BIOLOGY OF REPRODUCTION 55, 1243-1252 (1996)
Identification of Specific Relaxin-Binding Cells in the Cervix, Mammary Glands,
Nipples, Small Intestine, and Skin of Pregnant Pigs'
Gyesik Min3 and O.D. Sherwood 23, ,4
Department of Molecular and Integrative Physiology' and College of Medicine,4 University of Illinois at
Urbana-Champaign, Urbana, Illinois 61801
ABSTRACT
We previously demonstrated that relaxin promotes growth
and softening of the cervix and development of the mammary
glands in the pregnant pig. An important aspect of understanding relaxin's mechanism of action in these tissues is to identify
the specific cell type(s) that contains relaxin receptors, that is,
to identify those cells that initiate relaxin's effects. The objective
of the present study was to identify relaxin-binding cells in tissues known to respond to relaxin (cervix and mammary gland)
as well as in tissues suspected of being responsive to relaxin
(nipple, small intestine, and skin) in the pregnant pig. To accomplish that objective we developed an in vitro modification of an
immunohistochemical technique recently developed for identification of relaxin-binding cells. Two groups of pregnant gilts
were used: intact control (group C) and ovariectomized progesterone-treated (group OP). Group OP was ovariectomized on
Day 40 of gestation (Day 40) and treated with progesterone (50
mg/2 ml corn oil i.m., twice daily) until Day 110 to maintain
pregnancy. On Day 110, tissues from both groups were removed, cut into cubes (2-3 cm3), frozen in liquid nitrogen, and
cryosectioned (8 pm). Specific cell types that bind relaxin were
identified by sequential application of a biotinylated relaxin
probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. The study demonstrates for the first time that relaxin binds with specificity to
1) blood vessels (cervix, mammary glands, nipples, small intestine); 2) smooth muscles in small intestine (circular, longitudinal, muscularis mucosa); and 3) skin from sites other than the
mammary nipples (back, ear, thigh, leg). In addition, consistent
with previous findings in the rat, prominent labeling was observed in epithelial cells in the cervix, mammary glands, and
nipples; in smooth muscle cells in the cervix and mammary nipples; and in the skin of the nipples. There were no apparent
differences in relaxin binding between group C and group OP.
We conclude that the specific relaxin-binding cells in the cervix,
mammary glands, nipples, small intestine, and skin of the pregnant pig probably contain relaxin receptors and, therefore, mediate relaxin's effects in these tissues.
INTRODUCTION
The physiological roles of relaxin vary remarkably
among mammalian species during pregnancy [1]. Endogenous relaxin has well-established vital effects in pigs and
rats, but the physiological effects of relaxin differ in the
two species [1]. For example, relaxin's effects on the cervix
are estrogen-dependent in the rat [2-4] but not in the pig
[5]. Relaxin promotes marked growth of the mammary
gland parenchyma in pigs [6] but not in rats [7]. Relaxin
Accepted July 8, 1996.
Received May 17, 1996.
'This work was supported by USDA Grant AG 93-37203-9562 (to
O.D.S.).
2
Correspondence: Dr. O.D. Sherwood, Department of Molecular and
Integrative Physiology, University of Illinois at Urbana-Champaign, 524
Burrill Hall, 407 South Goodwin Avenue, Urbana, IL61801-3704. FAX:
(217) 333-1133; e-mail: [email protected]
promotes marked growth of the nipple in rats [7] but not
in pigs [6]. Relaxin is secreted but has no verified effects
in humans [1]. Relaxin does not appear to be produced in
sheep [8, 9]. The extraordinary diversity of relaxin's physiological roles among species makes it unjustifiable for investigators to extrapolate information concerning relaxin's
physiological roles and/or mechanisms of action from one
species to another.
In the pregnant pig, the corpora lutea are the source of
both the progesterone required for maintenance of pregnancy and the protein hormone relaxin [10-14]. Plasma relaxin
levels increase progressively and slowly from about 150
pg/ml on Day 6 to about 10 ng/ml on Day 110, and then
they surge to maximal levels during the two to three days
before parturition, which occurs on about Day 115 of pregnancy [15-19]. Circulating relaxin is a vital component of
the endocrine milieu during late pregnancy in pigs. Relaxin
promotes growth and softening of the cervix [20-22] and
thereby enables rapid and safe delivery of the fetuses [23,
24]. Relaxin also promotes development of the lobulo-alveolar (parenchymal) tissue of the mammary glands [6, 25].
Essentially nothing is known concerning the cellular
mechanism(s) of relaxin's action in pigs. A fundamental
step toward understanding how relaxin initiates its effects
is to identify specific cells that contain relaxin receptors.
The relaxin receptor has not been isolated in any species
to date. Until recently, investigators relied on radiolabeled
relaxin and receptor ligand affinities to probe for relaxin
receptors. Autoradiographic localization of specific relaxin
binding was described in the cervix, uterus, brain, and heart
of rats [26-29], and pubic symphysis, uterus, and ovary of
mice [30]. The only demonstration of relaxin binding in the
pig was by Mercado-Simmen et al. [31], who in 1982 reported specific relaxin binding in particulate membrane
preparations of the uterine and cervical tissues of the nonpregnant pig. However, the specific cell types that bind relaxin were not identified in these previous studies. Recently,
our laboratory developed a procedure whereby biotinylated
relaxin is injected i.v. in order to identify specific cells that
bind relaxin in the cervix, mammary gland, and nipples in
pregnant rats [32].
The objective of the present study was to identify relaxin-binding cells in tissues known to respond to relaxin (cervix and mammary gland) as well as in tissues suspected of
being responsive to relaxin (nipple, small intestine, and
skin) in the pregnant pig. To accomplish that objective, we
developed an in vitro modification of the immunohistochemical technique recently developed for identification of
relaxin-binding cells [32].
MATERIALS AND METHODS
Preparation of Biotinylated Relaxin
Porcine relaxin was isolated as described by Sherwood
and O'Byrne [33], and it was biotinylated by a modification
1243
1244
MIN AND SHERWOOD
[32] of the procedure described by Biillesbach and Schwabe
[34]. In brief, porcine relaxin was dissolved in 0.2 M Nmethylmorpholine-HCI buffer (pH 7.5) at a final concentration of 2 )pmol/ml. To supply the biotinylating reagent
in excess, 10 molar equivalents of biotinyl-e-aminocaproic
acid-N-hydroxysuccinimide ester (Sigma, St. Louis, MO)
in dimethylformamide at a concentration of 100 /xmol/ml
were added to the relaxin. The reaction mixture was stirred
at room temperature for 4 h, and the reaction was stopped
by the addition of acetic acid until a 1 M acetic acid solution was obtained. The contents of the reaction mixture
were separated from the biotinyl-e-aminohexanoyl-relaxin
(biotinylated relaxin) by ultrafiltration using an Amicon
model 402 stirred ultrafiltration apparatus with a Diaflo Ultrafilter type YM1 membrane (molecular weight cut-off
1000; Amicon, Beverly, MA). The N-methylmorpholineHCI buffer and acetic acid were replaced with PBS (0.01
M NaH 2PO 4 and 0.15 M NaCl, pH 7.4) in the ultrafiltration
unit. The biotinylated relaxin was stored at a final concentration of 9 nmol/ml at -70 0C.
Characterization of Biotinylated Relaxin
The mean number of biotin molecules per biotinylated
relaxin molecule was determined by a spectrophotometric
4'-hydroxyazobenzene-2-carboxylic acid (HABA) assay
according to the manufacturer's protocol (Pierce Chemical
Co., Rockford, IL). The bioactivity of the biotinylated relaxin was determined with the mouse interpubic ligament
bioassay [35]. In brief, immature female ICR mice (Harlan,
Indianapolis, IN), weighing 18-20 g, received s.c. injections of 5 gg estradiol cyclopentylpropionate (Upjohn, Kalamazoo, MI) in 0.1 ml sesame oil. On the seventh day after
estrogen priming, two groups of mice (n = 20/group) received s.c. injections of either 1 g of highly purified porcine relaxin [33] or 1 pxg of biotinylated relaxin in 0.2 ml
of the repository vehicle, 1% L-390 (Dykem, St. Louis,
MO), in PBS. A third group (vehicle control) received only
1% L-390 in PBS. Twenty-four hours after relaxin injections, the mice were killed by carbon dioxide inhalation,
and the distance between the pubic bones was measured.
Animals and Treatments for Immunohistochemistry
The animal experimentation described in this study was
approved by the University of Illinois Laboratory Animal
Care Advisory Committee. Six cycling cross-bred gilts (Pig
Improvement Co., Franklin, KY, 8 mo of age; -120 kg)
were obtained from the Swine Research Center at the University of Illinois-Urbana-Champaign. They were mated at
estrus (Day 0) and housed individually in confinement
crates throughout gestation. Gilts were fed a diet of corn
and soybean (12% protein) once daily and were allowed
free access to water. Gilts were assigned to one of two
groups (n = 3 per group): intact control (group C) and
ovariectomized progesterone-treated (group OP). Group OP
was used to eliminate the possibility that endogenous relaxin prevents binding of the biotinylated relaxin probe to
the relaxin receptor.
Surgery and Hormone Replacement Therapy for
Immunohistochemistry
Surgery was performed on animals of group OP on the
morning of Day 40 of pregnancy. Animals were anesthetized by administering 5 mg/kg BW Telazol (Fort Dodge
Laboratories, Inc., Fort Dodge, IA) and 2 mg/kg BW Rom-
pum (Mobay Corp., Shawnee, KS) i.m. in the neck. Each
animal was also administered 10 mg atropine sulfate (15
mg/ml; Anpro Pharmaceutical, Arcadia, CA), i.m. in the
neck. During surgery, animals were maintained at a surgical
plane of anesthesia using 0.5-3.0% halothane (Halocarbon
Laboratories, River Edge, NJ) and 2 liters O 2/min. Surgery
consisted of bilateral ovariectomy by ventral laparotomy.
Immediately after surgery and for 3 days thereafter, gilts
received an i.m. injection of 5 ml procaine-penicillin G (3
x 105 U/ml; Pfizer, New York, NY) in the neck. Group OP
gilts received i.m. injections in the neck of progesterone
(Sigma Chemical Co.; 50 mg/2 ml corn oil) beginning at
0600 h on Day 38 and continuing at 12-h intervals until
0600 h on Day 110. Previous studies showed that this dose
restored mean blood levels of progesterone to physiological
levels during pregnancy in ovariectomized gilts [19, 20].
Since estrogen is produced by the placenta during pregnancy in pigs, it was not administered.
Tissue Collection and Processing for
Immunohistochemistry
On Day 110 of gestation, gilts from both groups were
electrostunned and killed by exsanguination at the University of Illinois Meat Science Laboratory. Six different tissues-uterine cervix, mammary glands, nipples, small intestine (duodenum), skin (back, ear, thigh, leg), and abdominal skeletal muscle-were removed. Tissues were cut into
cubes (2-3 cm 3) and individually placed in peel-A-way
plastic embedding molds (Polysciences Inc., Warrington,
PA). The tissues were frozen with Tissue-Tek O.C.T. embedding compound (Miles Scientific, Elkhart, IN) in liquid
nitrogen and stored at -70°C until sectioning. Frozen sections (8 ,um) were cut on an HR Mark II cryostat (Slee
Medical Equipment Limited, London, England) at -20°C
and thaw-mounted on microscope slides coated with 0.2%
poly-L-lysine (Mr 300 000).
Immunohistochemical Localization of Biotinylated
Relaxin
The tissue slides were brought to room temperature, and
subsequent immunohistochemical procedures were performed at room temperature. Tissue slides were incubated
for 30 min in 50 mM glycine in PBS (pH 7.4), and then
incubated for 3 h with blocking buffer 1 (1% BSA fraction
V, 0.2% fish gelatin [Amersham, Arlington Heights, IL],
5% normal pig serum, and 2 mM NaN 3 in PBS). Tissue
slides were incubated for 3 h in incubation buffer 1 (1%
BSA fraction V, 0.2% fish gelatin, 1% normal pig serum,
and 2 mM NaN 3 in PBS) in four different ways. The first
treatment incubated each tissue with biotinylated relaxin
probe (3 [Lg/ml) in order to localize relaxin receptors. The
second treatment incubated each tissue with unmodified
porcine relaxin (3 pLg/ml). This treatment was used as a
negative control for the detector molecule, antibiotin antibody. The third treatment incubated each tissue with biotinylated relaxin plus a 2000-fold excess of porcine insulin
(ILETIN II; Eli Lilly, Indianapolis, IN) in order to determine hormonal specificity of binding of the biotinylated
relaxin probe. And the fourth treatment incubated each tissue with biotinylated relaxin plus a 2000-fold excess of
porcine relaxin [33] in order to determine whether there are
finite numbers of relaxin receptors in the tissue. Skeletal
muscle was used as a negative control tissue in order to
determine tissue specificity of the probe. After incubation,
tissue slides were rinsed for 2 h with five changes of wash
RELAXIN-BINDING CELLS IN PIG TISSUES
1245
FIG. 1. Localization of relaxirf-binding sites in the cervix of Day 110 intact pregnant pigs (group C). Relaxin binding was localized in cervices incubated
with biotinylated relaxin (A-C; B and C are a higher magnification of luminal and peripheral areas of A, respectively) but not in cervices incubated
with unmodified porcine relaxin (D). Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of
porcine insulin (E)but not in the presence of a 2000-fold excess of porcine relaxin (F). ep, epithelial cells; csm, circular smooth muscle; Ism, longitudinal
smooth muscle; by, blood vessels. Bar in A = 1970 m; D-F are the same magnification. Bar in B = 493 I.m; C is the same magnification.
buffer (1% BSA fraction V, 0.2% fish gelatin, and 2 mM
NaN 3 in PBS). The tissues were then postfixed for 10 min
in 2% glutaraldehyde in PBS, rinsed briefly with doubledistilled water, and incubated for 30 min in 50 mM glycine.
The tissues were then incubated for 4 h in blocking buffer
2 (1% BSA fraction V, 0.2% fish gelatin, 5% normal goat
serum, and 2 mM NaN 3 in PBS), and for 4 h in 800 ~xl of
antibiotin immunoglobulin G conjugated to 1 nm colloidal
gold (Auroprobe One anti-biotin, Amersham) diluted 1:20
with incubation buffer 2 (1% BSA fraction V, 0.2% fish
gelatin, 1% normal goat serum, and 2 mM NaN 3 in PBS).
The tissues were rinsed for 2 h with five changes of wash
buffer, and were postfixed in 2% glutaraldehyde for 10 min.
All slides were rinsed with copious amounts of doubledistilled water for 30 min before silver intensification of the
gold particles. Silver intensification was performed by incubating sections in IntenSE M silver solution (Amersham)
for 8 min at room temperature. The slides were rinsed with
copious amounts of double-distilled water for 10 min, and
the silver intensification step was repeated. The tissue sections were dehydrated in an ascending series of ethanol,
cleared in Clear-Rite 3 (Richard Allen, Richland, MI), and
coverslipped using mounting medium (Richard Allen).
Statistics
Data were analyzed by analysis of variance, and significant differences among groups were determined by t-test
for the bioassay [36].
1246
MIN AND SHERWOOD
FIG. 2. Localization of relaxin-binding sites in the cervix of Day 110 ovariectomized relaxin-deficient pregnant pigs (group OP). Relaxin binding was
localized in cervices incubated with biotinylated relaxin (A-C; B and C are a higher magnification of luminal and peripheral areas of A, respectively)
but not in cervices incubated with unmodified porcine relaxin (D). Tissue sections incubated with biotinylated relaxin showed binding in the presence
of a 2000-fold excess of porcine insulin (E)but not in the presence of a 2000-fold excess of porcine relaxin (F). ep, epithelial cells; csm, circular smooth
muscle; Ism, longitudinal smooth muscle; by, blood vessels. Bar in A = 2186 m; D-F are the same magnification. Bar in B = 350 Im ; C is the same
magnification.
RESULTS
Characterization of the Biotinylated Relaxin
Consistent with our previous study [32], there were 3.5
molecules of biotin per molecule of biotinylated relaxin, as
determined by the HABA assay. As there are only four
possible sites in relaxin that may react with the biotinyl-Eaminocaproic acid-N-hydroxysuccinimide ester [34], the
biotinylated relaxin probe contained nearly the maximum
possible number of incorporated biotin molecules. Also
consistent with our previous data [32], biotinylated relaxin
elicited a strong biological response, which did not differ
statistically [36] from that of unmodified porcine relaxin.
The mean ( SE) mouse interpubic ligament lengths (mm)
for vehicle control, 1 jIg of relaxin, and 1 ug of biotinylated
relaxin were 0.7 (+ 0.07), 2.4 (+ 0.17), and 2.2 ( 0.18),
respectively.
Immunohistochemical Localization of Biotinylated
Relaxin
Immunohistochemistry results from sections of cervix
obtained from intact pregnant gilts (group C) on Day 110
are shown in Figure 1. Prominent labeling of luminal epithelial cells and both circular and longitudinal smooth muscle cells of the cervix was observed in sections incubated
RELAXIN-BINDING CELLS IN PIG TISSUES
1247
FIG. 3. Localization of relaxin-binding sites in the mammary glands of Day 110 intact pregnant pigs (group C). Relaxin binding was localized in
mammary glands incubated with biotinylated relaxin (A) but not in mammary glands incubated with unmodified porcine relaxin (B). Tissue sections
incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of porcine insulin (C)but not in the presence of a 2000-fold
excess of porcine relaxin (D). aep, alveolar epithelial cells; lep, lactiferous duct epithelial cells; by, blood vessels. Bar = 199 Rm; all panels are the
same magnification.
with biotinylated relaxin (Fig. 1, A-C). Labeling of less
intensity was also observed in cervical blood vessels (Fig.
1, A-C). No signal was detected in cervical sections incubated with unmodified porcine relaxin (Fig. 1D). Binding
of the biotinylated relaxin to the cervix was hormone-specific and saturable (Fig. 1, E and F). Relaxin binding was
also associated with luminal epithelial cells, circular
smooth muscle cells, longitudinal smooth muscle cells, and
blood vessels in the cervix of pregnant gilts in which endogenous circulating relaxin had been removed by bilateral
ovariectomy (group OP; Fig. 2). There were no apparent
differences in relaxin binding (both intensity and distribution of labeling) either within each group or between group
C and group OP with the cervix or other tissues.
Immunohistochemistry results from sections of mammary glands obtained from intact pregnant gilts (group C)
on Day 110 are shown in Figure 3. Prominent labeling of
epithelial cells in both the lobulo-alveolar structures and
lactiferous ducts, and blood vessels was observed in sections incubated with biotinylated relaxin (Fig. 3A). It was
not possible to determine at the light microscopic level
whether biotinylated relaxin binds to the myoepithelial cells
surrounding the prominently labeled epithelial cells. No
signal was detected in sections incubated with unmodified
porcine relaxin (Fig. 3B). Binding of the biotinylated relaxin in the mammary glands was hormone-specific and
saturable (Fig. 3, C and D).
Immunohistochemistry results from sections of nipples
obtained from intact pregnant gilts (group C) on Day 110
are shown in Figure 4. Cell types containing relaxin-binding sites in the nipples were similar to those identified in
the cervix. There was prominent labeling of lactiferous duct
luminal epithelial cells, of both circular and longitudinal
smooth muscle cells, and of blood vessels (Fig. 4, A and
B). In the nipples, there was also prominent labeling of the
skin (Fig. 4A). No signal was detected in sections incubated
with unmodified porcine relaxin (Fig. 4C). Binding of the
biotinylated relaxin in the nipples was hormone-specific
and saturable (Fig. 4, D and E).
Immunohistochemistry results from sections of small intestine (duodenum) and skeletal muscle (abdominal) obtained from intact pregnant gilts (group C) on Day 110 are
shown in Figure 5. Prominent labeling of both circular and
longitudinal smooth muscle cells, and smooth muscle cells
of muscularis mucosa of the small intestine was observed
in sections incubated with biotinylated relaxin (Fig. 5A).
Low-intensity labeling was also observed in blood vessels
of the small intestine (Fig. 5A). No signal was detected in
sections incubated with unmodified porcine relaxin (Fig.
5B). Binding of the biotinylated relaxin in the small intestine was hormone-specific and saturable (Fig. 5, C and D).
Binding of the biotinylated relaxin was also tissue-specific.
No binding was observed when a putative nontarget tissue
for relaxin, skeletal muscle, was incubated with biotinylated
relaxin (Fig. 5E).
Immunohistochemistry results from sections of skin ob-
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MIN AND SHERWOOD
FIG. 4. Localization of relaxin-binding sites in the nipples of Day 110 intact pregnant pigs (group C). Relaxin binding was localized in nipples incubated
with biotinylated relaxin (A and B; B is a higher magnification of a luminal area of A) but not in nipples incubated with unmodified porcine relaxin
(C). Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of porcine insulin (D) but not in the
presence of a 2000-fold excess of porcine relaxin (E). ep, epithelial cells; csm, circular smooth muscle; Ism, longitudinal smooth muscle; by, blood
vessels; sk, skin. Bar in A = 1987 i.m; C-E are the same magnification. Bar in B = 500 pWm.
tained from the back of intact pregnant gilts (group C) on
Day 110 are shown in Figure 6. Prominent labeling with
the biotinylated relaxin was observed in the stratum granulosum and the malpighian layer (strata spinosum and germinativum) but not in the strata lucidum and corneum of
the epidermis (Fig. 6A). Prominent labeling was also observed in the external root sheath but not in the dermal root
sheath, internal root sheath, or hair of the hair follicles (Fig.
6A). No signal was detected in sections incubated with unmodified porcine relaxin (Fig. 6B). Binding of the biotinylated relaxin in the skin was hormone-specific and saturable (Fig. 6, C and D). Similar binding was also found in
skin obtained from the ear, thigh, and leg (data not shown).
DISCUSSION
The immunohistochemical technique used in this study
for identifying relaxin-binding cells is novel. Whereas the
original immunohistochemical model previously employed
in our laboratory simply involved injecting biotinylated relaxin into the rat [32], the present study established a highly
effective procedure for binding of biotinylated relaxin to
relaxin-binding cells in vitro on tissue sections. Optimization of conditions that reduced both deterioration of relaxinbinding sites and nonspecific binding of the biotinylated
relaxin probe was accomplished empirically. The in vitro
immunohistochemical technique described in this report is
an important contribution because the demands for large
RELAXIN-BINDING CELLS IN PIG TISSUES
1249
FIG. 5. Localization of relaxin-binding sites in the small intestine (duodenum) of Day 110 intact pregnant pigs (group C). Relaxin binding was localized
in small intestines incubated with biotinylated relaxin (A) but not in small intestines incubated with unmodified porcine relaxin (B). Tissue sections
incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of porcine insulin (C)but not in the presence of a 2000-fold
excess of porcine relaxin (D). Binding was not found in skeletal muscle incubated with biotinylated relaxin (E). csm, circular smooth muscle; Ism,
longitudinal smooth muscle; msm, muscularis mucosa smooth muscle; by, blood vessels. Bar = 500 ptm; all panels are the same magnification.
quantities of biotinylated relaxin make it prohibitively expensive to identify relaxin-binding cells in large animals
employing the in vivo method we developed for use in rats
[32].
The present study demonstrates for the first time that
relaxin binds with specificity to 1) blood vessels (cervix,
mammary glands, nipples, small intestine), 2) smooth muscles in small intestine (circular, longitudinal, muscularis
mucosa), and 3) skin from sites other than the mammary
nipples (back, ear, thigh, leg). In addition, consistent with
previous findings in the rat [32], prominent labeling was
observed in epithelial cells in the cervix, mammary glands,
and nipples; in smooth muscle cells in the cervix and mammary nipples; and in the skin of the nipples. The obser-
vation that relaxin binds to similar cell types in cervices,
mammary glands, and nipples in pigs and rats is important.
It informs us that the different physiological effects of relaxin on these three tissues in the two species, which were
described in the Introduction, are probably attributable to
factors other than differences in the specific cells that bind
relaxin.
The physiological significance of relaxin's binding to epithelial cells in the pig cervix, mammary glands, and nipples is not known, but the present and earlier observations
permit speculation. In the cervix, relaxin's actions on epithelial cells may be associated with cervical softening. Relaxin may promote epithelial cell secretion of paracrine factors into the stroma since relaxin promotes more dramatic
1250
MIN AND SHERWOOD
FIG. 6. Localization of relaxin-binding sites in the skin (back) of Day 110 intact pregnant pigs (group C). Relaxin binding was localized in skin
incubated with biotinylated relaxin (A)but not in skin incubated with unmodified porcine relaxin (B). Tissue sections incubated with biotinylated relaxin
showed binding in the presence of a 2000-fold excess of porcine insulin (C) but not in the presence of a 2000-fold excess of porcine relaxin (D). sc,
stratum corneum; sl, stratum lucidum; sg, stratum granulosum; ss, stratum spinosum; st, stratum germinativum; ml, malpighian layer; ed, epidermis; h,
hair; is, internal root sheath; es, external root sheath; ds, dermal root sheath; f, follicle. Bar = 500 m; all panels are the same magnification.
alterations in the extracellular matrix near the lumen than
near the periphery of the tissue [22]. In the mammary
glands, relaxin's actions on epithelial cells may be associated with both the marked lobulo-alveolar growth [6, 25]
and prominent secretions into the alveoli [25] that occur in
response to relaxin treatment. The possible importance of
relaxin's effects on the epithelial cells in the nipples is more
speculative. Relaxin's actions on the epithelial cells may
contribute to nipple growth.
For many years, relaxin's effects on stromal extracellular
matrix remodeling were postulated to be mediated at least
in part by a direct action(s) on stromal fibroblasts. Although
there is evidence that relaxin acts directly on human dermal
fibroblasts [37-39] and rat mesenchymal cells [40], there is
presently no evidence to support the idea that relaxin directly stimulates fibroblasts of the cervix, mammary glands,
or nipples of the pig or any other species. Studies at the
electron microscope level will be necessary to determine
whether there are specific relaxin-binding sites in the fibroblasts of these tissues.
Whereas it is well known that relaxin acts directly on
uterine myometrial smooth muscle to inhibit the frequency
and/or amplitude of uterine contractions in several mammalian species [1], the significance of relaxin's binding to
smooth muscle cells in other reproductive tissues such as
the cervix and nipples is unknown. Perhaps relaxin inhibits
smooth muscle contractility wherever smooth muscle occurs. For example, the present study demonstrated promi-
nent labeling in the muscularis mucosa, circular smooth
muscle, and longitudinal smooth muscle of the pig small
intestine (duodenum)-a nonreproductive tissue. This observation is consistent with an isolated and unconfirmed
earlier report [41] that rat relaxin markedly reduced the
strength and frequency of contractility of the rat small intestine (ileum).
This study demonstrated for the first time that relaxin's
binding was localized on blood vessels in the cervix, mammary glands, nipples, and small intestine. It remains to be
clearly demonstrated, however, whether relaxin binding is
associated with endothelial cells, smooth muscle cells, or
both types of cells. Positive labeling of vascular smooth
muscle cells is consistent with the earlier observations of
Del Mese and coworkers [42, 43] that in vivo topical administration of porcine relaxin triggered a prompt dilatation
of veins in the rat mesocecum. These authors postulated
that relaxin dilated the veins by reducing the contractility
of the smooth muscle cells surrounding the vessels. Relaxin's effects on vascular smooth muscle may also account
for the observations that endogenous relaxin promotes an
increase in blood vessel diameter in both the cervix [44]
and nipples [45] of the rat during pregnancy. The physiological significance of these observations is presently not
known. It may contribute to the increase in cervical water
concentration that occurs during the second half of pregnancy [1]. Also, enlargement of the blood vessels may enhance migration of nonresident cells into the cervix. It has
RELAXIN-BINDING CELLS IN PIG TISSUES
been reported that neutrophil invasion of the cervix occurs
in parallel with cervical ripening in the human [46, 47] and
guinea pig [48]. These neutrophils have been postulated to
release metalloproteinase enzymes that bring about degradation of the extracellular matrix of the cervix [47].
The demonstration that relaxin binds with specificity to
the skin surrounding the nipples and to skin removed from
other sites (back, ear, thigh, leg) is consistent with previous
reports describing biotinylated relaxin binding in the skin
of rat nipples [32, 49]. Binding of the biotinylated relaxin
in the skin was confined to the stratum granulosum and the
malpighian layer (strata spinosum and germinativum) of the
epidermis, and the external root sheath (an extension of
epidermal cells) of hair follicles. There was an apparent
lack of labeling on the keratinized squamous cells in the
strata corneum and lucidum of the epidermis, and on cells
in the dermal root sheath (connective tissue), internal root
sheath (Henle's layer, Huxley's layer, cuticle), and hair (cuticle, cortex, medulla) of the hair follicles. The physiological significance of relaxin's binding in specific cell types
of skin is not known. There is evidence that relaxin has
effects on the skin that may be of clinical importance in
humans with integumentary connective tissue disease. Pigs
were used in two previous studies because pig skin is similar to that of humans. In one of these studies, porcine relaxin was reported to increase the rate of tissue expansion
when administered into the skin over tissue expanders [50].
In the other study, intravenous administration of synthetic
human relaxin was reported to facilitate tissue expansion in
piglets [51]. Consistent with these findings, Unemori and
coworkers found that synthetic human relaxin decreased
collagen accumulation in vivo in two rodent models of fibrosis [40] and also decreased collagen synthesis by human
dermal fibroblasts in vitro [38, 39].
Although little is known concerning the regulation of
relaxin receptor expression, it has been suggested that relaxin receptor numbers in the uterine myometrium may be
up-regulated by estrogen in both the pig [31] and the rat
[52], and down-regulated by relaxin in the rat [52]. In this
study, endogenous circulating relaxin did not appear to occupy relaxin-binding sites to the extent that it interfered
noticeably with the binding of the biotinylated relaxin in
the cervix and other examined tissues (data not shown) of
the pregnant pig. Neither the cell types that bound relaxin
nor the relative intensity of the labeling differed noticeably
between intact pregnant pigs and pregnant pigs in which
endogenous circulating relaxin was removed by bilateral
ovariectomy. These results support the use of intact pregnant pigs as a model to identify specific relaxin-binding
cells in suspected target tissues for relaxin in the pig.
In summary, this study identifies specific relaxin-binding
cells in the cervix, mhmmary glands, nipples, small intestine, and skin of the pregnant pig. They are epithelial cells
in the cervix, mammary glands, and nipples; blood vessel
cells in the cervix, mammary glands, nipples, and small
intestine; smooth muscle cells in the cervix, nipples, and
small intestine; and both epidermal cells (strata granulosum, spinosum, and germinativum) and hair follicles (external root sheath) of the skin. We conclude that the specific
relaxin-binding cells probably contain relaxin receptors and
therefore mediate relaxin's effects in the cervix, mammary
glands, nipples, small intestine, and skin of the pregnant
pig.
ACKNOWLEDGMENTS
The authors thank the employees of the University of Illinois Swine
Research Center for their assistance with maintenance of animals, Mr. R.T.
1251
Gladin for his assistance with preparation of the photographs, and the
College of Medicine Word Processing Center for assistance with the preparation of the manuscript.
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