Table Of ContentZOOLOGICAL SCIENCE 10: 353-360 (1993) © 1993Zoological SocietyofJapan
Prolactin Binding Sites in Normal Uterus and the Uterus
with Adenomyosis in Mice
tlppawan slngtripop, takao mori, mln kyun park,
Koichi Shiraishi, Toshio Harigaya*
AND SEIICHIRO KaWASHIMA
Zoological Institute, Faculty ofScience, University ofTokyo, Tokyo 113,
and *Laboratory ofFunctionalAnatomy, Faculty ofAgriculture,
Meiji University, Kawasaki214, Japan
—
ABSTRACT Radioreceptor assay for mouse prolactin (mPRL) was developed in the mouse uterus.
The specificbindingof 125I]-mPRLtotheuterinepreparationswasdemonstrated andthe bindingwas
[
foundtohaveasimilaraffinitytothatintheliverpreparations. ThespecificPRL-bindingtotheuterine
preparationstendedtoincreaseafterestrogentreatment,whilethedifferencesamongthethreegroups,
diestrous intact, ovariectomized, and ovariectomized and estrogen-treated mice, were statistically not
significant. Immunohistochemical stainingofPRLrevealed that PRLimmunoreactivitywaspreferen-
tially localized in the myometrial cells. Pituitary grafting into the uterus induced a high incidence of
adenomyosisassociatedwiththeincreaseinplasmaPRLlevels. Furthermore,thePRL-bindingactivity
in the uterus with adenomyosis was significantly higher than that in normal uterus. These results
indicate that the elevation ofPRLbindingin the uterus, hyperprolactinemia, and the development of
adenomyosis are interrelated.
INTRODUCTION PRL-binding sites are detected in the uteri of rat
[34], rabbit [4], pig [36], sheep and cow [23], mink
Prolactin (PRL) is known to act on various [27] and human [5] by means of radioreceptor
organs, such as the brain, mammary gland, liver, assay. Inmice, PRL-bindingsiteshavebeenfound
ovary, testis andsomereproductivetractsinmam- in the brain, mammary gland, liver and kidney [3,
mals [1, 6, 20, 21, 26]. EvidenceofPRLeffectson 25]. However, the presence of PRL receptors in
these target organs involves the presence of spe- theuterushasnotyetbeenreportedinthisspecies,
cific PRL-binding sites. Estrogens have been in spite of evident synergistic effect of PRL with
found to increase the specific PRL-binding sites in estrogen on the mouse uterus.
the liver of rats in the presence ofintact pituitary While long-term exposure to estrogen is known
glands [22, 24]. In mice, however, Marshall etal. to induce uterine adenomyosis, a pathological dis-
[9] reported that ovariectomy increased the num- order of endometrial tissues defined as the out-
ber of liver PRL-binding sites, and exogenous growth of endometrial glands and stroma into the
estrogen administration resulted in a decrease in myometrium in some animal species [14, 15, 17,
the number. By contrast, estrogen administration 32], our experiments have demonstrated that in
increases the number of PRL-binding sites in the mice isologous anterior pituitary transplantation
mouse mammary gland [29]. Therefore, the reg- into the uterine lumen induces an early and a high
ulation of the number of PRL-binding sites is incidence of uterine adenomyosis associated with
variable among different organs. the elevation in blood PRL levels [11, 13]. Fur-
Previous studies have revealed that the specific thermore, we have foundthe accelerative effectof
dopamineantagonistwhichstimulatesPRLrelease
Accepted November 20, 1992 from the pituitary [30], and the inhibitoryeffect of
Received December 11, 1992 danazol which suppresses pituitary PRL release
354 T. Singtripop, T. Mori et al.
[31] on the development of adenomyosis. From strain purchasedfromJapan CLEAInc. wereused
these findings, we have proposed that PRL plays in this experiment. Three intact mice at diestrus
an important role for the development of ade- were killedbycervical dislocation at50daysofage
nomyosis, and have claimed that the uterus is one and used for PRL immunohistochemical staining.
of the target organs of PRL, suggesting the pre- Animal handlings were carried out in accord-
sence of PRL receptors in the mouse uterus. ance with the NIH Guide to the Care and Use of
In thepresentstudy,radioreceptorassayofPRL Laboratory Animals.
binding to the mouse uterus and liver was de-
veloped. In addition, the relationship between the Receptorpreparations
developmentofadenomyosisandthePRL-binding Receptor samples were prepared essentially fol-
ability of the mouse uterus was examined. lowing the method ofKelly etal. [7, 8] and Young
et al. [37]. Immediately after autopsy, the uteri
MATERIALS AND METHODS and liver from about 10-15 mice were separately
pooled on ice. Pooled tissues were weighed and
rinsed in5 ml ofice-cold homogenizingbuffer (pH
Animals and treatment mM mM
9.0) composed of 100 Tris, 150 NaCl, 50
ExperimentI Five-week-old female mice ofthe mM ethyleneglycol-bis-(B-aminoethyl ether)-N,
ICR/Jcl strain were purchased from Japan CLEA N-tetraacetic acid (EGTA), 50mM ethylene-
Inc. (Tokyo, Japan) and kept in light(12hr/day)- diaminetetraaceticacid (EDTA), 300 mMsucrose,
and temperature(25+0.5°C)-controlled animal 1 mM phenyl-methylsulfonyl fluoride (PMSF) and
room. Forty-five mice were ovariectomized at 8 aprotinin (400 kallikrin inhibitory units/ml), and
weeks of age. One day after ovariectomy, 20 out were chopped by a razor blade for 5 min on an
of the 45 mice were given a subcutaneous injec- ice-cold teflon plate. The chopped-tissues were
tions of 5fig estradiol-17^/3 in 0.1 ml sesame oil homogenized in cold homogenizing buffer (4ml/g
AM
dailyfor7daysat9:00 andkilledbydecapita- tissue fresh weight) by using a Polytron PT 3000
tion one hour after the last injection. Remaining homogenizer for three times at 20,000 rpm, 20sec
25 ovariectomized mice given no steroid injections each, in a glass tube in ice bath. After centrifuga-
were also killed at 9weeks ofage. In addition, 25 tion ofthe tissue homogenates at 3,750rpm for 15
intact mice at estrus were killed in the early min at 4°C, the precipitate was discarded and the
morning at 9 weeks of age. supernatant was recentrifuged at 33,000rpm for
Experiment II Fifty adult female mice of the 120min at4°C. Thesecondprecipitatewasused as
SHN strain were used in this experiment. The the receptor preparation and was resuspended in
experimental group of 24mice was given trans- 500fA ofcold assay buffer (pH7.4), containing 25
plantationofthe anteriorpituitarygland(AP) into mMTris, 10 mM MgCl2, 0.1% bovine serum albu-
the uterine lumen at 6 weeks of age and killed by min and 0.02% sodium azide. Protein measure-
decapitation at 20 weeks of age. The AP donors ment was performed in 10fA of the resuspension
were age-matched male littermates. The AP- by micro BCA protein assay reagent kit (Pierce
grafted mice which were at diestrus and marked Co., U.S.A). Remaining receptor preparations
—
subserosal nodules visible on the outer surface of were stored at 80°Cuntil radioreceptorassay for
the uterus, an advanced state ofadenomyosis [13] PRL.
were chosen for further analysis, and the AP-
mPRL
grafted mice which showed no sign ofthe develop- lodination of
ment ofnoduleswere discarded. Twenty six intact Mouse PRL (mPRL) (AFP6476C, donated by
mice which were killed by decapitation at diestrus Dr. A. F. Parlow) was radioiodinated with l25I
at 30 days or 20 weeks of age and showed no sign (Na125I, Radiochemical Centre, Amersham, UK)
of the development of nodules were used as con- in the presence of lactoperoxidase (Boehringer
trols. Mannheim GmbH, Germany) and hydrogen per-
Experiment III Female mice of the ICR /Jcl oxide according to the method of Miyachi et al.
Prolactin Receptors in Mouse Uterus 355
[10] with minor modifications. After the reaction, repeated twice in pooled samples of two groups
the radio-labeled hormone was separated from consisting of 10-15 mice each.
free radioiodine by gel filtration using a Sephadex
G-50 column (disposable plastic column; Whale Radioimmunoassay (RIA) of serum PRL
Sci. Inc. U.S.A). The specific activity of labeled In Experiment II in SHN mice, blood was
mPRL was about 50fid/fig. collectedatautopsybydecapitationandallowedto
clot at room temperature for one hour. After
Proceduresfor PRL-binding assay centrifugation at 3,000rpm for 20min, the serum
The procedures were basically the same as the was stored at —80°C. Serum PRL levels were
methodsofKellyetal. [7, 8] andYoungetal. [37]. determinedbyhomologousRIAusingmPRLRIA
For routine PRL-binding assay of liver samples of kit (reference preparation AFP6476C; rabbit anti-
ICR mice, 500fig ofprotein ofthe secondprecipi- mPRL serum, AFP131078; and mPRL,
tate ofliver homogenates in 100fA ofassay buffer AFP6476C, 125I]-labeled) donated by Dr. A. F.
[
was incubated with 125I]-mPRL (ca 55,000cpm, Parlow. Assay reaction mixture was incubated
[
0.5ng/100,ul) at 25°C in an assay tube (12x75 overnight at room temperature. PRL-antibody
barosilicate glass tube) in the presence or absence complexes were separated from unbound PRL by
of an excess amount (5jug) of unlabeled ovine centrifugationaftertheincubationfor3hratroom
PRL (oPRL, Sigma) for 18hrs. Incubation was temperaturewith goat anti-rabbit immunoglobulin
terminatedbyadditionof3 mlofcoldassaybuffer. serum, donatedbyDr. K. Wakabayashi. ThePRL
Unbound hormone was separated by centrifuga- concentrations assayed in 100fA serum were ex-
tion at 4,000rpm for 30min at 4°C. After aspira- pressed in terms of ng AFP6476C/ml.
tion, the radioactivity of precipitates was counted
by an autowell gamma counter (Aloka ARC-300). Immunohistochemistry ofPRL
Each assay was composed oftriplicate determina- In Experiment III, the uterus and kidney were
tions. In some experiments, recombinant mPRL dissectedoutandfixedin4% paraformaldehydein
(5/^g/tube) raised by Yamamoto et al. [35] was phosphate buffer(pH7.3) at 20°C overnight. Tis-
used as the competitor instead of oPRL. sues were dehydrated in alcohol, embedded in
The specificity of the binding was analyzed by paraffin, sectioned at 5fim, and mounted on glass
competitive displacement experiments. Specific slides previously coated with gelatin. The sections
binding of 125I]-mPRL was calculated as the dif- were deparaffinized and rinsed with 0.1 M phos-
[
ference between the binding in the absence and phate-buffered saline (PBS). Immunohistochem-
presence of various concentrations of unlabeled ical demonstration of PRL was carried out by
oPRL (5, 50, 500, 5,000, 50,000ng). All the Histofine SAB-PO (Nichirei Co. Tokyo) method.
reaction tubes contained 500fig of protein in 300 The sections were preincubated with goat normal
fA assay buffer and 55,000cpm of 125I]-mPRL serum diluted with PBS 10 times for 10min and
[
(100/A). The affinity of binding (Ka) and the washed briefly with a fresh PBS solution. Rabbit
dissociation constant (Kd) were determined from anti-mPRL (AFP6476C) at various dilutions
the Scatchard plots constructed from the binding (1:2,500, 1 :10,000 and 1 :50,000) in PBSwasused
displacement experiments [28]. as the primary antiserum. After incubation with
In some experiments, the receptorsampleswere the primaryantiserumovernight at room tempera-
pretreated with 4M MgCl2 to dissociate endoge- ture, the sections were washed with 0.1 M PBS
nously bound PRL from its binding sites before containing triton X-100 (PBST), and then incu-
assay following the method of Kelly et al. [8]. batedwith biotinylated anti-rabbit IgGfor 15 min,
However, routine assays were carried out without followed by incubation with peroxidase-
MgCl2 pretreatment, because MgCl2 treatment conjugated streptavidin for 20min. Following 15
was not effective in increasing PRL binding in min washing with PBST, the sections were incu-
either uterus or liver preparations (data not bated for 15 min with 0.05 % 3,3'-diaminobenzi-
shown). PRL-binding assays at each point were dine (DAB) tetrahydrochloride solution contain-
356 T. Singtripop, T. Mori et al.
ing 0.01% hydrogen peroxide and lightly counter- 24hrs. Incubation for 18hrs resulted in the max-
stained with hematoxylin. All these steps were imum specific binding of 125I]-mPRL to 500//g of
[
performed atroom temperature. The specificityof protein of liver preparations. When various
the method was examined by the use of diluted amounts of liver protein (125, 250, 500, 1,000,
normal rabbit serum 1 :50,000 or PBS instead of 2,000//g) and 55,000cpm of [125I]-mPRL were
primary antiserum. For another test for the anti- incubated in the presence or absence of 5/ug of
gen specificity, primary antiserum (1 :500dilution) unlabeled ofoPRL, the specific binding increased
was added with mPRL (400ng/ml antiserum), as a function protein concentration.
stirred for one hour at room temperature, and Figure 1 shows Scatchard plots drawn from the
incubated at 4°C overnight. To eliminate the data of displacement experiments in liver and
resolubilization of the antibody-antigen com- uterine preparations. From the plots, the equilib-
plexes, the mixture was centrifuged at 4,000rpm rium constant of dissociation (Kd) and the affinity
for 15 min, and the supernatant was used. The constant (Ka) were calculated: forliver Kd=0.048
sections incubated without primary antiserum or X10~8M, Ka=2.08xl09M"1 for uterus Kd=
;
with adsorbed antiserum showed no immuno- 0.054xlO"8M, Ka^l.SxlO^-1
.
staining. Thus, in this study the binding assays in both
liver and uterine preparations were routinely car-
Statistical analysis ried out under the following conditions; 500/ug of
The significance ofdifferences between any two protein for a one assay sample; 55,000cpm of
groups was evaluated by Student's t-test, where P 125I]-mPRL,withorwithout5 /ugunlabeledoPRL
[
<0.05 was considered as statistically significant. to obtain specific binding and 18hrs of incubation
at 25°C.
Ovariectomy increased 125I]-mPRL binding to
RESULTS [
the mouse liver preparations compared to that in
In order to determine the optimum incubation intact control mice at estrus (Fig. 2). Estrogen
time, 500/ug of liver protein and 55,000cpm of treatment to ovariectomized mice tended to de-
125I]-mPRL were incubated with or without 5/ug crease the binding to the liver preparations (Fig.
[
ofunlabeled hormone (oPRL) for3, 6, 12, 18 and 2), while the differences among the three groups
0.05 8 —._
„^8,
0.04 c
"a>
o 6
it 0.03 D.
"cO aE>
IoD 0.02 -
CD 4 -
TC5
0.01 - JD
O
O 2 -
a>
300 Q.
<z>
oPRLBound (pg/ml)
— estrus OX OX+E
-« uterinepreparation 2
-a— liverpreparation Fig. 2r.adioTahcteivsipteycifaidcdebdi)nditongliovfer[1p25rIep]a-rmaPtRioLns(%50o0fptgotoalf
Fig. 1. Scatchard plots of binding of [l25I]-mPRL to ICR mice at estrus, 7 days after ovariectomy (OX)
ICR mouse liverand uterine preparations, basedon and 7 daysofestrogen treatment following ovariec-
the data of the binding of [125I]-mPRL to the liver tomy (OX+E2). Fivepgunlabeled oPRLwasused
and uterine preparations of ICR mouse under var- toassessnon-specificbinding. The barsandvertical
ious concentrations of unlabeled oPRL. lines denote mean of two pooled samples.
Prolactin Receptors in Mouse Uterus 357
5
-
4 r B
o o
Q. Q-
OE) 3 OEl
T gO)
2 g
O £o
o o 1
cCQoD- 1 CCQOD_
n
estrus OX OX+E, control adenomyosis
Fig. 3. The specific bindingof[125I]-mPRL (% oftotal Fig. 4. Thespecific bindingof[125I]-mPRL(% oftotal
radioactivityadded) to uterine preparations (500fig radioactivityadded)touterinepreparationsofintact
protein) ofICRmouse atestrus , OXand OX+E2. control SHN mice and the 20-week-old mice with
Fivefig unlabeled oPRL was used to assess non- adenomyosis. Fivepig unlabeled oPRLwas used to
specific binding. The bars and vertical lines denote assess non-specific binding. The bars and vertical
mean of two pooled samples. For explanation of lines denote mean oftwo pooled samples.
abbreviations, refer to legend ofFig.2.
differences among the groups were again statisti-
were not statistically significant. cally not significant (Fig. 3).
In the uteri ofovariectomized mice, the specific The specific binding of 125I]-mPRL to the uter-
[
binding of [125I]-mPRL was lower than that in ine preparations from 20-week-old SHN mice at
intact mice at estrus (Fig. 3). Daily injections of5 diestrus with adenomyosis tended to be higher
fig estradiol for 7 days induced a slight increase in than that in age-matched control mice at diestrus,
the binding activity as compared to the prepara- where 5fig oPRL were used as the competitor to
tions from ovariectomized mice. However, the assess nonspecific binding. However, the differ-
encewasnotstatisticallysignificant(Fig. 4). When
5fig ofunlabeled recombinant mPRL was used in
c 100 T
CD
o
Q. -
D) I 80 -
E *
T
oc>
* o
C
!q
o concentration
o o
CD
Q.
CO
t 20
young adult adenomyosis * * l*l*
\—^
Fig. 5. The specificbindingof I25I]-mPRL (% oftotal n
radioactivityadded)touteri[nepreparationsofintact young adult adenomyosis
control 30-day-old or 20-week-old SHN mice and Fig. 6. Plasma PRL levels (ng AFP6476C) in intact
the 20-week-old mice with adenomyosis. The re- control 30-day-old or 20-week-old SHN mice at
combinant mPRL was used as competitor to assess diestrus and 20week-old SHN mice with adenomy-
non-specific binding. The bars and vertical lines osisintheiruteri. Thebarsandverticallinesdenote
denote mean oftwo pooled samples. */><0.05 (vs. means+S.E.M. (n=4-9). Significantly different
young & adult controls). from both intact controls: *T<0.01.
358 T. Singtripop, T. Mori et al.
/:",
VM §
Fig. 7. ImmunohistochemicallocalizationofmPRLinparaffinsectionsofthemousekidney. PRLisdetectedinthe
proximal convoluted tubules (P). Anti-mPRL (AFP6476C) at X2,500 dilution. Barrepresents 100^m.
Fig. 8. ImmunohistochemicallocalizationofmPRLinparaffinsectionsofthe mouseuterus. PRLisdetectedinthe
muscle layer (M). Anti-mPRL (AFP6476C) at X2,500 dilution. Barrepresents WOfan.
placeofoPRL, the specificbindingof 125I]-mPRL thatmaximumbindingof 125I]-mPRLto its recep-
[ [
touterinepreparationsinmicewithadvancedstate tors occurred at 18 hrs of incubation at 25"C. In
of adenomyosis (3.90+0.13%) was significantly addition, 500jugofsample protein and55,000cpm
higher than that in 30-day-old or 20-week-old of 125I]-mPRL with or without of 5fig unlabeled
[
normal mice (P<0.05, in eithercomparison) (Fig. oPRLwerefoundto be the optimumcondition for
5). the binding assay. Theseconditionswere basically
The serum PRL level is significantly higher in similar to those reported by previous investigators
pituitary-grafted 20-week-old mice bearing ade- usingrat liverpreparations [4, 5, 7, 23, 27, 34, 36].
nomyosis in their uteri than those in normal 30- According to Marshall et al. [9], ovariectomy in-
day-old or 20-week-old mice, (P<0.01, in either creased 125I]-mPRL binding to the liver prepara-
[
comparison) (Fig. 6). tions of female mice, and estrogen treatment de-
The specificity of antibody and immunohis- creased the binding, indicating the manifestation
tochemical reaction was tested by the sections of of down-regulation in the binding of PRL to the
mouse and rat kidneys, since the influence ofPRL liver by estrogen. In the present study, the same
on the kidney was well established [13, 16, 32]. tendency was observed in the PRL-binding to the
Immunoreactivity to mPRL was localized in some liver samples.
proximal convoluted tubules and the medulla of The present results indicate that specific PRL
mouse kidneys (Fig. 7). In the uterus, im- receptors were present in the uterus of mice.
munoreactivity to anti-mPRL serum (in either Scatchard plot analysis revealed that the affinityof
dilutions, X2,500, X10,000 or X50,000) was pre- binding of mPRL to mouse uterine preparations
ferentially localized in the muscle layers, and the was similar to that to liver preparations.
reactivity of other areas of the uterus was negligi- The level of specific PRL-binding in the uterus
ble (Fig. 8). was low in ovariectomized mice and tended to
increase after estrogen treatment, although these
differences were not statistically significant. The
DISCUSSION
present results that the changes in the PRL-
In the present experiments, optimum conditions binding activity were very slight under various
for the specific PRL-binding assay were deter- hormonal conditions may be due tothe insufficien-
mined by using mouse liver, because the assay cy in estrogen stimulation. Treatment by greater
methods in the liver have been well established. doses of estrogen for longer period might be
Assay data from the present experiments revealed needed. However, the present immunohistochem-
Prolactin Receptors in Mouse Uterus 359
ical observations revealed that the target cells of ACKNOWLEDGMENTS
PRL in the uterus were preferentially myometrial
This study was supported by Grants-in-Aid for Fun-
cells, confirming our previous suggestion [11, 16]. damental Scientific Research from the Ministry of
Because the amount of myometrial tissue in the Education, Science and Culture, Japan, to T.M. and
uterus constitutes only a small portion of the S.K., and Research Grants from Takeda Foundation to
organ, any hormonal influence on the number of T.M. and from Zenyaku Kogyo LTD. to S.K.. T.
PRL receptors in the myometrium might be too Singtripop is a recipient of the fellowship from Hitachi
Scholarship Foundation.
meager to get a significant difference. The authors thank Dr. K. Kohmoto and Dr. S. Sakai
In these experiments, oPRL was routinely used (Department of Animal Breeding, Faculty of Agricul-
to obtain non-specificbinding. When recombinant ture,UniversityofTokyo)fortheirtechnicaladvice. We
mPRLwasused, significantincreaseinPRLrecep- are grateful to Dr. A. F. Parlow (Pituitary Hormones
and Antisera Center Harbor-UCLA Medical Center,
tors was observed in the uterine preparations of California) for his generousgift ofmouse PRL RIA kit,
mice with adenomyotic changes. Therefore, re- and Dr. K. Wakabayashi (Institute of Endocrinology,
combinant mPRLmightbe abettercompetitorfor GunmaUniversity, Gunma,Japan)forprovidingusgoat
future study. anti-rabbit IgG serum.
PRL plays a key role in the development of
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