enhancement of the myometrial response to exogenous parathyroid hormone-related protein (PTHrP), and tissue localization of endogenous PTHrP and its mRNA in the virgin rat uterus

Oestrogen

V. Paspaliaris, S. J. Vargas, M. T. Gillespie, E. D. Williams, J.A. Danks, J. M. Moseley, M. E. Story, J. N. Pennefather, D. D. Leaver and T. J. Martin of Pharmacology, The University of Melbourne, Parkville, Victoria 3052, Australia *St Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia tDepartment of Pharmacology, Monash University, Clayton, Victoria 3168, Australia (Requests for offprints should be addressed to V. Paspaliaris)

Department

received

5 November 1991

ABSTRACT

Classical

pharmacological

studies have shown that

oestrogen dominance in humans and other animals

responsiveness of the uterus to many locally acting peptides. Parathyroid hormone-related protein (PTHrP) has been shown to be expressed in can

increase the

the pregnant and non-pregnant rat uterus and exogenous PTHrP is known to relax uterine contraction in vitro. We investigated whether oestrogen dominance can influence the responsiveness of the uterine horn to PTHrP, and further studied the localization of PTHrP mRNA and protein in the rat uterine horn using in\x=req-\ situ hybridization and immunohistochemistry. Exogenous PTHrP(1\p=n-\34) inhibited spontaneous and electrically induced contractions in uteri isolated from

non-cycling rats. Pretreatment of non-cycling rats with oestradiol-17\g=b\ increased uterine sensitivity to

2\m=.\6nmol/l and 7800 nmol/l in uteri from animals treated for 2 days with oestradiol-17\g=b\alone, 2 days with oestradiol-17\g=b\+1 day progesterone, 1 day with oestradiol-17\g=b\ alone and in untreated rats respectively. Similar EC50 values were obtained for electrically stimulated uteri. In agreement with these findings, uterine horns from cycling rats in pro-oestrous and oestrous phases of the cycle showed a higher responsiveness to PTHrP(1\p=n-\34)when compared with uterine horns taken from rats in metoestrus and dioestrus. PTHrP mRNA and protein were detected in the endometrial epithelium lining of the lumen and the endometrial glands, as well as in the myometrium of rats which were either pretreated for 2 days with oesuntreated. This study suggests that PTHrP may act in an autocrine and/or paracrine

tradiol-17\g=b\ or

modulate uterine

motility

PTHrP: EC50 values for inhibition of spontaneous contractions by PTHrP were 0\m=.\33nmol/l, 1\m=.\1nmol/l,

manner

to

Journal

of Endocrinology (1992) 134,

INTRODUCTION

ing mammary tissue (Thiede & Rodan, 1988), the chicken oviduct shell gland (Thiede & Leach, 1989), fetal sheep parathyroids (Rodda, Kubota, Heath et al 1988), areas of the central nervous system (Weir, Brines, Ikeda et al 1990) and recently in the occupied and unoccupied rat uterine horn (Thiede, Daifotis, Weir et al 1990; Thiede, Harm, Hasson & Gardner, 1991). The physiological roles of PTHrP have yet to be established, but its production in such a variety of tissues suggests that it may be a locally acting peptide.

Parathyroid hormone-related protein (PTHrP) was isolated (Moseley, Kubota, Diefenbach-Jagger et al 1987) and its cDNA cloned (Suva, Winslow, Wettenhall et al. 1987; Mangin, Webb, Dreyer et al 1988) from cancers derived from patients with humo¬ ral hypercalcaemia of malignancy. PTHrP protein and/or mRNA have been localized in sites as diverse as skin (Danks, Ebeling, Hayman et al 1989), lactat-

and function. 415\p=n-\425

Classical

pharmacological studies have shown that

pretreatment of animals with oestrogen can increase the responsiveness of the uterus to locally acting pep¬ tides such as oxytocin, vasoactive intestinal peptide (VIP) and relaxin, by increased receptor number and/ or

receptor coupling (Mercado-Simmen, Bryant-

Greenwood & Greenwood, 1982; Fuchs, Fuchs & Soloff, 1985; Ottesen, Larsen, Staun-Olsen et al 1985). Since amino-terminal fragments of PTHrP and parathyroid hormone have been shown to relax uter¬ ine smooth muscle (Shew, Yee & Pang, 1984; Shew, Yee, Kliewer et al 1991), we have investigated whether oestrogen and progesterone dominance can influence the sensitivity of the rat non-gravid uterine horn to PTHrP by treating immature rats with oes¬ trogen and/or progesterone and by following the oestrous cycle of mature rats. The mRNA of PTHrP in the rat uterus is thought to be confined to the myometrium, as was previously demonstrated with Northern blot analysis (Thiede et al. 1990, 1991). In this study, we used in-situ hybrid¬ ization and immunohistochemistry for the detection and localization of PTHrP mRNA and protein in uterine horns taken from non-cycling rats pretreated with oestradiol-17ß or untreated. MATERIALS AND METHODS

following

Cyding rats Vaginal swabs were taken from virgin female Sprague-Dawley rats (200 250 g) immediately after they were killed by cervical dislocation. The swab was smeared onto a glass slide which was then air dried and stained with méthylène blue. The four phases of the oestrous cycle: pro-oestrus, oestrus, metoestrus and dioestrus, were identified (Ham, 1969). The mid portion (2-3 cm long) of the uterine horns of each rat removed and mounted in 20 ml organ baths in a Krebs-Henseleit solution bubbled with 95% 02 and 5% C02 at 37 °C, pH 7-2. The Krebs-Henseleit solu¬ tion had the following composition (mmol/1) : NaCl, was

118; KC1, 4-7; NaHC03, 25; NaH2P04, 1-2; 2 ; CaCl2, 2- 5 ; glucose, 11 1. The iso¬

MgS04.7H20, 1

·

·

metric tension

was recorded using GRASS FT03 tension-displacement transducers and displayed on a GRASS polygraph. A resting tension of 0-5 g was applied to each uterine horn, and they were allowed to equilibrate for at least 30 min. All horns from the different phases of the oestrous cycle displayed spon¬

taneous contractions when set up. The effect of

Materials

The

The use of animals for this study was approved by the Animal Ethics Committee of the University of Melbourne; the guidelines for animal experimenta¬ tion set by the National Health and Medical Research Council of Australia were followed.

materials

were

used:

oestradiol-17ß,

progesterone, 3-aminopropyltriethoxysilane, Harris'

haematoxylin, fish gelatin

and Tween 20 (Sigma Chemical Co., St Louis, MO, U.S.A.) ; méthylène blue

(Bio-Rad, Richmond, CA, U.S.A.); [Asn Leu11]PTHrP(7 34) (Peninsula Laboratories Inc., Belmont, CA, U.S.A.); Tissue Tek OCT embedding medium (Miles Scientific, Mulgrave, Victoria, Australia); pGEM3 (Promesa Corp., Madison, WI, U.S.A.); pAM19 and [a- 5S]UTP (Amersham International pic, Amersham, Bucks, U.K.); DE-81 filters (What¬ man International, Singapore) ; RNase A (Boehringer Mannheim GmbH, Mannheim, Germany); K-5 liquid emulsion (Ilford, Notting Hill, Victoria, ,

Australia); single-well Lab-Tek tissue-culture chamber slides (Nunc Inc., Roskilde, Denmark) ; goat anti-rabbit immunoglobulins and goat peroxidase-

antiperoxidase (PAP) complex (Dako Corp., Carpintería, CA, U.S.A.). PTHrP peptides were diluted in 0-1% (w/v) bovine serum albumin and 10 mmol acetic acid/1 and stored at -20 °C in 10 pg/ 10 µ aliquots. Primary antiserum 8094 was raised in sheep against PTHrP(50-69) as described previously (Danks, Ebeling, Hayman et al 1990). Human PTHrP peptides were synthesized as previ¬ ously described (Kemp, Moseley, Rodda et al 1987).

PTHrP(l-34) on both spontaneous contractions and KC1 (30 mmol/l)-induced contractions was exam¬ ined.

Cumulative

concentration-response

(0-3 100 nmol PTHrP(l 34)/l)

curves

constructed for both forms of contraction. The results obtained with rats in the pro-oestrous and oestrous periods were pooled and compared with the pooled results obtained from rats in the metoestrous and dioestrous were

periods. Non-cycling rats Immature (non-cycling) female Sprague-Dawley rats (60-90 g) were treated for 2 successive days with oestradiol-17ß (200 pg/kg per day, s.c.) and then for 1 day with either progesterone (3 mg/rat per day, s.c; E(2)P) or vehicle (peanut oil, s.c. ; E(2)V) ; rats were also treated for 1 day with oestradiol-17ß (200 pg/kg per day, s.c; E(l)) or untreated. Rats were killed by cervical dislocation, on day 4 for E(2)P- and E(2)Vtreated rats and day 2 for E(l)-treated rats. The uter¬

ine horns were removed and mounted as described above. A resting tension of 1 g was applied to each horn compared with the 0- 5 g tension applied to horns taken from cycling rats since horns from immature rats under 1 g tension displayed better spontaneous contractions on trace. All horns from different treat¬ ments exhibited spontaneous contractions within the

time of equilibration. The effect of PTHrP(l-34) on spontaneous contractions and electrically evoked con¬ tractions was examined. The electrically evoked con¬ tractions were produced by electrical field stimulation delivered from platinum electrodes incorporated in tissue holders. Trains of pulses (2 ms, 20 Hz and 50 V) were applied for 5 s every 100 s using a GRASS S88 stimulator. Cumulative concentration-response curves for PTHrP(l 34) (0-03-30 nmol/1) were con¬ structed for both forms of contraction. KC1 stimula¬ tion was not adopted for uterine horns taken from non-cycling rats as electrical stimulation was easy to differentiate from spontaneous contractions on trace. The ability of the synthetic PTH and PTHrP recep¬

antagonist [Asn10, Leu"]-PTHrP(7-34) (Nutt, Caulfield, Levy et al 1990) to inhibit the effect of PTHrP( 1 -34) on both forms of contraction of uterine horns from E(2)V-treated rats was also examined. Concentration-response curves for PTHrP(l 34) (0-1-30 nmol/1) were constructed from experiments tor

in which the organ bath was washed after every concentration trial. [Asn10, Leu"]-PTHrP(7-34) (10 nmol/1) was added 2 min prior to each PTHrP

(1-34) addition.

Tissue collection and

preparation E(2)V-treated and untreated uteri from non-cycling

rats were removed and cut into 2-3 mm slices. Adja¬ cent slices were prepared for frozen or paraffin wax-

embedded sections. For frozen sections, rat uteri without prior fixation were mounted in Tissue Tek OCT embedding medium and frozen on the surface of a dry ice/hexane bath. Cryosections (5 pm) were cut, thaw-mounted onto glass slides coated with 2% (v/v) 3-aminopropyltriethoxysilane (AES), fixed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS), rinsed in PBS, dehydrated through graded alcohols, air dried and stored in the presence of a desiccant at 4 °C for 1 week. Tissue for paraffin wax sections was fixed in 10% (v/v) neutral-buffered formalin for 7-8 h, embedded in paraffin wax, sec¬ tioned at 5 pm and mounted on slides coated with 2% AES. In-situ hybridization was performed on serial frozen sections while immunohistochemistry was car¬ ried out on serial paraffin wax sections. Serial sections were also stained with haematoxylin and eosin for routine histological examination. In-situ

hybridization

Probe preparation Two DNA templates were used for probe synthesis. The first was a 407 bp Aval fragment of the plasmid pBRF61, representing —128 to +279 bp from the init¬ iating ATG of the coding region for human PTHrP.

fragment was subcloned into the Aval site of pGEM3, thus generating the plasmid pSMR158. The second was a 423 bp fragment, generated by polymer¬ ase chain amplification of the region +101 to +524 bp from the initiating ATG of PTHrP, with EcoRI clon¬ ing sites at the termini. This fragment was subcloned into the EcoRI site of pAM 19 thus constructing the recombinant plasmid pSMR194. Both DNA tem¬ plates were linearized with BamHI and transcribed with T7 RNA polymerase. The first and second DNA templates were used to generate the antisense (i.e. complementary to PTHrP mRNA) and sense (i.e. homologous to PTHrP mRNA) strands respectively. 35S-Labelled single-stranded RNA probes were syn¬ thesized using [a-35S]UTP (800 Ci/mmol) in T7 RNA polymerase transcription reactions. Transcription conditions were as described by Krieg & Melton (1987) with modifications suggested by the supplier. The specificity of the transcripts was confirmed by dot blot hybridization to either complementary or homologous single-stranded Ml3 DNA templates, containing human PTHrP cDNA sequences. The probe yield was measured by differential adsorption of the nucleic acid products onto the posi¬ tively charged surface of DE-81 filters ; transcript size was assessed by formaldehyde-agarose gel electropho¬ resis (Sambrook, Fritsch & Maniatis, 1989). The pro¬ bes were stored at 70 °C in 10 mmol Tris/1; 1 mmol EDTA/1, pH 8; 10 mmol dithiothrietol (DTT)/1 and used within 3 days following purification. This

—

Tissue localization Frozen sections were pretreated and prepared for hy¬ bridization as previously described by Zeller & Rogers (1989), with minor modifications. The sections were digested with 0-1 pg proteinase K/ml in 50 mmol Tris-HCl/1 (pH7-65) and 5 mmol EDTA/1. The slides were prehydridized for 16 h at 37 °C in hybrid¬ ization buffer consisting of 50% (v/v) deionized for¬ mamide, 0-6 mol NaCl/1, 50 mmol Na2HP04/l, 5 mmol EDTA/1, 1% skim milk powder, 100 µg denatured-salmon sperm DNA/ml, 5 Denhardt's solution, 0-5% (w/v) sodium dodeccyl sulphate and 10 mmol DTT/1. Following prehybridization, the slides were dehydrated through graded alcohols, air dried and used immediately for hybridization. The riboprobes were heated to 80 °C and diluted in hybridization buffer at 1-5 x 107c.p.m./ml. Probe (20 µ ) was applied to each slide and covered with 22 x 22 mm coverslips. Proportionally more probe was added to larger sections. Duplicate slides were hybridized with either the antisense (specific hybrid¬ ization to PTHrP mRNA) or sense probe (negative control). The slides were placed in a humid chamber and incubated at 50 °C for 16-18 h.

Following hybridization, the coverslips were dis¬ lodged by flotation in 4 x SSC prewarmed to 50 °C (1 x SSC is 0-15 mol NaCl/1, 0015 mol sodium citr¬ ate/1 ; pH 7). The slides were washed for 1 ·5 h at 50 °C in three changes of hybridization buffer (omitting probe, DNA, Denhardt's solution, skim milk powder and DTT). They were then incubated with 50 pg RNAse A/ml in 0-5 mol NaCl/1, 10 mmol Tris-HCl/1 (pH 7-6), 1 mmol EDTA/1 for 1 h at 37 °C. The slides

were washed in 2 SSC and 1 x SSC at 65 °C and 0-1 x SSC at room temperature prior to dehydration in graded alcohols containing 0-6 mol NaCl/1. For autoradiography, the slides were dipped in K-5 liquid emulsion diluted 1:1 (v/v) in 1 7% (v/v) glycerol in water at 45 °C, dried for 18 h at room temperature and humidity, dried for 2-3 h at room temperature in the presence of a desiccant and then exposed, desiccated at 4 °C for 21 days. The slides were developed using Ilford developer and fixed with Ilford Hypam fixer. The sections were lightly stained with Giemsa and examined by bright-light and darkfield microscopy to visualize silver grains at low ·

magnification.

As controls, COLO 16 cells,

a

human

epidermal

squamous carcinoma cell line that constitutively expresses high levels of PTHrP, and UMR 106 cells, a rat osteogenic sarcoma cell line that does not express

PTHrP, were co-cultured single-well LabTek tissue culture-chamber slides. After 36-48 h of culture (when approximately 80% confluent), the cells were fixed with 4% (v/v) paraformaldehyde in PBS for 30 min at room temperature. Fixation was stopped with 3 PBS. The slides were then rinsed in 1 PBS, dehydrated through graded alcohols and air dried. The slides were pretreated, prehybridized and hybrid¬ ized in parallel with the frozen sections. on

immunohistochemistry Peroxidase-antiperoxidase staining for PTHrP was carried out as described previously by Danks et al. (1989), and the PTHrP(50-69) antisera, raised in sheep against PTHrP(50-69), has previously been characterized (Danks et al. 1990). Positive and nega¬ PTHrP

tive controls were included in all experiments. Positive controls consisted of sections of normal skin, in which the spinous keratinocyte layer contained the PTHrP antigen (Danks et al. 1989). Negative method controls and tests for antibody specificity included the substi¬ tution of non-immune goat serum for the primary antiserum and unrelated immune sheep serum, and overnight preabsorption of human PTHrP(50-69) antiserum with PTHrP(50-69) (500 pg/ml). If the positive and negative controls did not give the expected result the experiment was disregarded. The intensity of immunoperoxidase staining was evaluated

with increasing dilutions of the primary antibody. The sections were assessed by three independent observers.

Analysis of data Relaxation of spontaneous, KC1- or electrically evoked contractions was expressed as a percentage of the maximal response obtained in the absence of PTHrP. Concentration response (% relaxation) curves were constructed for PTHrP(l-34) by pooling the data for each pair of uterine horns of the individ¬ ual experiments from a particular treatment or phase in the oestrous cycle. The molar concentrations pro¬ ducing 50% of the maximal response (EC50) were cal¬ culated, together with standard errors by linear regression analysis. Comparison of the potency of PTHrP in different treatment groups in non-cycling rats, in different stages of the oestrous cycle of cycling rats and in the presence and absence of antagonist were made by means of Student's unpaired f-test. P