction. The dose response curve












for rabbit placental relaxin was parallel to the dose response curve of

the porcine standard. With this assay, the CMC fraction was calculated

to have a biological activity of 21.6 U/mg. Electrofocusing of the CMC

peak resulted in the separation of four distinct fractions.

Human

Whereas it has been established that the ovary is a source of

relaxin in the pregnant human (Weiss et al., 1976; Weiss et al, 1977;

O'Byrne et al., 1978; Szalchter et al., 1980), characterization of

relaxin from the ovary has not been accomplished. On the other hand,

recent reports of isolation of a placental relaxin have been published

(Fields and Larkin, 1981; Yamamoto et al., 1981). These recent reports

support the earlier work of Dallenbach and Dallenbach-Hellweg (1964) who

found immunoreactive relaxin in the basal plate of the human placenta

using indirect immunofluorescence. Bigazzi et al. (1980) have demon-

strated relaxin production by the decidua capsularis in vitro and have

extracted relaxin from decidual tissue collected from term pregnancies.

Fields and Larkin (1981) first isolated and purified human placental

relaxin using the extraction technique of Griss et al. (1967). They

found human relaxin to be a peptide similar to porcine relaxin in molecu-

lar weight. The biological activity of an isolated Bio-Gel P-30 fraction

(6,000 mw) as determined by the mouse uterine motility assay, was 11.9

U/mg. The same fraction produced a linear response in the mouse inter-

pubic ligament bioassay, which was parallel to the porcine standard.

Electrofocusing of the active fraction produced one peak having an

isoelectric point of pH 11.4 and a biological activity of 45 U/mg. The

electrofocused fraction exhibited a continuous line of identity with no











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

decreased them.

In summary, work previous to 1950 indicated that relaxin was

present in the blood, uterus and placenta of pregnant guinea pigs. It

also showed that progesterone somehow stimulated production of relaxin











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

relaxin molecule?















MATERIALS AND METHODS

General Procedures

Experimental Design, Treatment of Animals and
Collection of Specimens

Guinea pigs obtained from a local vendor were housed in the

University of Florida Health Center Animal Resources Department, and had

access to food and water ad 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

manual palpation.











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.

Antirelaxin Sera

Antisera against highly purified porcine relaxin was produced in

New Zealand white rabbits as described by Larkin et al. (1977). In this

technique, 2 mg of a pig relaxin preparation (WL 150, 150 U/mg) obtained

from Warner Lambert, Inc., Morris Plains, NJ, were run on PAGE. The

bands were localized by fixing them in trichloroacetic acid (TCA) (15%),

and staining in 0.6% Coomassie blue in 15% TCA. Three bands were present

and were named Cl, C2, and C3; 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

two months.

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

Larkin, 1981).

Tissue Extraction

Preparation of crude uterine extracts was accomplished by utilizing

one of two methods. Initially, tissues were extracted with the acid-

acetone procedure of Griss et al. (1967). This procedure was employed

for the extraction of uteri taken from individual animals and the extract

was used for bioassay and RIA experiments. Recently, a new extraction

procedure for relaxin was reported by Walsh and Niall (1980). This

newer technique was employed to extract relaxin from uteri and the

resulting preparations were used in purification and characterization

studies. A more detailed account of these techniques is given below.

Griss procedure.--The extraction procedure of Griss et al. (1967),

was used for extraction of uteri utilized in bioassay and RIA experi-

ments. Cold extraction solution (acetone:water:hydrochloric acid, 5.0:

2.83:0.17 ratio) was added to minced frozen tissues at a ratio of 5:1

(ml/g), and homogenized in a Sorvall Omni-mixer at 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
VU CU
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.

Radioimmunoassay

Three different iodination methods were attempted in the devel-

opment of the homologous porcine RIA for guinea pig relaxin. Since

sufficient amounts of purified guinea pig relaxin were not available

for the RIA experiments, it became necessary to employ porcine relaxin

for both the immunization procedure and for iodination. The first two

procedures involved the use of the Bolton and Hunter reagent for the

iodination of porcine relaxin (NIH-RXN-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
Bo-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

different times.

Purification and Characterization of Guinea
Pig Relaxin

Purification

Gel filtration.--The Walsh and Niall (1980) procedure was utilized

to extract 118.50 g of uterine tissue from 5 late pregnant guinea pigs

and the crude extract from the ODS extraction (188.8 mg protein) was

suspended in 10 ml ammonium acetate buffer, pH 5.0. Protein was

determined by the 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

resin.

Ion exchange chromatography.--The active fraction from the Sephadex

column (35.5 mg) was applied to a 0.8 x 5 cm CMC column (CM-52) purchased

from Whatman Ltd., Springfield, England, and then equilibrated with

0.01 M ammonium acetate buffer, pH 5.0 until all unadsorbed material was

removed. The column was developed at a rate of 9 ml/hr with a linear

NaCl gradient (0.1 M to 0.3 M) in 0.01 M ammonium acetate buffer pH 5.0

to a final conductivity of 20 m Mho. Fractions (1.5 ml) were collected

every 10 min.

Characterization

Double immunodiffusion studies.--Double immunodiffusion plates

were employed as described by Clausen (1969) for the microtechnique.

Petri dishes of 12 x 60 mm (1.5% agar in 0.85% saline with 0.1% sodium

azide) were used. A center well was filled with 5 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

24 hr.











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

bioassay.

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

motility bioassay.

In vitro assay of antisera (Larkin et al., 1979).--Fifty micro-

liters of R19 antiserum were added to one of the tubes in the mouse

uterine motility bioassay, while the other tube received 50 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-

pared.














RESULTS

Detection of Guinea Pig Relaxin

Immunocytochemical Localization of Uterine Relaxin

The PAP immunocytochemical technique was used to examine the

ovary, placenta, uterus, spleen and liver of guinea pigs. The endo-

metrial gland cells (EGC) of the uterus was the only cell type that

showed heavy deposition of peroxidase reaction product (RP), indicating

the presence of relaxin. The ovary demonstrated weak staining, while

the liver, spleen and placentae did not stain. Therefore, subsequent

studies employed the uterus. Tissue samples were taken from guinea

pigs at different stages of pregnancy, during lactation and in ovari-

ectomized animals undergoing estrogen-progesterone treatments to deter-

mine periods of relaxin production.

Relaxin was not detected in sections of uteri taken from nonpreg-

nant ovariectomized control (no hormone treatment or estrogen treated

animals) or day 15 pregnant animals. Day 30 was the earliest stage of

pregnancy studied that showed accumulation of relaxin in the EGC. At

this stage, only a few glands contained relaxin (Figure 1). Control

(NRS treated) sections did not exhibit RP (Figure 2).

Hematoxylin and eosin (H & E) stained sections viewed at higher

magnification revealed that the EGC were low columnar cells with basally

located nuclei, and could be easily distinguished from uterine surface

epithelium (SE) (Figure 3). A section adjacent to that shown in

Figure 3 stained with R19 antiserum and the PAP technique demonstrated


40











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.





A











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
Uterine Relaxin

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).

Characterization

Double immunodiffusion studies.--Analyses of a Bio-Gel P-30

relaxin fraction (6 mg/ml) from the guinea pig uterus (day 60 of

pregnancy) tested against R19 antiserum to porcine relaxin and NIH-

RXN-P1 porcine relaxin showed a single precipitin line with no spurring

(Figure 34).












Two dimensional gel electrophoresis.--Carboxymethyl cellulose

purified guinea pig relaxin tested in a two dimensional gel electro-

phoresis system showed that guinea pig relaxin migrated in the same mw

range as did NIH-RXN-P1 relaxin, which is known to have a mw of 6,000

(Figure 35). An equilibrium isoelectric point of a molecule cannot be

resolved with NEPHGE. Nevertheless, it was apparent that the guinea

pig relaxin molecule did not migrate as far in the first dimension

(pH 6.9) as did NIH-RXN-P1 porcine relaxin (pH 7.2), indicating that the

guinea pig relaxin molecule had a lower pH than the porcine molecule

(Figure 35). This observation supported data from the CMC column run,

which showed guinea pig relaxin eluting earlier (7 m Mho range) than

NIH-RXN-P1 porcine relaxin (10 m Mho range (Figure 33)). Figure 35 is

a representation of a slab gel, made up of a composite of two separately

run gels.

Mouse interpubic ligament assay.--A Sephadex G-50 6,000 mw fraction

from guinea pig uterine extracts was tested in the mouse interpubic

ligament assay (Figure 36). A positive response was obtained as noted

by linear response to log-dose of the Sephadex fraction. The data

indicate a valid assay according to the following criteria: (1) parallel-

ism existed between the guinea pig relaxin fraction and the NIH-RXN-P1

porcine relaxin standard, and (2) a lambda value (standard error/slope)

of less than 0.4 was obtained. The specific activity of the Sephadex

fraction was calculated to be 0.8 U/mg protein. The mouse uterine motil-

ity assay of the same fraction gave a biological activity of 1.5 U/mg.

The best fit curve for the NIH-RXN-P1 porcine relaxin was y = 1.2 (log x)

+ 0.83, with a standard error (SE) = 0.086 and a lambda value of 0.08.











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.














DISCUSSION

R19 Antiserum: Detection of Guinea Pig Relaxin

A major portion of this dissertation utilized techniques which

employed antirelaxin serum (R19). R19 was produced in rabbits against

highly purified porcine relaxin (Larkin et al., 1977). This antiserum

has been shown to crossreact with relaxin from different species; i.e.,

cow (Fields et al., 1980), human (Fields and Larkin, 1981), and rabbit

(Fields et al., 1981). R19 also has the ability to detect guinea pig

relaxin, as demonstrated by the following observations from the current

study: (1) Double immunodiffusion agar plate assays showed a reaction

of identity when partially purified uterine relaxin from day 60 preg-

nant guinea pigs was tested against highly purified pig relaxin and

R19. (2) R19 was shown to be effective in inhibiting the action

of guinea pig relaxin using the in vitro mouse uterine motility anti-

serum assay. (3) The immunoperoxidase labeling data from different

stages of gestation correlated well with bioassay and radioimmunoassay

data of crude uterine extracts from these stages; that is, the stages

demonstrating the greatest degree of labeling were also the stages

with the highest relaxin levels (days 45, 60 of pregnancy and late

pregnant animals). (4) All the controls in the immunolocalization

experiments were negative, including the observation that immunoperoxi-

dase labeling was eliminated when the R19 antiserum was absorbed with

purified porcine relaxin standard (NIH-RXN-P1) prior to tissue incubation.











Detection of Guinea Pig Relaxin with the
PAP Technique

The R19 antiserum discussed in the previous section was employed

in immunocytochemical studies on sections of uteri from pregnant and

lactating guinea pigs, as well as in uteri of guinea pigs induced to

undergo pelvic relaxation with estrogen and progesterone treatments.

The immunocytochemical studies show an increase during pregnancy in

RP localized in the EG. This suggests that there is an increase in the

accumulation of relaxin in these cells, supporting the contention that

the EG are the sites for relaxin production. Of the intervals evalu-

ated, day 30 was the first stage of pregnancy where a small amount of

immunoperoxidase labeling (relaxin) was noted in some EG. At this

stage, very few glands were labeled and in those glands that did label,

the reaction production was detected in only a few of the gland cells.

In uterine tissue from day 48 animals almost all of the EG appeared to

contain relaxin, but even at this stage, not all of the EGC of a given

gland were labeled. Thus it appears that there exists a gradual build up

of relaxin by the EGC. This is most clearly seen in day 60 and late

pregnant stages where dense deposits of RP were localized in the apical

cytoplasm of the EGC. In uteri from lactating animals (3 days post-

partum), relaxin was absent from most of the EG, and a high incidence

of mitotic figures were noticed in the glandular epithelium.

Results from experiments involving nonpregnant, ovariectomized-

hormone treated animals provide strong evidence for a non-ovarian source

of relaxin. When ovariectomized animals were treated with estrogen,

no RP was seen localized over the EGC. When ovariectomized animals

were treated with estrogen and progesterone, RP was seen localized over











the EGC. These results agree with and extend the results of Zarrow

(1948), who noted that hysterectomized and ovariectomized guinea pigs

did not undergo pelvic relaxation or contain serum relaxin when treated

with a similar course of steroid injections. It is not known how

estrogen and progesterone induce the accumulation or synthesis of

relaxin in the EGC.

Detection of Guinea Pig Relaxin with
Radioimmunoassay

There have been no publications to date that report on the produc-

tion of antisera to purified guinea pig relaxin. All studies dealing

with radioimmunological detection of relaxin in the guinea pig have

employed antiporcine relaxin sera (Sherwood et al., 1975; O'Byrne and

Steinetz, 1976; Bryant-Greenwood and Greenwood, 1979; Boyd et al., 1981;

Nagao and Bryant-Greenwood, 1981). It seems logical that differences

in the specificity and sensitivity of the various antisera could be

responsible for the wide variations in relaxin levels reported between

different laboratories which have studied relaxin in the guinea pig.

For example, the present RIA (employing R19 antiserum) detected porcine

relaxin as well as relaxin from guinea pig uterine extracts, but not

guinea pig serum relaxin. This antiserum was produced in rabbits to

highly purified porcine realxin. Sherwood et al. (1975) also reported

that an antiserum produced in rabbits to highly purified porcine relaxin

detected only porcine relaxin. These investigators were not able to

detect relaxin in sera of pregnant guinea pigs. This is in agreement

with the present study. However, Sherwood et al. (1975) did not report

attempts to detect relaxin in extracts of relaxin containing tissues.











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
3 Units











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

pig.

Endometrial Glands and Their Role in Relaxin Production

EG classically have been assumed to play a role during pregnancy

where they serve a nutritive and/or supportive role to the preimplanta-

tion embryo (Finn, 1977). In the mouse, the end result of glandular

differentiation is the secretion of periodic acid Schiff positive

material from the EG into the uterine lumen, and it has been shown that

estrogen and progesterone together can induce uterine glandular secre-

tions (Finn, 1971; Finn and Martin, 1971). In the pig, Bazer (1975)

has shown that a uterine specific purple protein is secreted by the

glandular endometrial epithelium throughout pregnancy. Most animals

studied have been shown to produce uterine specific proteins, especially

during the early stages of pregnancy (Aitken, 1979). Animals with an











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

women.

One of the most interesting problems remaining to be investigated

is how relaxin enters the systemic circulation from the EG and what

course it follows to reach the target tissues. Studies to identify

target organs for relaxin become very important when one considers the

possibilities of local effects of relaxin.

Relaxin may be released into the uterine lumen, and make its way

back through the endometrial stroma into the bloodstream. While the

findings in the present study did not indicate direct secretion of

relaxin into the uterine stromal compartment, this cannot be ruled out.

Work in other systems by Bazer and Thatcher (1977) suggested an

endocrine-exocrine mechanism for the release of prostaglandin F 2-alpha











(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

for parturition?

It is hoped that the research presented in this dissertation will

amplify the knowledge of relaxin physiology in the guinea pig, and

advance an understanding of the parturition process in this animal as

well as in other species.















BIBLIOGRAPHY


Abramowitz, A. A., W. L. Money, M. X. Zarrow, R. V. N. Talmage, L. H.
Kleinholz, and F. L. Hisaw. (1944) Preparation, biological assay
and properties of relaxin. Endocrinology, 34: 103-114.

Abramson, D., E. Hurwitt, and G. Lesnik. (1937) Relaxin in human
serum as a test of pregnancy. Surg. Gynecol. Obstet., 65: 335-339.

Aitken, R. J. (1979) Uterine proteins. In: Oxford Reviews of
Reproductive Biology. (A. A. Finn, ed.) Clarendon Press, Oxford,
England.

Anderson, M. L., and J. A. Long. (1978) Localization of relaxin in the
pregnant rat. Bioassay of tissue extracts and cell fractionation
studies. Biol. Reprod., 18: 110-117.

Anderson, M. L., J. A. Long, and T. Hayashida. (1975) Immunofluorescence
studies on the localization of relaxin in the corpus luteum of
the pregnant rat. Biol. Reprod., 13: 499-504.

Arakari, R. F., R. G. Kleinfeld, and G. D. Bryant-Greenwood. (1980)
Immunofluorescence studies using antisera to crude and to purified
porcine relaxin. Biol. Reprod., 23: 153-159.

Barr, A. J., and J. H. A. Goodnight. (1976) Users Guide to the Statisti-
cal Analysis System. North Carolina State University, Raleigh,
N. C.

Bazer, F. W. (1975) Uterine protein secretions: Relationship to
development of the concepts. J. Anim. Sci., 41: 1376-1382.

Bazer, F. W., and W. W. Thatcher. (1977) Theory of maternal recognition
of pregnancy in swine based on estrogen controlled endocrine
versus exocrine secretion of Prostaglandin F2-alpha by the uterine
endometrium. Prostaglandins, 14: 397-401.

Belt, W. D., L. L. Anderson, L. F. Cavazos, and R. M. Melampy. (1971)
Cytoplasmic granules and relaxin levels in porcine corpora lutea.
Endocrinology, 89: 1-10.

Bigazzi, M., E. Nardi, P. Bruni, and F. Petrucci. (1980) Relaxin in
human decidua. J. Clin. Endocrinol. Metab., 51: 939-941.

Blundell, T. (1979) Conformation and molecular biology of polypeptide
hormones. I. Insulin, insulin-like growth factor and relaxin.
TIBS. March, 1979: 51-54.











Bolton, A. E., and W. M. Hunter. (1973) The labelling of proteins to
high specific radioactivities by conjugation to a 125I-containing
acylating agent. Biochem. J., 133: 529-539.

Boyd, S., J. Z. Kendall, N. Mento, and G. D. Bryant-Greenwood. (1981)
Relaxin immunoactivity in plasma during the reproductive cycle of
the female guinea pig. Biol. Reprod., 24: 405-414.

Brouha, L. (1933) Recherches sur la mobilisation de la symphyse
pubienne chez le cobaye impubere. Compte Rendu Soc. Biol., 113:
406-408.

Bryant, G. D. (1972) The detection of relaxin in porcine, ovine and
human plasma by radioimmunoassay. Endocrinology, 91: 1113-1117.

Bryant, G. D., and W. A. Chamley. (1976) Changes in relaxin and pro-
lactin immunoactivities in ovine plasma following suckling. J.
Reprod. Fertil., 46: 457-459.

Bryant, G. D., M. E. A. Panter, and T. Stelmasiak. (1975) Immunore-
active relaxin in human serum during the menstrual cycle. J.
Clin. Endocrinol. Metab., 41: 1065-1069.

Bryant, G. D., J. F. Sassin, E. D. Weitzman, S. Kapen, and A. Frantz.
(1976) Relaxin immunoactivity in human plasma during a 24-hour
period. J. Reprod. Fertil., 48: 389-392.

Bryant, G. D., and T. Stelmasiak. (1974) The specificity of radio-
immunoassay for relaxin. Endo. Res. Commun., 1: 415-433.

Bryant-Greenwood, G. D., and F. C. Greenwood. (1979) Specificity of
radioimmunoassays for relaxin. J. Endocrinol., 81: 239-247.

Castro-Hernandez, A. (1976) Isolation and purification of bovine
luteal polypeptides with relaxin hormone activity (Doctoral
Dissertation, University of Florida).

Catchpole, H. R. (1969) Hormonal mechanisms during pregnancy and
parturition. In: Reproduction in Domestic Animals. (H. H. Cole
and P. T. Cupps, eds.) Academic Press, New York.

Challis, J. R. G., R. B. Heap, and D. V. Illingsworth. (1971) Concentra-
tions of oestrogen and progesterone in the plasma of non-pregnant,
pregnant and lactating guinea pigs. J. Endocrinol., 51: 333-345.

Clausen, J. (1969) Immunochemical Techniques for the Identification and
Estimation of Macromolecules. (T. S. Work, E. Work, eds.)
American Elsevier, New York, p. 521.












Dallenbach, F. D., and G. Dallenbach-Hellweg. (1964) Immunohistologische
untersuchungen zur lokalisierung des relaxins in menschlicher
plazenta and decidua. Virch. Arch. Path. Anat., 337: 301-316.

Dallenbach-Hellweg, G., J. V. Battista, and F. D. Dallenbach. (1965)
Immunohistological and histochemical localization of relaxin in
the metrial gland of the pregnant rat. Am. J. Anat., 117:
435-450.

Fevold, H., F. L. Hisaw, and R. K. Meyer. (1930) The relaxative hor-
mone of the corpus luteum, its purification and concentration.
J. Am. Chem. Soc., 52: 3340-3348.

Fields, M. J., P. A. Fields, A. Castro-Hernandez, and L. H. Larkin.
(1980) Evidence for relaxin in corpora lutea of late pregnant
cows. Endocrinology, 107: 869-875.

Fields, P. A., and L. H. Larkin. (1979) Isolation of rat relaxin.
Anat. Rec., 193: 537.

Fields, P. A., and L. H. Larkin. (1981) Purification and immunohisto-
chemical localization of relaxin in the human term placenta. J.
Clin. Endocrinol. Metab., 52: 79-85.

Fields, P. A., L. H. Larkin, and R. J. Pardo. (1981) Purification of
relaxin from the placenta of the rabbit. Ann. N. Y. Acad. Sci.
380: 76-86.

Finn, C. A. (1977) The implantation reaction. In: Biology of the
Uterus (R. M. Wynn, ed.) Plenum Press, New York.

Finn, C. A., and L. Martin. (1971) Endocrine control of the prolifer-
ation and secretion of uterine glands in the mouse. Acta Endocrinol.
Suppl., 155: 139.

Frieden, E. H., and W. C. Adams. (1977) The response to endogenous
relaxin of guinea pigs refractory to porcine relaxin. Proc. Soc.
Exp. Biol. Med., 155: 558-561.

Frieden, E. H., and F. L. Hisaw. (1953) The biochemistry of relaxin.
Rec. Prog. Horm. Res., 8: 333-372.

Frieden, E. H., and L. Yeh. (1977) Evidence for a "pro-relaxin" in
porcine relaxin concentrates. Proc. Soc. Exp. Biol. Med., 154:
407-411.

Fugo, N. W. (1943) Relaxation of the pelvic ligaments of castrate
hysterectomized guinea pigs induced by progesterone. Proc. Soc.
Exp. Biol. Med., 54: 200-201.











Griss, G., J. Keck, R. Engelhorn, and H. Tuppy. (1967) The isolation
and purification of an ovarian polypeptide of uterine-relaxing
activity. Biochim. Biophys. Acta, 140: 45-54.

Hall, K. (1960) Relaxin. J. Reprod. Fertil., 1: 368-384.

Harkness, M. L. R., and R. D. Harkness. (1956) Changes in the foetal
membrane during pregnancy in the rat. J. Physiol., 129: 788.

Harkness, M. L. R., and R. D. Harkness. (1957) Changes in the physical
properties of the uterine cervix of the rat during pregnancy.
J. Physiol., 148: 524-547.

Hisaw, F. L. (1926) Experimental relaxation of the pubic ligament of
the guinea pig. Proc. Soc. Exp. Biol. Med., 23: 661-663.

Hisaw, F. L. (1927) Experimental relaxation of the symphysis pubis of
the guinea pig. Anat. Rec., 37: 126.

Hisaw, F. L., and M. X. Zarrow. (1950) The physiology of relaxin.
Vit. and Horm., 8: 151-178.

Hisaw, F. L., M. X. Zarrow, W. L. Money, R. V. N. Talmage, and A. A.
Abramowitz. (1944) Importance of the female reproductive tract
in the formation of relaxin. Endocrinology, 34: 122-134.

Horst, M. N., S. M. M. Basha, G. A. Baumbach, E. H. Mansfield, and R. M.
Roberts. (1980) Alkaline urea solubilization, two-dimensional
electrophoresis and lectin staining of mammalian cell plasma
membrane and plant seed proteins. Anal. Biochem., 102: 399-408.

Hunter, W. M., and F. C. Greenwood. (1962) Preparation of iodine-131
labelled human growth hormone of high specific activity. Nature,
194: 495-496.

Isaacs, N., R. James, H. Niall, G. Bryant-Greenwood, G. Dodson, A. Evans,
and A. C. T. North. (1978) Relaxin and its structural relation-
ship to insulin. Nature, 271: 278-281.

James, R., H. Niall, S. Kwok, and G. Bryant-Greenwood. (1977) Primary
structure of porcine relaxin: Homology with insulin and related
growth factors. Nature, 267: 544-546.

John, M. J., B. W. Borjesson, J. R. Walsh, and H. Niall. (1981)
Limited sequence homology between porcine and rat relaxins: Impli-
cations for physiological studies. Endocrinology, 108: 726-729.

Kendall, J. Z., C. G. Plopper, and G. D. Bryant-Greenwood. (1978)
Ultrastructural immunoperoxidase demonstration of relaxin in
corpora lutea from a pregnant sow. Biol. Reprod., 18: 94-98.











Krantz, J. C., H. H. Bryant, and C. J. Carr. (1950) The action of
aqueous corpus luteum extract upon uterine activity. Surg.
Gynecol. Obstet., 90: 372-375.

Kroc, R. L., B. G. Steinetz, and V. L. Beach. (1959) The effects of
estrogens, progestagens, and relaxin in pregnant and non-pregnant
laboratory rodents. Ann. N. Y. Acad. Sci., 75: 942-980.

Larkin, L. H. (1974) Bioassay of rat metrial gland extracts for
relaxin using the mouse interpubic ligament technique. Endocrin-
ology, 94: 567-570.

Larkin, L. H., P. A. Fields, and R. M. Oliver. (1977) Production of
antisera against electrophoretically separated relaxin and immuno-
fluorescent localization of relaxin in the porcine corpus luteum.
Endocrinology, 101: 679-683.

Larkin, L. H., P. A. Fields, and R. J. Pardo. (1981) Mouse uterus bio-
assay for relaxin. In: Relaxin. (G. D. Bryant-Greenwood, H. D.
Niall and F. C. Greenwood, eds.) Elsevier, North Holland.

Larkin, L. H., C. A. Suarez-Quian, and P. A. Fields. (1979) In vitro
analyses of antisera to relaxin. Acta Endocrinol., 92: 568-576.

Loumaye, E., B. Teuwissen, and K. Thomas. (1978) Characterization of
relaxin radioimmunoassay using Bolton-Hunter reagent. Gynecol.
Obstet. Invest., 9: 262-267.

Lowry, 0. H., N. J. Roseborough, A. L. Farr, and R. J. Randall. (1951)
Protein measurement with the folin phenol reagent. J. Biol. Chem.,
193: 265-275.

MacLennan, A. H., R. C. Green, G. D. Bryant-Greenwood, F. C. Greenwood,
and R. F. Seamark. (1980) Ripening of the human cervix and
induction of labor with purified porcine relaxin. Lancet, Feb. 2,
1980, pp. 220-223.

Marcus, G. J. (1974) Mitosis in the rat uterus during the estrus cycle,
early pregnancy, and early pseudopregnancy. Biol. Reprod. 10:
447-452.

Markwell, M. A. K., and C. F. Fox. (1978) Surface-specific iodination
of membrane proteins of viruses and eukaryotic cells using 1, 3,
4, 6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. Biochem.,
17: 4807-4817.

Mathieu, P. H., J. Rathier, and K. Thomas. (1981) Localization of
relaxin in human gestational corpus luteum. Cell Tiss. Res., 219:
213-216.












Nagao, R., and G. D. Bryant-Greenwood. (1981) Evidence for a uterine
relaxin in the guinea pig. In: Relaxin. (G. D. Bryant-Greenwood,
H. D. Niall and F. C. Greenwood, eds.) Elsevier, North
Holland.

Noall, M. W., and E. H. Frieden. (1956) Variations of sensitivity of
ovariectomized guinea pigs to relaxin. Endocrinology, 5: 659-664.

O'Byrne, E. M., F. F. Flitcraft, W. K. Sawyer, J. Hochman, G. Weiss, and
B. G. Steinetz. (1978) Relaxin bioactivity and immunoactivity
in human corpora lutea. Endocrinology, 102: 1641-1644.

O'Byrne, E. M., W. K. Sawyer, M. C. Butler, and B. G. Steinetz. (1976)
Serum immunoreactive relaxin and softening of the uterine cervix
in pregnant hamsters. Endocrinology, 99: 1333-1335.

O'Byrne, E. M., and B. G. Steinetz. (1976) Radioimmunoassay (RIA) of
relaxin in sera of various species using an antiserum to porcine
relaxin. Proc. Soc. Exp. Biol. Med., 152: 272-276.

Pardo, R., L. H. Larkin, and P. A. Fields. (1980) Immunocytochemical
localization of relaxin in endometrial glands of the pregnant guinea
pig. Endocrinology, 107: 2110-2112.

Porter, D. G. (1972) Myometrium of the pregnant guinea pig: The
probable importance of relaxin. Biol. Reprod., 7: 458-464.

Porter, D. G. (1979) Relaxin: Old Hormone, new prospect. In: Oxford
Reviews of Reproductive Biology, Vol. 1 (C. A. Finn, ed.) Clarendon
Press, Oxford, England.

Reinig, J. W., D. N. Lambert, C. Schwabe, L. K. Gowan, B. G. Steinetz,
and E. M. O'Byrne. (1981) Isolation and characterization of
relaxin from the sand tiger shark (odontaspis taurus). Endocrin-
ology, 109: 537-543.

Sanders, M. M., V. E. Groppi, Jr., and E. T. Browning. (1980) Resolu-
tion of basic cellular proteins including histone variants by
two-dimensional gel electrophoresis: Evaluation of lysine to
arginine ratios and phosporylation. Anal. Biochem., 103: 157-
165.

Sar, M., and W. E. Stumpf. (1974) Cellular and subcellular localization
of 3H-progesterone or its metabolites in the oviduct, uterus,
vagina and liver of the guinea pig. Endocrinology, 94: 1116-1125.

Schwabe, C., and S. A. Braddon. (1976) Evidence for one essential
tryptophan residue at the active site of relaxin. Biochem.
Biophys. Res. Commun., 68: 1126-1132.












Schwabe, C., J. K. McDonald, and B. G. Steinetz. (1976) Primary
structure of the A-chain of porcine relaxin. Biochem. Biophys.
Res. Commun., 70: 397-405.

Schwabe, C., J. K. McDonald, and B. G. Steinetz. (1977) Primary
structure of the B-chain of porcine relaxin. Biochem. Biophys.
Res. Commun., 75: 503-510.

Schwabe, C., B. G. Steinetz, G. Weiss, A. Segaloff, J. K. McDonald,
E. O'Byrne, J. Hochman, B. Carriere, and L. Goldsmith. (1978)
Relaxin. Rec. Prog. Horm. Res., 34: 123-211.

Sherwood, 0. D. (1979) Purification and characterization of rat
relaxin. Endocrinology, 104: 886-892.

Sherwood, 0. D., and V. E. Crnekovic. (1979) Development of a homo-
logous radioimmunoassay for rat relaxin. Endocrinology, 104:
893-897.

Sherwood, O. D., P. A. Martin, C. C. Chang, and P. J. Dziuk. (1977a)
Plasma relaxin levels in pigs with corpora lutea induced during
late pregnancy. Biol. Reprod., 17: 97-100.

Sherwood, O. D., P. A. Martin, C. C. Chang, and P. J. Dziuk. (1977b)
Plasma relaxin levels during late pregnancy and at parturition in
pigs with altered utero-ovarian connections. Biol. Reprod.,
17: 101-103.

Sherwood, 0. D., and E. M. O'Byrne. (1974) Purification and character-
ization of porcine relaxin. Arch. Biochm. Biophys., 160: 185-196.

Sherwood, 0. D., K. R. Rosentreter, and M. L. Birkhimer. (175) Develop-
ment of a radioimmunoassay for porcine relaxin using I labelled
polytyrosyl-relaxin. Endocrinology, 96: 1106-1112.

Steinetz, B. G., V. L. Beach, R. L. Kroc, N. R. Stasilli, R. E. Nussbaum,
P. J. Nemith, and R. K. Dun. (1960) Bioassay of relaxin using
a reference standard: A simple and reliable method utilizing
direct measurement of interpubic ligament formation in mice.
Endocrinology, 67: 102-115.

Sternberger, L. A. (1979) Immunocytochemistry. John Wiley and Sons,
New York.

Stumpf, W. E. (1968) Subcellular distribution of 3H-estradiol in rat
uterus by quantitative autoradiography--a comparison between 3H-
estradiol and 3H-norethynodrel. Endocrinology, 83: 777-782.












Sutcliffe, R. G., D. J. H. Brock, L. B. V. Nicholson, and E. Dunn.
(1978) Fetal- and uterine-specific antigens in human amniotic
fluid. J. Reprod. Fertil., 54: 85-90.

Szalchter, N., E. O'Byrne, L. Goldsmith, B. G. Steinetz, and G. Weiss.
(1980) Myometrial inhibiting activity of relaxin-containing
extracts of human corpora lutea. Am. J. Obstet. Cynecol., 136:
584-586.

Walsh, J. R., and H. D. Niall. (1980) Use of an octadecylsilica
purification method minimizes proteolysis during isolation of
porcine and rat relaxins. Endocrinology, 107: 1258-1260.

Warembourg, M. (1974) Radiographic study of the guinea pig uterus
after injection and incubation with H-progesterone. Endocrinology,
94: 665-670.

Weiss, G., E. M. O'Byrne, J. A. Hochman, L. T. Goldsmith, I. Rifkin, and
B. G. Steinetz. (1977) Secretion of progesterone and relaxin by
the human corpus luteum at midpregnancy and at term. Obstet.
Gynecol., 50: 679-681.

Weiss, G., E. M. O'Byrne, J. A. Hochman, B. G. Steinetz, L. Goldsmith,
and J. G. Flitcraft. (1978) Distribution of relaxin in women
during pregnancy. Obstet. Gynecol., 52: 568-570.

Weiss, G., E. M. O'Byrne, and B. G. Steinetz. (1976) Relaxin: A
product of the corpus luteum of pregnancy. Science, 194: 948-949.

Yamamoto, S., S. C. M. Kwok, F. C. Greenwood, and G. D. Bryant-Greenwood.
(1981) Relaxin purification from human placental basal plates.
J. Clin. Endocrinol. Metab., 52: 601-607.

Zarrow, M. X. (1947) Relaxin content of blood, urine and other tissues
of pregnant and postpartum guinea pigs. Proc. Soc. Exp. Biol.
Med., 66: 488-491.

Zarrow, M. X. (1948) The role of the steroid hormones in the relaxation
of the symphysis pubis of the guinea pig. Endocrinology, 42: 129-
140.

Zarrow, M. X., E. G. Holmstrom, and H. A. Salhanick. (1955) The con-
centration of relaxin in the blood serum and other tissues of
women during pregnancy. J. Clin. Endocrinol. Metab., 15: 22-27.

Zarrow, M. X., and J. A. McClintock. (1966) Localization of 131I
labelled antibody to relaxin. J. Endocrinol., 36: 377-387.









71


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


Zarrow, M. X., and B. Rosenberg.
rabbit. Endocrinology, 53:


(1953) Sources of relaxin in the
593-598.















APPENDIX 1
TABLES









































+1


'A
-4

0n

11%4O
49o







000







.4





+1
49







ci000










0000




r49%00
N 049q



-4~O






O'
40N .




.4 .


-4
A
















V4













N
+1




co










-4
r4










N N N






Go 00

cc it 00






0 N 0 A

-4





S0 0 -4
1-^ 1
OMlO


+
aM




N



04
N




















+1







CM" N


m










O
M I


.41





















N00.
9---0
<---4-t.t "













0(M N
p^S^' M


+1
0






























+1
49
9MM N0 N


















r-






9 a




















--4-- N





















*U,
* -
44I4-
.Sto:vu


+1






49




















Nl






0



.4
49^ '
A



















o
00-4.














S0 00M








n ON













IC
i35


+4
U
we
















0




-o



2
wse
u









S0
,"
















Cv











MU
01
* 0




















'C
2 ,



eC'











4-
4,






0

z 74
u
u



Cc






0li -4 *
0 1 0 1 1 0 I 0



to I ae I 1
r I i





0 0'l 04 I I C I c +



1 I I I N I* I
0* I 3 I I I 'I 0S 3 | 3
S0 0 + +| +( I


I X +I I rI +V < 3
C rI I I I 3 E 3




I t I I I
) I I I I I -. 0





p I -. I I I c
I I n I I C I








I I I I o

-'1 I 06 I I O I <
II I I II I -0 0 e l
I I I I ( I '0





co I 1 1 I a3 a r I a, 0 0 3



CA I I 4 I A I9 0 v; 41 -
I 7 I 0 I 0




II TI C
I40 I I Ig
SI I I .







m I I -.l- io Coio I 0- i o 3 q






a a
Ci i l
























CO < o a




cu
4 '-I I I I I -







































a a
Ef1 -4 0 3 I f 7 Ir -a
l I' U I o- I I I I
UI4 I I I SC

I I I 1 I 03
I I e a














I I I I 0 UC


1 I I V
-U I I II



















1-











-4

































(N 0
A-, Al,

0 -r


< 6
-j
41



41
























-13
-a



4.2.
0










. -3











I CC
SO
C
i















I-
u
41














0
U
Clr
ra
uP























01
5
























u.1
B






















14
1s


0' 0
* 0




















0 0

ao o
00 ,


c c



o r
CC -I




- 0 41



O u C a

0 w
0 X a



f. u
4, )C


u






*o



S S
4 1
0



s i

fa F F
0 0
s c c
41.1




= s

0 0


o E E
41 0 0
4 U U







a a a









,u 0.

.0 0
0 0
0 0




41 c






















I U
*3 C C

0 0



3 u u
.0 0 4 C
u o
5u 0 0 0

0 0 0 0
4a -








*0 -

41- 0 0
-u 4


1 4 u u
UO 03 0

1l. 4. U U






0 -i a I







0o a 0o
-i 0 -< -


o @A

o
4 in












Table 4. Physiochemical characteristics of guinea pig
uterine relaxin. The source of relaxin was an
ODS* purified uterine preparation.


Reduced Activity


No Change


Dithiothrietol 24x

Heating at 700 C 2x

Trypsin 24x

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.















APPENDIX 2
FIGURES

















Figures 1-4 represent sections of uteri taken from guinea pigs
on day 30 of pregnancy.

1. Transverse section of uterus stained using the PAP tech-
nique with R19 antiserum (1:500 dilution). Arrow endometrial
glands exhibiting RP; arrowhead endometrial glands lacking RP.
X 40.

2. Section adjacent to that shown in Figure 1 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo-
metrial glands (arrows). X 40.

3. Hematoxylin and eosin stained section. X 500.

4. Section stained using the PAP technique with R19 anti-
serum (1:500 dilution). Note that not all endometrial gland cells
demonstrate RP. Clear areas in basal regions of the endometrial
gland cells represent unstained nuclear profiles. X 500.

















L '




L
..


L_ ..



S





.- r ":,'. a
r. I .









se *












4




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 ,.:^ -


-r


a
;LS ."
.. ,
".':.*

. -..,


4... *
5 1+-. .-


'1


k"


















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.

















*O1
a'
S

I
* a


/


.
O a9
" rpgip
ol


ip a



* '


-*


0.1


ro r


or


W


it
* *


,
. lrr .


t r


S..-






'-l

bE
a 'Tr


ca

-,^f


^.* \ "V

i' ^ ^
L,


.- .



1 -a
ii 't-
*r I t-

-;' ^10


- q%


ID















Figures 13-16 represent sections of uteri taken from late
pregnant guinea pigs.

13. Transverse section of the guinea pig uterus from late
pregnant animals (day 65 or 66 of pregnancy) and stained using the
PAP technique with R19 antiserum (1:500 dilution). Note all of the
endometrial glands exhibit RP. X 40.

14. Section adjacent to that shown in Figure 13 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo-
metrial glands. X 40.

15. Hematoxylin and eosin stained section. Continuity
between the lumen of an endometrial gland and the lumen of the
uterus can be seen in the lowest of the three gland profiles. Note
the difference in cytoplasmic and nuclear staining densities between
the EGC and cells of the uterine lumen epithelium. X 500.

16. Section adjacent to that shown in Figure 15 stained
using the PAP technique with R19 antiserum (1:500 dilution). Note
that some of the endometrial gland cells have RP distributed through-
out the cytoplasm, some have no RP and in some cells (lower gland)
the RP is localized in a specific supranuclear region. Note also
that the extent of the gland can be determined by the region where
deposition of RP ceases in cells that are continuous with the
uterine lumen epithelium. This pattern of deposition of RP
corresponds with differences in staining noted in H & E stained
tissue (Figure 15). X 500.








85







T- -


A* -.


I0" L



I ", 16 .
I'. .. ,15
L


a





.1 1














i.:*"



a.


r l

pt.


,w
se
. 14
















Figures 17-20 represent sections of uteri taken from lactating
guinea pigs.

17. Transverse section of guinea pig uterus taken from lac-
tating animals (3 days postpartum) and stained using the PAP
technique with R19 antiserum (1:500 dilution). Arrow endometrial
glands exhibiting RP; arrowhead endometrial glands lacking RP.
X 40.

18. Section adjacent to that shown in Figure 17 treated with
normal rabbit serum (1:500 dilution). Note lack of RP over endo-
metrial glands (arrows). X 40.

19. Hematoxylin and eosin stained section. Note the large
number of mitoses in the glands (arrows). X 500.

20. Section stained using the PAP technique with R19 anti-
serum (1:500 dilution). Note that RP is located in only a few
cells of the gland and that the pattern of deposition of RP is
variable from cell to cell. X 500.















Vs.N


.7&


;! ~ ~ 4'. 4k~ .


*3 At ar

At?

7j(. *,


a.


tI
* '
4;,


" .-


"....,


1 .


18


A t..


20















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.















aP /
o


00.'

l4"" ,
-' + (


9 a 21


A.


N22


a M-


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.









91






80



70



S60
4-


I 50
0



S40



<6 50
>-
- 40

I-


Z
x




0
Co, 10
0-
z


30 45 60 Ip lac

STAGE OF PREGNANCY/ LACTATION




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