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J Biol Chem, Vol. 274, Issue 53, 37982-37989, December 31, 1999

Rat Decidual Prolactin
IDENTIFICATION, MOLECULAR CLONING, AND CHARACTERIZATION^*

Anne Prigent-Tessier, Christian Tessier, Mitsuko Hirosawa-Takamori, Catherine Boyer, Susan Ferguson-Gottschall, and Geula Gibori

From the Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Establishment and maintenance of pregnancy require the activity of a highly specialized maternal tissue, the decidua. It is well established that the human decidua synthesizes and releases prolactin. However, in the rat, no study has been able to demonstrate the production of prolactin by the decidua. In this report, we established for the first time using Northern blot analysis and reverse transcription-polymerase chain reaction, Western blot analysis, immunocytochemistry, and enzyme-linked immunosorbent assay, that a defined cell population located in the rat antimesometrial decidua expresses prolactin mRNA, as well as synthesizes and secretes this hormone. By reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, we cloned a full-length cDNA for rat decidua prolactin, whose sequence was identical to that of pituitary prolactin. Our results also showed that pituitary prolactin appeared to down-regulate decidual prolactin levels. Under these circumstances, inhibition of pituitary prolactin secretion led to a rise in both decidual prolactin mRNA and protein expression. Moreover, addition of exogenous prolactin to primary decidual cells in culture also caused a marked decrease in decidual prolactin mRNA expression. Finally, treatment of primary decidual cells with steroid hormones or 8-bromo-cAMP revealed a differential regulation of decidual prolactin expression from that of pituitary suggesting a tissue-specific regulation of prolactin gene expression, possibly through the use of an alternative promoter in rat decidua.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In all mammalian species, pregnancy is accompanied by remarkable changes in the uterine environment that allow maternal/fetal adaptation to pregnancy without damage to the mother or rejection of the fetus. This requires profound reorganization of the different tissues forming the uterus. Growth and differentiation of the decidua is the earliest adaptation to pregnancy by the uterus (1-3). In humans, decidualization normally occurs with each menstrual cycle and the formation of the decidual tissue depends primarily on levels of progesterone and estradiol in the circulation (4). However, in other species, including the rat, decidualization requires, in addition to adequate levels of these hormones, an exogenous trigger, which may be either the contact of the blastocyst with the endometrium or artificial stimulation of the uterus (5).

Decidual cell products in maternal target tissues such as ovary and uterus control changes that are essential for successful pregnancy (1, 2, 6, 7). The action of the decidua on ovarian and uterine tissues is due, in part, to a hormone related to pituitary prolactin (PRL)¹ named decidual luteotropin (DLt; Refs. 7-19). Using decidual cell secretory products, this hormone was shown to bind to the PRL receptor on both ovarian (15, 18, 20) and decidual cells (19). Because this decidual hormone was not recognized at this time by a rat pituitary PRL antibody (anti-rPRL 1C-1, provided by the National Institutes of Health; Ref. 18), it was concluded that DLt is a different member of the PRL gene family, a group of proteins structurally related to pituitary PRL (21, 22). Expression of DLt examined by radioreceptor and immunoblot assays was found to be confined to a defined cell population located in the antimesometrial endometrium, initiated shortly after implantation and terminated after day 14 (15, 17, 18). Two PRL-related proteins were shown by two different groups to be expressed by the rat decidua and termed PRL-like protein B (PLP-B; Ref. 23) and decidual PRL-related protein (dPRP; Ref. 24). This prompted Croze and co-workers (25) and Roby et al. (24) to suggest that these proteins could be candidate genes encoding DLt. However, further investigations failed to demonstrate that these PRL-related hormones bind to the PRL-R (26, 27) and their biological actions are still not clear. DLt, identified 20 years ago (8-11), is actually the only decidual hormone that can bind to the PRL-R and maintain both luteal progesterone production by the ovary and ₂-macroglobulin by the decidua, two well established actions attributed to pituitary PRL (15, 18-20). However, the cDNA encoding this protein possessing characteristics resembling PRL has never been identified. Therefore, our project was to determine the sequence of this luteotropic factor gene expressed by the rat decidua.

In this study, we describe the molecular cloning, characterization, and regulation of rat decidual prolactin (rdPRL). We demonstrate, for the first time, that a defined cell population located in the antimesometrium site of the rat decidua produces and secretes PRL. Sequence analysis of rdPRL cDNA established its structural identity with pituitary PRL. Moreover, our results show that pituitary PRL down-regulates the expression of rdPRL both in vivo and in cell culture and that the regulation of rdPRL mRNA expression differs markedly from that of pituitary PRL.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Tissue culture medium RPMI 1640, antibiotic-antimycotic solution, non-essential amino acids, and sodium pyruvate were obtained from Mediatech (Washington, DC). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT). Progesterone, 17--estradiol, 8-bromo-cyclic AMP, CB-154 (2-Br--ergocriptine), phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, aprotinin, and all other reagent grade chemicals were purchased from Sigma. The tetramethyl rhodamine (TRITC) and horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). 4',6-Diamidino-2-phenylindole was obtained from Vector Laboratories (Burlingam, CA). Acrylamide and bisacrylamide were obtained from Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak Co. (Rochester, NY), respectively. ExTaq DNA polymerase was purchased from Panvera (Madison, WI). The oligonucleotides used as primers for sequencing and RT-PCR analysis, and the 3' RACE system were obtained from Life Technologies, Inc. GeneScreen plus nylon membranes were purchased from NEN Life Science Products and [-³²P]deoxycytidine triphosphate (dCTP) was from Amersham Pharmacia Biotech. Ovine PRL (PRL-18, 30 IU/mg), rPRL RP3 (AFP-4459B), and polyclonal anti-rPRL IC-4 antibody (AFP-1753080191281) were kindly supplied by NIDDK, National Institutes of Health (Besthesda, MD).

Animal Model-- Pseudopregnancy was induced in Holtzman Harlan Sprague-Dawley-derived female rats by mating them with vasectomized male rats on the afternoon of proestrus at the Harlan facilities (Madison, WI). The day a vaginal plug was found was considered day 1 of pseudopregnancy. Rats were housed in a controlled environment (22-24 °C) and kept under a photoperiod of 14 h of light and 10 h of darkness with free access to standard rat chow and water. Animal care and handling were conformed with National Institutes of Health guidelines for animal research. The experimental protocols were approved by the Institutional Animals Care and Use Committee.

Decidualization of uterine endometrium was induced in pseudopregnant rats under ether anesthesia by scratching the antimesometrial surface of both uterine horns with a hooked needle on day 5 of pseudopregnancy. Rats were sacrificed at different stages of pseudopregnancy (days 8-15) by overdose of ether. Decidualized uterine horns were isolated and washed thoroughly in ice-cold phosphate-buffered saline (PBS) to remove excess blood. The antimesometrial decidual tissue was separated from the mesometrial tissue, as described previously (28). Total or antimesometrial and mesometrial decidual tissues were frozen in liquid nitrogen and kept at 80 °C until nucleic acid and protein isolation.

Primary Decidual Cell Culture-- Decidual tissue, obtained from 3-5 pseudopregnant rats (day 9 of pseudopregnancy), was incubated under mild agitation in a water-jacketed cell stir (Wheaton Scientific, Millville, NJ) containing RPMI 1640 medium supplemented with collagenase (50 units/ml), dispase (2.4 units/ml), and deoxyribonuclease (200 units/ml) for 1 h at 37 °C. At the end of the incubation, dispersed cells were filtered through a nylon mesh to remove undigested tissue and centrifuged at 3000 rpm for 10 min. The cell pellet was gently resuspended in RPMI 1640 medium supplemented with 10% FBS, antibiotic-antimycotic solution (2×), non-essential amino acids (1×), sodium pyruvate (1×), and D-glucose (0.45%). Viable decidual cells, determined by the trypan blue exclusion method, were seeded in six-well plates at 1.5-2 × 10⁶ cells/well and cultivated in a humidified atmosphere containing 5% CO₂, at 37 °C. After allowing the cells to attach for 3-4 h, the unattached blood cells were removed with sterile PBS. The decidual cells were then treated for 12 h with various concentrations of PRL, progesterone, estradiol, or 8-bromo-cAMP in RPMI 1640 phenol-free medium supplemented with 1% dextran charcoal-treated FBS. At the end of the treatment, the cells were washed twice with ice-cold PBS and frozen at 80 °C until RNA extraction.

Isolation of Total RNA and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis-- Total RNA from frozen decidual tissue was purified using TRI reagent (Sigma) according to the manufacturer's instructions, whereas total RNA from primary decidual cells was isolated by one-step guanidinium-thiocyanate-phenol chloroform extraction procedure (29).

For mRNA analysis by RT-PCR, one or two micrograms of total RNA were reverse transcribed at 42 °C using the Advantage RT-PCR kit (CLONTECH, Palo Alto, CA) following manufacturer's instruction. The reaction mixture (20 µl) containing random hexamer primers (20 pmol), oligo(dT)₁₈ primer (20 pmol), reaction buffer (1×) (50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl₂), deoxynucleoside triphosphate (dNTP, 0.5 mM), RNase inhibitor (20 units), and Moloney murine leukemia virus-reverse transcriptase (200 units) was increased to 100 µl with DEPC-treated water at the end of the reverse transcription reaction. 5-10 µl of this solution was used for amplification of gene products using a touch-down PCR protocol described below. The reaction mixture containing specific oligonucleotide primers (20 pmol), [-³²P]deoxy-CTP (2 µCi of 3000 Ci/mmol), dNTP (150 µM), and ExTaq DNA polymerase (0.8 unit) was added to each tube containing the RT product. The final volume was increased to 40 µl with 1× PCR buffer (ExTaq buffer Panvera, Madison, WI). Two sets of amplification cycles were utilized. In the first five cycles, annealing temperature (4 °C + annealing temperature of the primer) for 5 min was followed by denaturation temperature (92 °C) for 1 min. In the second set of amplification, for 20-30 cycles depending on each PCR-amplified product, annealing temperature of the primer for 35 s was followed by extension temperature (71 °C) for 40 s and another denaturation temperature (92 °C) for 40 s. The conditions were such that the amplification of the products was in the exponential phase, and the assay was linear with respect to the amount of input RNA. Reaction products were electrophoresed on 8% polyacrylamide non-denaturing gel. Each PCR reaction included rat ribosomal protein L19 mRNA used as internal control. After autoradiography, data were analyzed using a Molecular Dynamics PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Sunnyvale, CA). For the detection of the rat PRL mRNA expressed by the decidua, we designed oligonucleotide primer pairs based on the sequence of the rat pituitary prolactin gene: (5'-ATGAACAGCCAGGTGTCAGCCCG-3' and 5'-CTTCATGGATTCCACCTAGTC-3', 403-base pair (bp) fragment; Ref. 30).

The other sets of primers were as follows: L19, 5'-CTGAAGGTCAAAGGGAATGTGC-3' and 5'-GGACAGAGTCTTGATGATCTCG-3' (198-bp fragment, Ref. 31); PRL receptor long form, 5'-AAAGTATCTTGTCCAGACTCGCTG-3' and 5'-AGCAGTTCTTCAGACTTGCCCTT-3' (279-bp fragment; Ref. 32).

Molecular Cloning of the rat PRL cDNA by PCR and RACE-- Total RNA from antimesometrial decidua was isolated and RT-PCR was performed using specific oligonucleotide primers based on the sequence of the rat pituitary PRL gene. A sense oligonucleotide corresponding to the first 23 nucleotides of the coding region (5'-ATGAACAGCCAGGTGTCAGCCCG-3') was combined with an antisense oligonucleotide corresponding to the 383-403 nucleotides of the coding region (5'-CTTCATGGATTCCACCTAGTC-3'). The predicted size of the PCR-amplified product was 403 bp. The PCR products from three independent experiments were electrophoresed on a 0.7% agarose gel. Only one band was detected by ethidium bromide at 403 bp. The cDNA fragments were extracted from the agarose gel, purified, and reamplified by PCR using the specific oligonucleotide primers for rat pituitary PRL containing four CUA repeats for subcloning into the CLONEAMP pAMP10 vector (Life Technologies, Inc.). DH5-competent cells were then transformed with the vector. Clones were selected for DNA sequencing from both strands by the dideoxy-chain termination method (Perkin Elmer Corp., Foster City, CA). Sequencing was performed by the DNA Sequencing Facility of the University of Chicago. Sequence analysis was carried out using MacMollyTetra computer software (Soft Gene, Berlin, Germany). To determine the complete sequence of the rdPRL coding region, RACE was done as described by Frohman et al. (33). The first strand cDNA was synthesized using an adapter primer supplied with the 3'-RACE system (Life Technologies, Inc.) and 5 µg of total RNA from antimesometrial tissue used as template. Polymerase chain reaction amplification was done using universal primer from the 3'-RACE system and a PRL gene-specific primer 1 (GSP1) (TAGCTACTCCTGAAGACA) corresponding to the 269-286 nucleotides of the coding region of the pituitary rat PRL gene. Then, the resulting product was amplified again using a PRL gene-specific primer 2 (GSP2), downstream of the GSP1 corresponding to the 313-333 nucleotides of the coding region containing four CUA repeats for subcloning (CUACUACUACUAGTTCTTTTGAACCTGATC) and the universal primer. GSP2 was designed to overlap 49 nucleotides of the sequence given by the 403-bp PCR-amplified product cloned and sequenced first. After amplification, the 3'-RACE products from two independent experiments were directly cloned into the pAMP10 vector. DH5-competent cells were then transformed with the vector. Clones were selected for DNA sequencing from both strands by the dideoxy-chain termination method. Sequence analysis was carried out using MacMollyTetra computer software.

Northern Blot Analysis-- Poly(A)⁺ mRNA (10 µg) from the decidua was isolated by the oligo(deoxythymidine)-cellulose method using an Ambion isolation kit (Austin, TX). Total RNA (3 µg) from the pituitary was purified using TRI reagent (Sigma). RNA was fractionated through a 1% agarose gel containing 0.74 M formaldehyde and transferred to a GeneScreen nylon membrane by overnight capillary blotting with 10× sodium chloride-sodium citrate buffer (SSC buffer, 1× = 150 mM sodium chloride and 15 mM sodium citrate, pH 7.0). Membranes were baked at 80 °C under vacuum for 2 h. The 403-bp PCR-amplified decidual product cloned into pAMP10 as described previously was used to synthesize the -³²P-labeled riboprobe with T7 RNA polymerase as outlined by the vendor of the in vitro transcription system (Promega, Madison, WI). RNA blot hybridization with cRNA probe was performed at 42 °C in 50% deionized formamide, 4× SET (1× SET = 150 mM sodium chloride, 20 mM Tris, pH 7.8, 1 mM EDTA), 0.2% polyvinylpyrrolidone, Ficoll, bovine serum albumin, and 8% dextran sulfate. The final oligonucleotide probe concentration was 2 × 10⁷ cpm/ml. Blots were hybridized for 24-36 h, then washed with 1× SSC (containing 0.1% sodium dodecyl sulfate or SDS) at 25 °C for 15 min, followed by 0.2× SSC (containing 0.1% SDS) at 42 °C for 15 min and finally 0.2× SSC (containing 0.1% SDS) at 55 °C for 15 min. The resultant blots were exposed to Kodak X-Omat film (Kodak) using intensifying screens at 80 °C.

Immunocytochemistry-- Primary decidual cells, obtained from adult female pseudopregnant rats (day 9 of pseudopregnancy), were grown on sterile cover glass (13 mm diameter) in four-well plastic culture dishes (Nunc), and fixed for 10 min in 4% paraformaldehyde solution in 0.1 M PBS (pH 7.2) at room temperature. After rinsing in Tris-buffered saline (TBS, pH 7.6), they were incubated first for 15 min in 10% bovine serum albumin, 0.1% Triton X-100, and 0.2% Tween 20 in TBS and thereafter overnight at 4 °C with the polyclonal anti-rPRL antibody (dilution 1:500) in TBS with 1% bovine serum albumin. Single primary antibody was detected by incubation for 3 h at room temperature with TRITC-conjugated anti-rabbit IgGs (1:200). Primary decidual cells were mounted in Vectashield medium containing a counterstain for DNA, 4',6-diamidino-2-phenylindole and observed with a Zeiss LSM 510 laser scanning confocal microscope (Oberkochen, Germany) equipped with a 40× water-immersion objective lens (NA 1.2). The specificity of immunostaining was checked by omitting the primary antibody incubation step from the procedure. All such controls were free of staining.

Western Blot Analysis-- Decidual tissues were homogenized in a lysis buffer (PBS containing 2% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin and leupeptin, 1 µg/ml pepstatin) with a Polytron homogenizer (Brinkmann Instruments, Ontario, Canada) and were centrifuged at 10,000 × g for 10 min. An aliquot of the supernatant was kept for protein measurement. Equal amounts of total proteins (30-40 µg/lane) were dissolved in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromphenol blue) and were heated at 100 °C for 10 min. Proteins were separated on 15% SDS-PAGE according to a modified Laemmli method (34), using 100 mM Tris-Tricine with 0.1% SDS, which allows reduction of the gel acrylamide concentration and improved migration of low molecular weight proteins. Proteins were electrophoretically transferred to nitrocellulose filters in carbonate buffer (35). The blots were incubated overnight at 4 °C in 5% non-fat dry milk to block unspecific binding. Blots were washed, incubated for 4 h at room temperature with the polyclonal anti-rPRL antibody (1:2000), washed again, and incubated with a horseradish peroxidase-conjugated anti-rabbit IgGs (1:6000) for 1 h. Protein-antibody complexes were visualized using the enhanced chemiluminescence Western blotting detection system (ECL, Amersham Pharmacia Biotech). The band densities were determined by scanning densitometry (Kodak).

PRL Assay-- PRL was measured in the conditioned medium of antimesometrial decidual explants (100 mg) incubated for 3, 9, and 24 h in RPMI serum-free medium using an enzyme immunometric assay designed for the quantitative measurement of rat PRL according to the instructions of manufacturer (ALPCO, Windham, NH). The sensitivity of the system was 0.6 ng/ml, and the inter- and intra-assay coefficients of variations were 7.2% and 4.4%, respectively.

Statistics-- Data were examined by one-way analysis of variance, followed by Duncan's multiple range test. A level of p < 0.05 was accepted as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nucleotide and Deduced Amino Acid Sequences of rdPRL-- Specific sense and antisense oligonucleotide primers based on the sequence of the pituitary rat PRL gene were used to amplify, by RT-PCR, a 403-bp fragment from decidua total RNA. The PCR-amplified products from three independent experiments were directly cloned into the pAMP10 vector. Four clones were selected for each experiment and sequenced on both strands using the dideoxy-chain termination method. All clones were identical and showed 100% homology to rat pituitary PRL. To determine the complete sequence of the rdPRL protein cDNA, we performed the 3' RACE using a PRL gene-specific primer (3' RACE primer) chosen to overlap 49 nucleotides of the sequence given by the 403-bp PCR-amplified decidual product. The 3'-RACE products from two independent experiments were directly cloned into the pAMP10 vector. Five of the clones obtained for each experiment were selected for DNA sequencing. The DNA sequence of all five clones were identical to rat pituitary PRL. The rdPRL cDNA sequence (817 bp) shown in Fig. 1 contains an open reading frame of 678 bp encoding 227 amino acids and a 3'-untranslated region of 139 bp that are identical to rat pituitary PRL.

View larger version (66K): [in this window] [in a new window]   Fig. 1.   Nucleotide and amino acid sequences of rdPRL. Translation begin at the initiation codon, ATG (nucleotides 1-3), and continues until the termination codon, TAA (nucleotides 679-681). The nucleotides and amino acids are numbered from the ATG; the specific primers used to determine the complete sequence of the PRL cDNA are underlined.

Characterization of rdPRL Expression-- Northern blot analysis in Fig. 2 showed that the labeled cRNA probe synthesized as described under "Experimental Procedures" specifically hybridized to decidual PRL mRNA and migrated similarly to pituitary mRNA, which is a transcript of 0.9 kilobases in size. To detect decidual PRL, it was necessary to use poly(A)⁺ mRNA, whereas total RNA was sufficient to detect pituitary PRL. However, considering the large size of the decidua in relation to that of the pituitary (~1 g versus ~50 mg), the total level of decidual PRL mRNA appears substantial.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Expression of rdPRL mRNA in the decidua. Poly(A)+ mRNA was isolated from total decidual tissue on day 12 of pseudopregnancy. Ten micrograms/lane poly(A)+ and 3 µg of total rat pituitary RNA used as positive control were electrophoresed on 1% agarose formaldehyde gel, blotted onto GeneScreen nylon membrane, and hybridized with a 32P-labeled cRNA probe synthesized from a 403-bp PCR-amplified decidual product cloned into pAMP10 as described under "Experimental Procedures." The position of the RNA marker, run on the same gel, is shown on the left panel. One transcript is detected in the decidual tissue at approximately the same level as the pituitary transcript. Results show representative autoradiograms from three independent experiments.

Developmental Expression of rdPRL in Pseudopregnant Rats-- To determine whether decidual PRL expression is temporally associated with particular stages of pseudopregnancy, we first examined rdPRL mRNA levels by RT-PCR using specific oligonucleotide primers for rat pituitary PRL. Results shown in Fig. 3A reveal that rdPRL mRNA was expressed in the decidua throughout pseudopregnancy, although with a different pattern of expression. rdPRL was expressed at low levels early in pseudopregnancy (days 8 and 9) and increased until day 12 when it became remarkably abundant. It declined thereafter and disappeared on day 15 of pseudopregnancy at a stage when extensive cell death occurs in the decidua and principally in the antimesometrial decidua. Because PRL-like activity is found only in cells located in antimesometrial decidua (7, 18, 36), we examined whether rdPRL expression is also confined to this cell population. As shown in Fig. 3B, rdPRL mRNA was expressed only in the antimesometrial decidua. No mRNA was detected in the mesometrial decidua.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Developmental expression of rdPRL mRNA in the rat decidua throughout pseudopregnancy. Total RNA was isolated from total or antimesometrial and mesometrial decidual tissues at different stages of pseudopregnancy and analyzed by RT-PCR using specific primers for rat PRL as described under "Experimental Procedures." Data were quantified by densitometry and corrected using L19 as internal standard. The mRNA levels for each day of pseudopregnancy are graphically represented in A (total decidual tissue) or B (antimesometrial (AM) and mesometrial (M) decidua) as the mean ± S.E. (n = 3) of values expressed as percentage of the maximum ratio (rdPRL/L19) considered as 100%.

Expression and Secretion of PRL by Rat Decidual Tissue-- Western blot analysis shown in Fig. 4 (A and B) revealed that the rat decidua expresses a protein immunologically similar to rat pituitary PRL (Pit PRL, provided by National Institutes of Health) with an apparent molecular mass of 23 kDa. The pattern of rdPRL protein expression (Fig. 4A) was similar to its mRNA expression and was highly expressed around day 12 of pseudopregnancy but was barely detectable in early pseudopregnancy and disappeared from the tissue on day 15. Western blot analysis shown in Fig. 4B confirmed that rdPRL protein was expressed only in the antimesometrial decidua tissue with a maximum expression on day 12 of pseudopregnancy and was not detected in the mesometrial tissue. Finally, immunocytochemistry performed on both antimesometrial and mesometrial decidual cells confirmed the expression of rdPRL only in the large antimesometrial cells (Fig. 4C).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Tissue localization and developmental expression of rdPRL immnunoreactive proteins in the rat decidua. A, total decidua was obtained on different days of pseudopregnancy. B, mesometrial and antimesometrial decidua were separated as described under "Experimental Procedures." Equal amounts of total proteins were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis using the rat PRL polyclonal antibody (anti-rPRL IC-4 provided by the National Institutes of Health). A stand- ard of rat pituitary PRL depicted as Pit (5 ng ib panel A and 2 ng in panel B) was used as positive control. The normalized protein levels are graphically represented in panel A as the percentage of maximal PRL expression. Results are mean ± S.E. of three different experiments. C, both antimesometrial and mesometrial decidual cells were grown on sterile cover glass and processed by immunocytochemistry as indicated under "Experimental Procedures." A negative control obtained by omitting the primary antibody incubation step from the procedure was free of staining (b). A positive signal was indicated by the red staining. Panels c and d represent a higher magnification of antimesometrial (c) and mesometrial (d) decidual cells observed in panel a. The antimesometrial cells expressed high rdPRL demonstrated by the red staining, whereas mesometrial cells expressed no rdPRL and are free of staining as observed in the negative control in b. Scale bars, 20 µm.

To examine whether PRL produced by the antimesometrial decidua was secreted, antimesometrial tissue from days 12 and 13 of pseudopregnant rats were cultured in serum-free medium for 3-24 h and PRL secreted into the medium was examined using Western blot analysis and ELISA. As shown in Fig. 5, PRL was secreted by the decidua on every day of pseudopregnancy examined.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Characterization of secreted rdPRL by Western blot analysis and ELISA. Antimesometrial decidua were maintained in culture up to 24 h in serum-free conditions at day 12 and 13 of pseudopregnancy. Conditioned media were concentrated using Centriplus concentrators and submitted to Western blotting analysis (left) using the rat PRL polyclonal antibody as described under "Experimental Procedures." A standard of rat pituitary PRL (Pit PRL; 0.5-2 ng) was used as positive control. rdPRL accumulation was measured by ELISA (right) in conditioned media at different time points and days of pseudopregnancy. Results are mean ± S.E. of three different experiments.

Regulation of rdPRL Expression by PRL-- To examine the regulation of decidual PRL, we developed a primary decidual cell culture. Time-course analysis (Fig. 6) revealed a steady increase in rdPRL mRNA levels between 0 and 48 h of culture. A drop in mRNA expression was seen thereafter. Interestingly, decidual cells expressed much higher levels of rdPRL and lower levels of PRL receptor than decidual explants when maintained 12 h in culture (Fig. 7, A and B). The high expression of rdPRL in cells that express little PRL-R suggested to us that PRL may inhibit its own expression. To examine this possibility, we cultured primary decidual cells in the presence of exogenous PRL. As shown in Fig. 8, PRL treatment in culture caused a dose-related down-regulation of rdPRL mRNA levels.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Time course of decidual PRL mRNA expression by primary decidual cells. Decidual cells obtained from day 9 pseudopregnant rats were cultured up to 96 h in RPMI medium supplemented with 1% FBS. PRL mRNA was analyzed by RT-PCR. The upper panel depicts one representative autoradiogram, and the lower panel represents the normalized mRNA levels as the mean ± S.E. (n = 6). *, significantly different compared with values measured at time 0.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Expression of PRL and PRL-R in decidual tissue (DT) and primary decidual cells. Total RNA was purified from decidual tissue, depicted DT, dissected from day 9 pseudopregnant animals and from primary decidual cells, depicted Cells, isolated from day 9 pseudopregnancy rats that were cultured for 12 h in RPMI 1640 phenol-free medium supplemented with 1% dextran charcoal-treated FBS. rdPRL (A) or PRL-R (B) mRNA were analyzed by RT-PCR as described under "Experimental Procedures." One representative autoradiogram is shown in the upper panels. The densitometric analysis represents the normalized mRNA levels as the mean ± S.E. (n  3). *, significantly different compared with decidual tissue.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effect of PRL on rdPRL mRNA expression in primary decidual cells. Primary decidual cells were isolated and cultured in RPMI medium supplemented with 1% FBS for 12 h in the presence of different doses of ovine PRL. Total RNA was prepared and subjected to RT-PCR analysis, as described under "Experimental Procedures." RT-PCR products were visualized by autoradiography and normalized to the amount of the L19 mRNA internal control. The upper panel depicts one representative autoradiogram (n  3) and the lower panel the densitometric analysis (mean ± S.E. of values expressed as percentage of the control, which was considered 100%).*, significantly different compared with vehicle-treated control.

To examine whether pituitary PRL regulates the expression of the decidual PRL in vivo, day 9 and 11 pseudopregnant rats with decidualized uteri were treated with CB-154, a dopaminergic agonist that is known to block pituitary PRL secretion, and decidual tissues were collected 24 h later. Both levels of rdPRL mRNA (Fig. 9A) and protein (Fig. 9B) were examined. The results revealed that a significant increase in both rdPRL RNA and protein expression occurs 24 h after inhibition of pituitary PRL secretion. CB-154 had no such inhibitory effect when added to decidual cells in culture.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Effect of CB-154 on rdPRL mRNA and protein expression. Adult rats were injected with a dopaminergic agonist 2-bromo- -ergocriptine (CB-154; 2 mg/kg of body weight) on day 9 and 11 of pseudopregnancy. Decidual tissues were collected 24 h later and used for nucleic acid and protein isolation. A, rdPRL mRNA was analyzed by RT-PCR. The upper panel depicts one representative autoradiogram, and the lower panel represents the normalized mRNA levels as the mean ± S.E. (n = 3).*, significantly different compared with vehicle-treated controls on day 10 or 12 of pseudopregnancy, respectively. B, decidual proteins (30 µg/lane) were separated on a 15% SDS-PAGE gel, transferred to nitrocellulose, and immunoreacted with PRL antiserum as described under "Experimental Procedures." The upper panel depicts one representative autoradiogram, and the lower panel represents the normalized protein levels as the mean ± S.E. (n = 3).*, significantly different compared with vehicle-treated controls on day 10 or 12 of pseudopregnancy, respectively.

Effect of Steroid Hormones on rdPRL mRNA in Primary Decidual Cells-- Because steroid hormones are essential for the formation and survival of the decidual tissue, we examined the role of progesterone and estradiol on rdPRL expression. Whereas progesterone caused an up-regulation of rdPRL mRNA expression in a dose-dependent manner (Fig. 10A), estradiol had no stimulatory effect (Fig. 10C). Interestingly, co-treatments with PRL and progesterone (which alone was able to increase rdPRL expression) or with PRL and estradiol (which alone had no effect on rdPRL expression) had no effect on the prolactin-induced decrease in rdPRL expression (Fig. 10, B and D).

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Effect of progesterone, estradiol, and PRL on rat decidual PRL mRNA expression in primary decidual cells. Total RNA was obtained from primary decidual cells (day 9 of pseudopregnancy) treated 12 h with different doses of progesterone (P) or estradiol (E2) alone, or in combination at 0.1 µg/ml for P and 1 ng/ml for E2 with 1 µg/ml PRL. The upper panels depict representative autoradiogram (n  3), and the lower panels show the densitometric analysis (mean ± S.E. of values expressed as percentage of the control, which was considered 100%).*, significantly different compared with vehicle-treated controls.

cAMP-induced Expression of rdPRL mRNA by Primary Decidual Cells in Culture-- Because the activators of the signaling pathway implicated in the cAMP/protein kinase A system are the principal regulators of decidual PRL expression in humans (37-39), we looked at the expression of rdPRL in primary decidual cells treated with cAMP for 12 h. We found that cAMP was able to dose-dependently increase the rdPRL mRNA expression (Fig. 11). The highest dose of cAMP used (100 µM) resulted in a 1.8-fold increase in rdPRL expression.

View larger version (21K): [in this window] [in a new window]   Fig. 11.   cAMP effect on rdPRL mRNA expression in primary decidual cells. rdPRL mRNA was analyzed by RT-PCR after incubation of primary decidual cells (day 9 of pseudopregnancy) for 12 h with different doses of 8-bromo-cAMP. One representative autoradiogram is shown in the upper panel. The lower panel represents the densitometric analysis from three independent experiments (mean ± S.E. of values expressed as percentage of the control, which was considered 100%). *, significantly different compared with vehicle-treated controls.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this report, we provide the first evidence that the rat decidua expresses the pituitary PRL gene and produces and secretes PRL similarly to human where the PRL gene is also expressed in both anterior pituitary and decidua (40-44). rdPRL gene expression and protein secretion are confined to a defined cell population located in the antimesometrial site of the uterus. Decidualization of the endometrial stroma, induced by either the blastocyst in pregnant rats or by artificial stimuli in pseudopregnant rats, gives rise to at least two major cell populations located in opposite sides of the uterus. The cells that decidualize in the antimesometrial region become more extensively differentiated than the cells in the mesometrial region, which undergo only limited differentiation. We and others have established that the two decidual cell populations differ not only in their morphology, but also by the genes they express and the putative roles they play in pregnancy (18, 36, 45-48). The antimesometrial cells that decidualize first also degenerate first. Because decidual tissue of either pregnant or pseudopregnant rats is similar in its formation, regression, and secretory capacity, the pseudopregnant rat has been extensively used as a model to study this organ in the absence of contaminating trophoblast cells. Our finding that rdPRL gene is expressed only in the antimesometrial cells confirms our previous reports (7, 18, 49) that these cells are the site of the PRL-like hormones secreted by the decidua. Interestingly the antimesometrial cells express not only rdPRL but also two other members of the PRL-family, dPRP (24, 49-51) and PLP-B (23, 25, 26), suggesting that during decidualization only this cell population acquires the factors needed for this cell-restricted expression. Mesometrial decidual cells may be either unable to express these PRL-related genes or they may be actively prevented from expression by signals originating from the antimesometrial cells. Such a paracrine regulation of mesometrial cell genes expression by antimesometrial cell products has been shown previously (19, 36, 45). Our results, however, do not support such a possibility since neither rdPRL nor dPRP (49) could be detected in mesometrial tissues or cells cultured in the absence of antimesometrial cells. These two decidual cell types may have unique tissue-specific factors or may be subjected to differentiation signals that activate different genes during development.

Developmental studies revealed that rdPRL is expressed at low levels early in development, which may explain why no study has ever shown the expression of the PRL gene in the decidua especially in early pregnancy. The rdPRL mRNA drop by day 15 is most probably due to the extensive apoptosis that occurs in these cells at this stage (52, 53).

Although the rat decidua expresses three members of the PRL family of hormones, only rdPRL has total homology with pituitary PRL and may therefore act similarly to PRL in regulating both ovarian (12, 54-57) and decidual functions (19). dPRP and PLP-B which have 37% (24) and 44% (23) homology, respectively, to pituitary PRL are unable to bind to the PRL receptor (26-27) and have no clear functions at this time. dPRP was recently shown to associate with heparin-containing molecules and to accumulate in the extracellular matrix (51). Additional studies (27) have also shown that heterologous expression of dPRP in Chinese hamster ovary cells significantly increased the ability of Chinese hamster ovary cells to form tumors following transplantation into athymic mice. dPRP is expressed in much higher levels than PLP-B (49) and rdPRL, although the rat decidua is able to secrete nanogram amounts of rdPRL. This may be due to differential regulation of these genes. Indeed, results of this investigation indicate clearly that rdPRL is down regulated by PRL whereas PRL has no such inhibitory effect on dPRP (36). Results obtained both in vivo and in vitro indicate that pituitary PRL and also the locally produced rdPRL limit rdPRL mRNA expression and the ability of the decidua to secrete PRL. A decrease in both forms of the PRL-R correlates with an increase in rdPRL expression and inhibition of pituitary PRL secretion caused an increase in both rdPRL mRNA expression and rdPRL secretion. Moreover, addition of exogenous PRL to primary decidual cells in culture also caused a decrease in rdPRL mRNA expression. It is of interest to note that decidual production of PRL-like hormones was first noted in rats in which pituitary secretion of PRL was prevented (8-10). In these studies, inhibition of pituitary PRL secretion caused a precipitous drop in luteal progesterone secretion in pseudopregnant rats without decidual tissue, whereas no change in progesterone secretion was seen in either pseudopregnant or pregnant rats with decidual tissue. Removal of the decidual tissue in these rat models caused a precipitous decline in progesterone levels. rdPRL appears to compensate for any deficiency in pituitary PRL and to be able to sustain ovarian secretion of progesterone and the maintenance of pregnancy in the total absence of PRL. On the other hand, the pattern of plasma pituitary PRL in early pregnancy consists of two surges each day, one nocturnal and one diurnal, which terminate at midpregnancy, the last surge being observed on the morning of day 11. The early termination of PRL surges during pregnancy, which correlates with the increased expression of PRL in the rat decidua, supports the contention of an inhibitory effect of pituitary PRL on decidual PRL (58). Moreover, although pituitary PRL and most probably rdPRL limit rdPRL expression in the decidua, they do not totally prevent its expression. The mechanism by which PRL down-regulates rdPRL expression is still undetermined. The 5'-flanking region of the rat PRL gene, a cell-specific element of the proximal promoter designated footprint II (FPII), functions as a dominant repressor element restricting the expression of the PRL gene in pituitary cells (59). When FPII was either deleted or specifically mutated, a 20-fold activation of the rat PRL promoter was observed in non-pituitary cell types. Whether FPII serves as a specific element for rdPRL inhibition by PRL remains to be investigated. The up-regulation of rdPRL by progesterone and cAMP, but not by estradiol, indicate that the regulation of rdPRL expression differs from that of the pituitary PRL (60, 61) suggesting a tissue-specific regulation of PRL gene expression (62), possibly through the use of alternative promoter in rat decidua.

In summary, results of this investigation clearly revealed that the pituitary PRL gene is expressed in both anterior-pituitary and decidua of the rat as it is in human. Limitation for in vivo experimentation in humans has reduced the possibility of investigating and understanding the role of decidual PRL in reproduction. This report provides evidence that the rat can be used as an experimental animal model to study the role of PRL produced by the decidua.

	ACKNOWLEDGEMENTS

We are grateful to NIDDK and the National Hormone and Pituitary Program (National Institutes of Health) for the rPRL RP3 and the antibody against rPRL, to R. Clepper for animal care, to L. Alaniz-Avila for photography, and to Vivian Rogala for preparation of the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HD-12356 (to G. G.) and a grant from the Ernst Schering Research Foundation (to C. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprints requests should be addressed: Dept. of Physiology and Biophysics (M/C 901), University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612-7342. Tel.: 312-996-7688; Fax: 312-996-1414; E-mail: ggibori@uic.edu.

	ABBREVIATIONS

The abbreviations used are: PRL, prolactin; rat decidual prolactin, rdPRL; PRL-R, prolactin receptor, dPRP, decidual prolactin-related protein; PLP-B, prolactin-like protein B; DLt, decidual luteotropin; bp, base pair(s); FPII, footprint II; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; RTreverse transcription, PCR, polymerase chain reaction; FBS, fetal bovine serum; TBS, Tris-buffered saline; RACE, rapid amplification of cDNA ends; TRITC, tetramethylrhodamine B isothiocyanate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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