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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 4 1711-1718
Copyright © 2000 by The Endocrine Society

Original Studies

Localization and Signaling of the Prolactin Receptor in the Uterus of the Common Marmoset Monkey

A. Dalrymple and H. N. Jabbour

Medical Research Council Reproductive Biology Unit, Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9ET

Address all correspondence and requests for reprints to: Dr. H. N. Jabbour, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: h.jabbour{at}ed-rbu.mrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study investigated the expression and signaling pathway of PRL and its receptor in the non-pregnant uterus of the common marmoset monkey. Immunohistochemistry localized PRL expression to the stromal compartment of the endometrium. Expression was minimal during the proliferative phase and was up-regulated during the mid to late secretory phase of the ovulatory cycle. In situ hybridization and immunohistochemistry localized expression of the PRL receptor to the glandular epithelium of the endometrium. Similar to that of PRL, PRL receptor expression was minimal during the proliferative phase and was dramatically up-regulated during the secretory phase. The temporal pattern of PRL receptor gene expression in the marmoset uterus across the cycle was further confirmed by ribonuclease protection assay. The roles of Janus kinase-2 (JAK2) and signal transducer and activator of transcription-1 (STAT1) in the intracellular signaling pathway of PRL were also assessed in the mid to late secretory phase. JAK2/STAT1 proteins were localized in the glandular epithelial compartment, and both proteins were temporally phosphorylated in response to PRL. Finally, the pattern of expression of the interferon regulatory factor-1 (IRF-1) gene and the effect of PRL on transcription of IRF-1 were investigated during the mid to late secretory phase. IRF-1 expression in the marmoset uterus was encoded by a protein of 48 kDa and was localized to the glandular epithelial compartment, as was observed for the PRL receptor and JAK2/STAT1 proteins. Moreover, incubation of mid to late secretory uterine tissue with PRL for 1 and 3 h resulted in 0.4 ± 0.2- and 2.4 ± 0.5-fold (P < 0.05) inductions of the IRF-1 gene, respectively. These studies confirm the expression of both PRL and its receptor in the uterus of the marmoset monkey. Expression of both genes is up-regulated during the mid to late secretory phase of the ovulatory cycle. PRL function in the marmoset uterus is linked to the JAK/STAT signaling pathway, leading to the regulation of expression of PRL-responsive genes such as IRF-1. The site of expression of PRL, PRL receptors, and IRF-1 in the marmoset uterus suggest that PRL may influence glandular epithelial function and direct gene transcription in these cells in a paracrine fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRL IS SYNTHESIZED by human endometrial stromal cells during the mid to late secretory phase of the menstrual cycle. PRL synthesis commences with the first signs of decidualization and subsequently decreases at menses (1). In the event of pregnancy, PRL synthesis increases after implantation, reaches a peak at 20–25 weeks of pregnancy, and decreases toward term (2, 3). Decidual PRL has an identical amino acid sequence as pituitary PRL (4), which confers similar chemical, immunological, and biological characteristics (5). However, decidual PRL uses an alternative promoter located about 6 kb upstream of the pituitary PRL start site (6). Alternative promoter sites confer cell-specific regulation of transcription of the PRL gene in the pituitary and uterus (6, 7).

The exact function of PRL in the non-pregnant and pregnant uterus has not been clarified. In the non-pregnant human uterus, PRL receptors have been localized to the stromal and glandular compartments of the mid to late secretory phase endometrium (8, 9). In the event of pregnancy, PRL receptor expression is maintained and is localized to the decidua, chorionic cytotrophoblast, placental trophoblast, and the amniotic epithelium (10). The coordinated temporal pattern of expression of both PRL and its receptor in the non-pregnant and pregnant uterus suggest that PRL may be involved in the establishment and maintenance of pregnancy. PRL function in the human uterus is linked in part to phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) proteins. JAK/STAT proteins have been co-localized with the PRL receptor in the glandular compartment of the endometrium and are temporally phosphorylated in the glandular epithelial cells after stimulation with PRL (8). These data have prompted the suggestion that PRL may influence epithelial cell function/differentiation and direct gene transcription in the glandular compartment of the human endometrium. Genes up-regulated within endometrial glands are unknown, but may function to promote implantation and/or trophoblast proliferation (8). The importance of PRL in the implantation process has been demonstrated in female mice with a homozygous null mutation in the PRL receptor gene. In this model, female mice are sterile, and their uteri are refractory to implantation (11).

The following study was designed to establish whether PRL is associated with normal uterine function in a non-human primate species, the common marmoset monkey. The pattern of expression of the PRL and PRL receptor genes was investigated in the uterus of the non-pregnant marmoset monkey. In addition, PRL intracellular signaling in the non-pregnant uterus was analyzed by investigating the phosphorylation of JAK2 and STAT1 proteins after short term culture of marmoset uterine tissue with PRL. The effect of exogenous PRL on the transcription of interferon regulatory factor-1 (IRF-1) was assessed using uterine tissues collected during the mid to late secretory phase of the ovulatory cycle. The demonstration of regulation of IRF-1 expression by PRL in the uterus may provide some insight into the possible target genes and diverse functions that PRL regulates in the endometrium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue collection

Animals used for these studies were adult female marmoset monkeys (Callithrix jacchus) housed at the Medical Research Council Reproductive Biology Unit Primate Center. All procedures were in agreement with the Animals (Scientific Procedures) Act of 1986. The ovulatory cycles of the animals were monitored by twice weekly measurements of plasma progesterone concentration (12) for a number of cycles before use. The proliferative phase was defined as the period when the plasma progesterone concentration was 12 nmol/L or less. Day 1 of the secretory phase was when the progesterone concentration rose above 30 nmol/L and was followed by a sustained increase. The secretory phase has three time points: early secretory phase (days 2–4), midsecretory phase (days 8–10), and late secretory phase (days 14–20). Tissues used in this study were from animals within the proliferative phase or the mid to late secretory phase (days 10–16 of the secretory phase; day 0 being the estimated day of ovulation). At the required stage of the cycle, animals were killed, and uteri were promptly removed. Uterine samples for in situ hybridization (proliferative phase, n = 4; mid to late secretory phase, n = 4) were submerged in Tissue-Tek (Miles, Inc., Slough, UK), snap-frozen in isopentane precooled with dry ice, and stored at -70 C until required. Uterine tissues collected for ribonucleic acid (RNA; proliferative phase, n = 3; mid to late secretory phase, n = 3) or protein (mid to late secretory phase, n = 4) extraction were snap-frozen on dry ice and stored at -70 C until required. For immunohistochemistry, uterine samples (proliferative phase, n = 4; early secretory phase, n = 4; mid to late secretory phase, n = 4) were fixed by immersion in Bouin’s reagent, followed by processing and paraffin embedding. For the in vitro culture experiments, mid to late secretory phase uterine samples (total n = 6) were finely dissected, washed in phosphate-buffered saline (PBS; Sigma, Dorset, UK), and incubated in the required volume of RPMI 1640 medium (Sigma) before stimulation as detailed below.

In situ hybridization

Complementary RNA (cRNA) probes for in situ hybridization were produced with the Riboprobe In Vitro Transcription Systems Kit (Promega Corp., Southampton, UK) as described previously (8), using 1 µg sense or antisense linearized plasmid containing the full-length marmoset PRL receptor complementary DNA (cDNA) (13), 50 µCi [{alpha}-33P]UTP (NEN Life Science Products, Hounslow, UK), and 20 U of the appropriate RNA polymerase (T7 or SP6, Promega Corp.). cRNA probes were then hydrolyzed in 100 mmol/L NaHCO3 (pH 10.5) at 65 C for 10 min, precipitated and resuspended in diethylpyrocarbonate-treated H2O, and the activities of two 1-µL aliquots were analyzed by liquid scintillation spectroscopy. The average of the samples was determined, and the volume of the radiolabeled probes required to give 1 x 106 cpm was calculated. Uterine cryostat sections (5 µm) were thaw-mounted onto ribonuclease (RNase)-free 3-aminopropyltriethooxy saline (Tespa, BDH, Poole, UK)-coated slides. Tissue sections were fixed for 5 min in 4% (vol/vol) formaldehyde in PBS, acetylated in 0.25% acetic anhydride, subsequently dehydrated with increasing concentrations of ethanol (60%, 80%, and 95%), and air-dried.

Hybridization of sense or antisense cRNA probes to uterine tissue was performed overnight at 55 C in hybridization buffer (9%, wt/vol) dextran sulfate, 45% deionized formamide, 540 mmol/L NaCl, 54 mmol/L sodium citrate, 44.6 mmol/L dithiothreitol, 0.9 x Denhardt’s solution, and 500 µg/mL transfer RNA) with a probe concentration of 1 x 106 cpm. Surplus and nonspecifically bound cRNA probe were subsequently removed from the tissue by washing in 4 x SSC (single strength SSC contains 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.0) followed by 2 x SSC for 15 min at room temperature. The tissue was then incubated at 37 C for 30 min in 2 x SSC containing 10 µg/mL RNase A, followed by washing with 4 x SSC, 2 x SSC, and 0.1 x SSC for 30 min each at room temperature. Tissue sections were dehydrated with increasing concentrations of ethanol (60%, 80%, and 95%), air-dried, and dipped in NTB3 emulsion (Eastman Kodak Co., Cambridge, UK) at 45 C in the dark. Emulsion-coated slides were stored in a humidified light-proof box for 1 h before storage at 4 C with silica gel for 4 weeks. After this period, slides were developed in the dark using D19 developer (Eastman Kodak Co.) and fixed with Polymax fixer (Eastman Kodak Co.) at 15 C.

RNase protection assay

RNA was extracted from uterine tissue using Tri-Reagent (Sigma). Antisense cRNA probes were produced as previously described (8). The PRL receptor cRNA probe was generated from a 310-bp cDNA fragment (515–833 bp) of the marmoset PRL receptor cDNA. To control for variability in RNA loading, an 18S antisense cRNA probe was prepared from 18S cDNA (Ambion, Inc., AMS Biotechnology, Oxon, UK) as previously described (8). The activities of two 1-µL aliquots of each cRNA probe were determined by liquid scintillation spectroscopy. The averages obtained were used to determined the volume of the radiolabeled probe required to give 2 x 105 cpm for the PRL receptor cRNA probe and 2 x 104 cpm for the 18S cRNA probe.

The RNase protection assay (RPA) was performed using the RPA III kit (Ambion, Inc.). Briefly, total RNA (50 µg) from proliferative phase uteri (n = 3), mid to late secretory phase uteri (n = 3), and yeast RNA (n = 2) were precipitated with both cRNA probes, resuspended in hybridization buffer, heated to 95 C for 5 min, and incubated overnight at 42 C. The next day, nonhybridized, single stranded RNA was digested in all uterine RNA samples and one of the yeast controls by the addition of 5 U RNase A and 200 U RNase T1 in RNase buffer. RNase buffer alone was added to the other yeast sample. All samples were incubated at 37 C for 30 min. Yeast RNA was used as a reaction control in the presence or absence of RNase digestion to establish the specificity of the hybridization reaction and the size of the unprotected RNA fragment. Samples were then precipitated, resuspended in loading buffer, heated to 95 C for 4 min, and loaded onto a 5% (vol/vol) denaturing polyacrylamide gel. Subsequently, the gel was dried at 80 C and exposed to an autoradiographic film (Eastman Kodak Co.) or quantified using a phosphorimager (Storm, Molecular Dynamics, Inc., Sunnyvale, CA).

Immunohistochemistry

Uterine sections (5 µm) were floated onto glass slides coated with a 2% solution of 3-aminopropyltriethoxy silane in acetone and dried overnight at 50 C. Sections were dewaxed with Histoclear (National Diagnostics, Hull, UK), rehydrated with decreasing concentrations of ethanol (100%, 96%, and 70%) followed by washes (two, 5 min each) in Tris-buffered saline (50 mmol/L Tris-HCl, pH 7.4, and 0.85% NaCl). The nitro blue tetrazolium detection method (14) was used with the PRL, PRL receptor, JAK2, and STAT1 antibodies and the diaminobenzidine method with the IRF-1 antibody (8). The antibodies used were rabbit polyclonal anti-human PRL (1:150 dilution; A0569, DAKO Corp., High Wycombe, UK), anti-rat PRL receptor (R120; 1:25 dilution; donated by Dr. P. M. Ingleton, University of Sheffield, Sheffield, UK), anti-mouse JAK2 (1:25 dilution; sc-278, Autogen Bioclear, Wiltshire, UK), anti-human STAT1 (1:50 dilution; sc-346, Autogen Bioclear), and anti-human IRF-1 (1:50 dilution; sc-497, Autogen Bioclear). Control sections were incubated with non-immune rabbit serum (DAKO Corp.) or non-immune rabbit IgG (Autogen Bioclear) at the same protein or IgG concentration as the primary antibody. Color reaction was developed by incubation in Tris-MgCl2 buffer [100 mmol/L Tris (pH 9.5), 100 mmol/L NaCl, and 50 mmol/L MgCl2] containing the substrates for alkaline phosphatase [337.5 µg/mL nitro blue tetrazolium, 175 µg/mL X-phosphate (5-bromo-4-chloro-3-indolylphosphate), and 1 mmol/L levamisol] or in 50 mmol/L Tris-HCl (pH 7.4) containing 0.05% (wt/vol) diaminobenzidine and 0.01% hydrogen peroxide.

In vitro tissue culture and Western blotting for JAK2 and STAT1

Uterine tissues collected during the mid to late secretory phase (n = 3) were resuspended in 5 mL RPMI 1640 medium (Sigma) and then split into five equal aliquots. One aliquot was snap-frozen in dry ice at the beginning of the experiment and used as a control. The other four aliquots were cultured with 400 ng human PRL (hPRL SIAFP-B2, donated by NIDDK, NIH) at 37 C for 5, 10, 15, and 20 min and then stored at -70 C. Proteins were extracted as described previously (8). Proteins (50 µg) from each sample were immunoprecipitated overnight at 4 C with 5 µg mouse monoclonal anti-phosphotyrosine antibody (Affiniti, Exeter, UK) and precipitation buffer [1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.4), 1 mmol/L ethylenediamine tetraacetate, 0.2 mmol/L sodium vanadate, and 0.2 mmol/L phenylmethylsulfonylfluoride]. Immunoprecipitated phosphorylated proteins were isolated by incubation with 50 µL Dynabeads M-450 rat anti-mouse IgG2 (DynAl, Wirral, UK) and were separated using a DynAl MPC magnet. Proteins were then washed three times in PBS containing 0.1% BSA, resuspended in 20 µL sample buffer [125 mmol/L Tris-HCl (pH 6.8), 4% SDS, 2.5% dithiothreitol, 20% glycerol, and 0.05% bromophenol blue], boiled for 5 min at 95 C, and run on a 7.5% polyacrylamide resolving gel. Proteins were transferred onto a polyvinylidene difluoride membrane (PVDF; Millipore Corp., Watford, UK) and subjected to immunoblot analysis. Membranes were blocked overnight at 4 C in blocking buffer [5% (vol/vol) normal donkey serum diluted in washing buffer (50 mol/L Tris-HCl, 150 mmol/L NaCl, and 0.05% (vol/vol) Tween-20)]. Subsequently, membranes were incubated for 2 h at room temperature with JAK2 (1:50) or STAT1 (1:20) antibodies (as used for immunohistochemistry) diluted in blocking buffer. Membranes were subsequently incubated for 1 h with donkey anti-rabbit secondary IgG antibody conjugated to horseradish peroxidase (1:2000; Amersham Pharmacia Biotech, Aylesbury, UK). Proteins were revealed by chemiluminescence (ECL kit, Amersham Pharmacia Biotech) following the manufacturer’s instructions.

Western blotting for IRF-1

Western blotting was conducted to determine the expression of IRF-1 protein in the marmoset uterus. Proteins were extracted as described previously (8) from uterine tissue collected during the mid to late secretory phase (n = 4). Mid to late secretory phase animals only were used for the IRF-1 studies, as the data indicated that PRL receptors were only expressed during the mid to late secretory phase. A total of 50 µg of each protein sample were immunoprecipitated with anti-phosphotyrosine antibody (as before). Proteins were loaded on a 7.5% polyacrylamide resolving gel and transferred to a PVDF membrane. The membrane was immunoblotted using a rabbit polyclonal anti-human IRF-1 antibody (1:75 dilution, as used for immunohistochemistry).

In vitro tissue culture and induction of IRF-1 expression by PRL

Uterine samples collected from mid to late secretory phase animals (n = 3) were finely dissected, resuspended in 5 mL RPMI 1640 medium (Sigma), and split into five equal aliquots. One aliquot (time zero) was immediately snap-frozen at -70 C in dry ice and used as a control. The other four aliquots were incubated in RPMI 1640 medium (Sigma) containing cycloheximide (10 µg/mL; Sigma) with or without human PRL (400 ng/mL) for 1 or 3 h at 37 C. After the specified incubation period, the samples were snap-frozen in dry ice and stored at -70 C until required. RNA was extracted using Tri-Reagent (Sigma) and was used for RPA to assess the up-regulation of IRF-1 expression by PRL. Antisense cRNA probes were produced as detailed previously (8), using a HindIII-linearized plasmid containing a 260-bp cDNA fragment (829–1089 bp) of the human IRF-1 cDNA and 18S cDNA. Total RNA (4 µg) from each sample and yeast RNA was hybridized with 2 x 105 cpm IRF-1 cRNA probe and 2 x 104 cpm 18S control probe. The relevant amounts of IRF-1 and 18S, expressed as relative density units, were quantified using a phosphorimager. The 18S ribosomal standard was used to correct loading variations in RNA between treatments. The background IRF-1 expression level was calculated from time zero; this value was subtracted from all treated samples. The fold induction of IRF-1 expression, at both time points, was calculated by dividing the value for the PRL-treated sample by the value for the untreated sample. The data were analyzed by one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The temporal pattern of expression of the PRL and PRL receptor genes were investigated in the marmoset uterus across the ovulatory cycle using an array of techniques. PRL expression, as assessed by immunohistochemistry, was minimal during the proliferative (Fig. 1AGo) and early secretory phases (data not shown) and was up-regulated during the mid to late secretory phase of the cycle (Fig. 1BGo). Expression of the PRL gene was localized to the stromal compartment of the endometrium (Fig. 1BGo). No PRL immunostaining was detected in the glandular compartment of the uterus. PRL receptor expression was assessed by RPA (Fig. 2Go) in situ hybridization (Fig. 1Go, D–I) and immunohistochemistry (Fig. 1Go, J–L) using tissue collected across the ovulatory cycle. PRL receptor expression was minimal during the proliferative and early secretory (data not shown) phases and was up-regulated during the mid to late secretory phase of the ovulatory cycle. However, in contrast to PRL, PRL receptor expression was localized predominantly to the glandular epithelial cells (Fig. 1Go, G and K, in situ hybridization and immunohistochemistry, respectively).



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  		Figure 1. Expression of the PRL and PRL receptor genes in the marmoset endometrium. Minimal PRL immunostaining is observed during the proliferative phase (A), whereas intense immunostaining is apparent in the stromal compartment during the mid to late secretory phase (B). C, Mid to late secretory phase endometrium incubated with non-immune sera (negative control). PRL receptor expression was investigated by in situ hybridization (D–I) and immunohistochemistry (J–L). D and E demonstrate that the PRL receptor is not expressed during the proliferative phase (darkfield and lightfield, respectively). F, A proliferative phase section incubated with the sense probe (negative control). G and H demonstrate PRL receptor expression in the glandular epithelial cells during the mid to late secretory phase (darkfield and lightfield, respectively). I, Mid to late secretory phase section incubated with the sense probe (negative control). Limited PRL receptor immunostaining was observed during the proliferative phase (J), whereas intense immunostaining was evident within the glandular epithelium during the mid to late secretory phase (K). L, Mid to late secretory endometrium incubated with non-immune sera (negative control). Scale bars, 50 µm.

 


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  		Figure 2. RPA conducted using 50 µg total RNA collected during the ovulatory cycle and a 310-bp homologous PRL receptor cRNA probe. The integrity and the relative amount of total RNA were determined using a ribosomal 18S cRNA probe. P, Proliferative phase (n = 3); S, mid to late secretory phase (S1, S2, and S3 are days 11, 13, and 16 of the secretory phase, respectively).

 
The role of the JAK/STAT signal transduction proteins in the intracellular signaling pathway of PRL was also assessed in the marmoset uterus. Only mid to late secretory phase uterine tissue was used in these experiments, as this was the phase in which PRL and PRL receptors were expressed in the uterus. Both JAK2 and STAT1 were localized by immunohistochemistry (Fig. 3Go, A and C, respectively) to the glandular epithelial compartment, as was observed for the PRL receptor. In addition, the association of the JAK2/STAT1 proteins with PRL intracellular signaling was investigated after short term culture of marmoset uterine tissue with PRL. Stimulation of mid to late secretory phase uterine tissue with PRL resulted in rapid phosphorylation of JAK2. A reactive band of approximately 125 kDa corresponding to JAK2 was detected in samples after stimulation with PRL for 10 min (Fig. 4AGo). Similarly, STAT1 phosphorylation was observed after stimulation of mid to late secretory uterine tissue with PRL. Reactive bands of approximately 84/91 kDa corresponding to the two variant forms of STAT1 were detected within 15 min of stimulation with PRL (Fig. 4BGo).



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  		Figure 3. Immunolocalization of JAK2 (A), STAT1 (C), and IRF-1 (E) in the mid to late secretory phase endometrium. All proteins are localized to the glandular epithelium, as was observed for the PRL receptor. B, D, and F are sections incubated with non-immune rabbit IgG (negative controls) for JAK2, STAT1, and IRF-1 respectively. Scale bar, 50 µm.

 


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  		Figure 4. Tyrosine phosphorylation of JAK2 and STAT1 by PRL in mid to late secretory phase uterine tissue. Uterine tissue was incubated with 400 ng/mL human PRL for 0, 5, 10, 15, and 20 min. Proteins were immunoprecipitated with anti-phosphotyrosine antibody and subsequently immunoblotted with JAK2 and STAT1 antibodies. JAK2 phosphorylation was detected after 10 min (A), whereas STAT1 phosphorylation was detected after 15 min (B) of stimulation with human PRL.

 
Western blot analysis initially confirmed expression of IRF-1 in the mid to late secretory phase marmoset uterus. In the marmoset uterus, IRF-1 is encoded by a protein of approximately 48 kDa (Fig. 5Go). Moreover, IRF-1 expression in the secretory uterus was localized to the glandular epithelial cells (Fig. 3EGo), as was observed for the PRL receptor and its signaling proteins JAK2/STAT1 (Fig. 3Go, A and C). To investigate the effect of PRL on expression of the IRF-1 gene, marmoset uterine tissue was incubated in the presence or absence of 400 ng/mL PRL for 1 or 3 h. IRF-1 expression was similar after 1-h culture in the presence or absence of PRL (24.7 ± 13.9 vs. 44.7 ± 28.3 arbitrary units for uterine tissue cultured in the presence or absence of PRL, respectively). However, culture of uterine tissue with PRL for 3 h resulted in an increase in IRF-1 expression (152.7 ± 60.1 vs. 58.0 ± 10.3 arbitrary units for uterine tissue incubated in the presence or absence of PRL, respectively; Fig. 6AGo). The fold induction of IRF-1 expression was significantly increased (P < 0.05) after stimulation with PRL for 3 h (Fig. 6BGo; 0.4 ± 0.2- vs. 2.4 ± 0.5-fold induction after stimulation with PRL for 1 and 3 h, respectively; mean ± SEM).



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  		Figure 5. Western blot analysis conducted using 50 µg protein isolated from mid to late secretory phase marmoset uteri (n = 4; S1, S2, S3, and S4 are days 10, 11, 14, and 16 of the ovulatory cycle, respectively). The proteins were immunoprecipitated with an anti-phosphotyrosine antibody, loaded onto a 7.5% polyacrylamide gel, transferred to PVDF membrane, and subjected to immunoblot analysis with an IRF-1 antibody. IRF-1 in the marmoset uterus is encoded by a 48-kDa protein.

 


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  		Figure 6. The effect of PRL on IRF-1 gene expression in the mid to late secretory phase marmoset uterus. IRF-1 expression in each sample was analyzed by RPA using 4 µg total RNA and a 260-bp human IRF-1 cRNA probe. The integrity and the relative amount of total RNA were determined using a ribosomal 18S cRNA probe. A, Representative RPA from one animal (day 15 of the ovulatory cycle). Lane 1, RNA from untreated tissue (control); lanes 2 and 3, RNA from tissue incubated with or without PRL for 1 h, respectively. Lanes 4 and 5, RNA from tissue incubated with or without PRL for 3 h, respectively. B, The fold induction of IRF-1 expression was significantly increased (P < 0.05) after stimulation with PRL for 3 h (B; 0.4 ± 0.2- vs. 2.4 ± 0.5-fold induction after stimulation with PRL for 1 and 3 h, respectively; mean ± SEM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented herein demonstrate that both the PRL and PRL receptor genes are temporally expressed in the marmoset uterus during the ovulatory cycle. Expression of both genes was minimal during the proliferative phase and xincreased during the mid to late secretory phase. However, PRL and PRL receptor expression were localized to the stromal and glandular epithelial compartments, respectively. Similar results have been documented recently in the nonpregnant human (8) and baboon (15) endometrium. In the human endometrium, both PRL and PRL receptor expression are temporally up-regulated during the mid to late secretory phase of the menstrual cycle (8). In contrast, in the baboon only PRL expression is temporally expressed across the menstrual cycle; PRL receptor expression is similar during the proliferative and secretory phases (15). The temporal expression of PRL during the secretory phase by the endometrial stromal cells has been extensively studied (16) and is thought to be indirectly influenced by progesterone (17, 18). However, the factors influencing PRL receptor expression in the endometrium are not completely understood. The pattern of expression of the PRL receptor gene in the marmoset and human endometrium suggests that progesterone may also regulate PRL receptor gene expression. However, in the endometrium this is unlikely to be a direct regulatory effect, because progesterone receptors do not localize to the glandular epithelial cells during the mid to late secretory phase (19, 20), and these cells have been shown to be the major site of the PRL receptor expression. A positive effect of progesterone on PRL receptor expression has been reported previously in breast cancer cell lines (21) and primary mammary epithelial cells (22). It is also possible that PRL may direct the transcription of its own receptor in the marmoset endometrium. This is supported by the close temporal expression of both genes in the marmoset and human uterus (8). PRL has been demonstrated to induce the expression of its receptor in human endometrial stromal cells (23) and other tissues, including pituitary adenomas (24), lactating mammary gland (25, 26), and hypothalamus (27).

PRL relays its physiological response on target cells by binding to its membrane-bound receptor and subsequently activating JAK2 and different STAT proteins, including STAT1 and STAT5 (reviewed in Ref. 28). Activated STAT proteins subsequently translocate to the nucleus and up-regulate the transcription of target promoters, such as ß-casein (29), ß-lactoglobulin (30), whey acidic protein (31), {alpha}2-macroglobulin (32), and IRF-1 (33), which mediate differentiative or mitogenic effects of PRL. The effects of the different STAT proteins may vary with the target genes being analyzed. Comparative studies have illustrated that STAT5 is a positive regulator of PRL-induced transcription of the ß-casein (34) and {alpha}2-macroglobulin (35) promoters, whereas, PRL-induced transcription of the IRF-1 promoter is promoted by STAT1 and inhibited by STAT5 (34). The data obtained in this study demonstrate that PRL signaling in the marmoset uterus is linked to the JAK/STAT signaling pathway in vivo. JAK2 and STAT1 proteins co-localized with the PRL receptor in the uterine glandular epithelium. Moreover, both JAK2 and STAT1 proteins were rapidly phosphorylated after stimulation of marmoset uterine tissue with exogenous PRL. Similar in vivo phosphorylation of JAK/STAT proteins after stimulation with PRL has been reported recently in a number of tissues, including rat ovaries (36), rat mammary gland (37), and human endometrium (8). These data strongly suggest similar intracellular signaling of the PRL receptor in the marmoset and human uterus. Such a suggestion is supported further by comparative assessment of the cDNA and amino acid sequences of the marmoset and human PRL receptors. Both species share approximately 94% and 90% homologies in their cDNA and protein sequences, respectively. Moreover, all of the amino acid sequences that are essential for ligand binding and signaling are conserved between the two species (13).

In this study we also investigated the expression of IRF-1 and the effect of PRL on transcription of the IRF-1 gene in the marmoset uterus. IRF-1 is a known PRL-responsive gene that is activated by STAT1 (38). The expression of IRF-1 gene in mid to late secretory phase marmoset uterus was first confirmed by Western blotting and immunohistochemistry. IRF-1 was encoded by a 48-kDa protein, as has been observed in other cell types (39). Moreover, IRF-1 expression was colocalized with the PRL receptor and its JAK2/STAT1 signaling proteins. The role of PRL in the expression of IRF-1 in the marmoset uterus was investigated by conducting short term culture of marmoset uterine explants with exogenous PRL. The culture studies confirm that expression of IRF-1 in the mid to late secretory marmoset uterus is regulated by PRL. The sites of expression of PRL, PRL receptor, and IRF-1 in the marmoset endometrium suggest that transcription of IRF-1 is regulated by PRL in a paracrine fashion. PRL expression in the marmoset endometrium is localized to the stromal compartment, whereas co-localization of expression of the receptor and IRF-1 is restricted to the glandular epithelial compartment. The exact role of PRL in the uterus is still not clarified. The site and temporal pattern of expression of PRL suggest that it influences glandular epithelial cell function and differentiation at the time of predicted conception and trophoblast implantation. Additionally, the role and target genes of the PRL-induced IRF-1 in the uterine glandular epithelium remain to be ascertained. In other biological models, IRF-1 influences the transcription of numerous target genes that facilitate cellular growth, apoptosis, adhesion, neoplastic transformation, viral resistance, and differentiation (40, 41). It is reasonable to predict that in the glandular epithelium PRL, via IRF-1, regulates the expression of an array of genes that would regulate a multitude of functions associated with the establishment of pregnancy.

In conclusion, this study confirms the expression of PRL and its receptor in the marmoset uterus. The expression of both genes is minimal during the proliferative and early secretory phases. However, both PRL and PRL receptor gene expression are up-regulated during the mid to late secretory phase of the ovulatory cycle. The function of PRL in the marmoset uterus is partly linked to the JAK/STAT signal transduction pathway. Moreover, this study confirmed that IRF-1 is a PRL-responsive gene within the glandular epithelial compartment. The target genes for PRL function via IRF-1 in the marmoset uterus remain to be elucidated. Overall, these data strongly suggest that PRL is involved in uterine function of non-human primate species such as the marmoset monkey and outline the suitability of this species for investigating the role of PRL in the primate implantation process.


   Acknowledgments
 
We thank Mr. K. D. Morris and the staff at the Medical Research Council primate center for animal care and assistance, Dr. H. Fraser for assistance with tissue collection, Mrs. F. Pitt for performing the progesterone RIA, and Dr. J. Brooks and Ms. Sheila Boddy for advice and technical assistance. We also thank the NIH for the supply of human PRL.

Received October 13, 1999.

Revised December 1, 1999.

Accepted December 9, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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