Developmental Expression and Hormonal Regulation in Preg

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 11 Printed in U.S.A.

The Different Forms of the Prolactin Receptor in the Rat Corpus Luteum: Developmental Expression and Hormonal Regulation in Pregnancy* C. M. TELLERIA†, T. G. PARMER†, L. ZHONG, D. L. CLARKE, C. T. ALBARRACIN, W. R. DUAN, D. I. H. LINZER, AND G. GIBORI‡ Department of Physiology and Biophysics (C.M.T., T.G.P., L.Z., C.T.A., W.R.D., G.G.), College of Medicine, University of Illinois, Chicago, Illinois 60612; and Department of Biochemistry, Molecular Biology, and Cell Biology (D.L.C., D.I.H.L.), Northwestern University, Evanston, Illinois 60208 ABSTRACT The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20a-hydroxysteroid dehydrogenase (20a-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most

dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20a-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20a-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression. (Endocrinology 138: 4812– 4820, 1997)

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marked and selective inhibition in 20a-HSD gene expression (4). 20a-HSD is a 37-kDa enzyme responsible for the catabolism of progesterone to the inactive progestagen, 20adihydroprogesterone (5, 6). This inhibitory action of PRL on 20a-HSD gene expression is essential for maintaining high levels of progesterone during pregnancy. Whether or not the dephosphorylation of EF2 and the decreased expression of 20a-HSD induced by PRL are due to different signaling mechanisms in the corpus luteum, perhaps through different receptors, remains unknown. Indeed, in the rat, two distinct PRL receptors (PRL-R) have been identified (7–9). These PRL-R have been classified as long (PRL-RL) and short (PRLRS) depending upon the length of the cytoplasmic domain. Both receptor forms have identical extracellular and transmembrane domains and a unique intracellular sequence of 57 and 358 amino acids for the short and long forms, respectively (10). Signaling through these two forms of the receptor appears to differ. Whereas the JAK/Stat pathway and tyrosine phosphorylation are involved in PRL signaling through the PRL-RL (11–13), the signaling mechanism through the short form is unknown. Our finding that PRL inhibition of 20a-HSD gene expression appears not to involve tyrosine phosphorylation (14) and our recent discovery of a novel luteal protein that can associate with the intracellular domain of the PRL-RS in an in vitro system (15) led

HE role of PRL in the maintenance of corpus luteum function in the pregnant rat is well established. PRL not only maintains luteal production of progesterone but also stimulates, in synergy with estradiol, overall protein synthesis leading to luteal cell hypertrophy (1). The PRL-mediated up-regulation of protein synthesis and progesterone production by the corpus luteum appears to be due, at least in part, to PRL action on the phosphorylation state and gene expression of two key proteins: elongation factor-2 (EF2) and 20a-hydroxysteroid dehydrogenase (20a-HSD). PRL causes the dephosphorylation and activation of EF2, a 100-kDa protein that plays an important role in peptide elongation and is an essential component of the protein synthetic machinery (2). When dephosphorylated, EF2 catalyzes the translocation of peptidyl-transfer RNA on ribosomes leading to increased elongation (3). Whereas PRL appears to stimulate overall protein synthesis acting at the translational level, it causes a Received April 28, 1997. Address all correspondence and request for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. * This work was supported by NIH Grants HD-11119 (to G.G.), FIC 1F05TW05241 (to C.M.T.), and HD-21921 (to D.I.H.L.) † These authors contributed equally to this work. ‡ NIH Merit Awardee (HD-11119).

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THE PRL-RECEPTOR IN THE RAT CORPUS LUTEUM

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FIG. 1. Comparison of the two forms of the PRL-R mRNA in the follicle and corpus luteum of the pregnant rat. Total RNA was isolated from follicles and corpora lutea of day 14 pregnant rats, reverse transcribed into single-stranded complementary DNA, and amplified with oligonucleotide primers as described in Materials and Methods. One primer was specific to the common region of both the short and long form of the receptor while the other primer contained a sequence unique for each receptor mRNA. Included in each reaction was a pair of oligonucleotide primers for the L19 ribosomal mRNA. Data were quantified by densitometry and corrected using L19 as an internal standard. Normalized mRNA levels are graphically represented in the right panel as the mean 6 SEM (n 5 3).

us to suggest that PRL action on the rat corpus luteum may involve different forms of receptors. However, before examining the role of the two forms of the receptor in PRL signaling, we first had to establish whether indeed the rat corpus luteum of pregnancy expresses PRL-RL and PRL-RS messenger RNA (mRNA). Since the corpus luteum undergoes changes during pregnancy, including a dramatic hypertrophy particularly at midpregnancy and a marked drop in progesterone secretion late in pregnancy, and since the maintenance and function of the corpus luteum are dependent on an interplay of pituitary, ovarian, and placental hormones for its existence (1), it is also important to characterize the developmental and hormonal regulation of the PRL-R expression throughout pregnancy. We were specially interested in determining whether one of the two forms of the PRL-R decline at the end of pregnancy, since at this stage the corpus luteum becomes nonresponsive to PRL and PRL-like hormone from placental origin. Materials and Methods Chemicals Acrylamide and bis-acrylamide were obtained from Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak (Rochester, NY), respectively; Taq DNA polymerase was purchased from Perkin-Elmer Corp.

FIG. 2. Developmental expression of PRL-RL and PRL-RS mRNA in the rat corpus luteum during pregnancy. Corpora lutea were dissected from ovaries of rats at different stages of pregnancy, and total RNA was isolated and analyzed by RT-PCR as described in Materials and Methods. Data were quantified by densitometry and corrected using L19 as an internal standard. Normalized mRNA levels for each day of pregnancy are graphically represented in panel B as the mean 6 SEM (n 5 3). (Foster City, CA); [a-32P]deoxycytidine triphosphate (dCTP) was from Amersham Corp. (Arlington Heights, IL); the oligonucleotides used as primers in the RT-PCR analysis were obtained from Life Technologies (Grand Island, NY). Genistein was obtained from ICN Biomedicals (Aurora, OH). Ovine PRL (oPRL) was provided by the NIADDK (ovine PRL-18, 30 IU/mg, Bethestha, MD). Antibiotic-antimycotic mixture and FBS were obtained from GIBCO-BRL (Gaithersburg, MD) and Hyclone (Logan, UT) respectively. DMEM-Ham’s F12 (DMEM/F12), 8-bromocAMP, human CG (hCG), and all other reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals Pregnant (day 1 5 sperm positive) and immature female (day 26 of age) Sprague-Dawley rats were obtained from Sasco Animal Labs (Madison, WI). They were kept under controlled conditions of light (lights on 0700 –1900 h) and temperature (22–24 C) with free access to standard rat chow and water. Animals were handled according to the

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procedures approved by the Institutional Animal Care and Use Committee.

Experimental procedures For the developmental studies, rats were obtained at various stages of pregnancy from days 4 to 22 (day of parturition) and at the day after parturition. Corpora lutea were dissected from the ovaries under a stereoscopic microscope. Follicles were also obtained from day 14 pregnant rats. All tissues were frozen in liquid nitrogen and stored at 280 C until processed for RNA or protein. Blood samples were collected for assays of serum progesterone and 20a-dihydroprogesterone levels. To determine the effect of PRL on PRL-R mRNA in corpora lutea, pregnant rats were hypophysectomized on day 3 of pregnancy and injected sc with 125 mg PRL (NIDDK oPRL-18, 30 IU/mg twice daily) in 50% polyvinylpyrrolidone, pH 9.0, for 4 days. To neutralize circulating LH at the end of pregnancy, rats were injected sc with 1 ml LH antiserum generated in a mare against bovine LH as used previously (16, 17). Control rats were injected with normal horse serum. Corpora lutea were removed on the morning of day 22 of pregnancy, before parturition, which usually occurs on the afternoon of day 22 in this rat strain.

Granulosa cell culture Maturation of preovulatory follicles was stimulated by treatment of immature rats at day 28 of age with 0.15 IU hCG sc twice daily for 2 days (18). Luteinization of these preovulatory follicles was subsequently achieved by an ovulatory dose (10 IU) of hCG on the third day via the tail vein. Luteinized granulosa cells were harvested from preovulatory follicles 7 h after the iv injection of hCG. Briefly, follicles were incubated sequentially in DMEM/F12 1:1, containing 6 mm EGTA and 0.5 m sucrose, respectively, and granulosa cells were harvested by needle pricking the follicles. The cells were plated in 60-mm culture dishes at 8 3 105 cells/ml and incubated at 37 C under 95% air/5% CO2 atmosphere in DMEM/F12 containing 15 mm HEPES, 1% FBS, 100 IU/ml penicilin G, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. After 72 h of incubation, the medium was changed, cells were treated during 12 h with different hormones or reagents, and total RNA was extracted.

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primer (59-AGCAGTTCTTCAGACTTGCCCTT-39) or a PRL-RS specific primer (59-TTGTATTTGCTTGGAGAGCCAGT-39) corresponding to the first 23 nucleotides of the unique cytoplasmic coding region. For 20a-HSD mRNA analysis, oligonucleotide primers of 21 nucleotides were designed based on the sequence of the rat 20a-HSD gene (22) (59-CAACCAGGTAGAATGCCAATCT-39 and 59-TTCGAGCAGAACTCATGGCTA-39). In each reaction, an additional pair of 21 nucleotides (59-CTGAAGGTCAAAGGGAATGTG-39 and 59-GGACAGAGTCTTGATATCTC-39) specific to the rat ribosomal protein L19 mRNA was included for use as an internal control (23). The predicted sizes of the PCR-amplified products were 279 bp for both PRL-RL and PRL-RS, 440 bp for 20a-HSD, and 194 bp for L19. RNA samples were assayed for DNA contamination by PCR without prior reverse transcription. One microgram of total RNA was reverse transcribed at 37 C using random hexamer primers (Pharmacia, Piscataway, NJ) and Moloney murine leukemia virus-RT (Life Technologies, Grand Island, NY) in a 20-ml reaction mixture. The reaction mixture was added to tubes containing specific oligonucleotide primer (50 pmol each) for amplification of either form of the PRL-R or 20a-HSD complementary DNAs. A mix containing the oligonucleotide primers for L19 mRNA (50 pmol each), Taq DNA polymerase (2.5 U), and [a-32P]deoxy-CTP (2 mCi of 3000 Ci/mmol) was added to each tube, and the final volume was increased to 90 ml with 1 3 PCR buffer [20 mm Tris (pH 8.4), 50 mm KCl and 2.5 mm MgCl2 ]. The samples were overlaid with light mineral oil, and PCR was carried out for 20 cycles with an annealing temperature of 65 C in a Perkin-Elmer/ Cetus Thermal Cycler (Norwalk, CT), except for the experiments shown in Figs. 1 and 2, where a 60 C annealing temperature was used. 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 a 8% polyacrylamide nondenaturing gel. After autoradiography, data were ana-

RNA isolation and RT-PCR analysis Total RNA from frozen corpora lutea was purified by homogenization in guanidinium thiocyanate and centrifugation through a cesium chloride cushion (19), whereas total RNA from cultured cells was isolated by a one-step guanidinium-thiocyanate-phenol-chloroform extraction procedure (20). For detection of long and short form PRL-R mRNAs by RT-PCR, three oligonucleotide primers were synthesized as described earlier (21). To detect each form of PRL-R mRNA, a sense strand oligonucleotide from the common extracellular domain coding region (59-AAAGTATCTTGTCCAGACTCGCTG-39) was combined with either a PRL-RL specific

FIG. 3. PRL-RL and PRL-RS protein in corpora lutea of early and late pregnant rats. Corpora lutea were isolated from days 4 and 22 of pregnancy, and equal amounts of protein (200 mg) were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot using polyclonal antiserum to the PRL-R as described in Materials and Methods. The images depicted were obtained from the same blot.

FIG. 4. Expression of PRL-RL, PRL-RS, and 20a-HSD mRNA in the rat corpus luteum at the end of pregnancy, the day of parturition, and on the day after parturition. Total RNA was isolated from corpora lutea obtained on days 19, 20, 21, 22 (day of parturition), and on the day after parturition (indicated as PP), and subjected to RT-PCR analysis as described in Materials and Methods The right panel represents the densitometric analysis of the data normalized against the ribosomal protein L19 mRNA used in each reaction as internal standard and expressed as the mean 6 SEM (n 5 3). Values of luteal PRL-RL, PRL-RS, and 20a-HSD mRNA obtained on days 21, 22, and after parturition were significantly different (P , 0.05) from that observed on days 19 and 20 of pregnancy.


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RIAs Serum progesterone concentrations were measured using a commercially obtained kit (Diagnostic Products Corporation, Los Angeles, CA). The sensitivity of the assay was 0.02 ng/ml, and the inter- and intraassay coefficients of variation were 5% and 6%, respectively. Serum 20adihydroprogesterone was assayed after hexane extraction using a highly specific antiserum kindly provided by Dr. Quadri (Department of Anatomy/Physiology, Kansas State University, Manhattan, KS). The sensitivity of the assay was 0.01 ng/assay tube, and the inter- and intraassay coefficients of variation were less than 10%.

Results Developmental expression of luteal PRL-RL and PRL-RS throughout pregnancy

FIG. 5. Serum progesterone and 20a-dihydroprogesterone concentrations at the end of pregnancy. Sera were obtained from the same animals used in Fig. 4. Values are expressed as the mean 6 SEM (n 5 5). Values obtained on days 21, 22, and after parturition were significantly different (P , 0.05) from that measured on days 19 and 20 of pregnancy.

lyzed using a Molecular Dynamics PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Sunnyvale, CA). The intensity of the PRL-RL, PRL-RS, and 20a-HSD signals were normalized to that of the ribosomal protein L19 internal control. Data were examined by one-way ANOVA followed by Duncan’s multiple range test. When appropriate, Student’s t test was used. A level of P , 0.05 was accepted as statistically significant.

Immunoblot analysis Isolated corpora lutea were suspended in 2 ml ice-cold homogenization buffer containing 50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and 250 mm sucrose followed by homogenization in a Potter-Elvejhem homogenizer. Total luteal homogenates were stored at 280 C and aliquots assayed for protein content (24). Equivalent amounts of protein (200 mg) were separated through 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes by electrotransfer, stained with Ponceau S (to quantify protein loaded), and blocked with 5% nonfat milk in Trisbuffered saline (TBS; pH 7.5) containing 0.1% Tween 20. Blots were then incubated overnight with a 1:1000 dilution of a polyclonal antiserum generated to both forms of the PRL-R (25), washed, and incubated for 1 h with a horseradish-peroxidase-labeled antirabbit IgG. After extensive washing, blots were developed using an enhanced chemiluminescence Western blotting detection system (Amersham Corp, Arlington Heights, IL) and exposed for 5–30 sec to x-ray film.

RT-PCR analysis was used in this study to determine changes in PRL-R mRNA levels in the corpus luteum throughout pregnancy since mRNA levels for the different forms of PRL-R have been reported to be of low abundance in the whole rat ovary (9) and are difficult to quantify using Northern analysis. To compare mRNA levels of PRL-RL and PRL-RS, we used oligonucleotide primers designed to amplify a 279-bp fragment for each of the two receptor mRNAs (21). In each reaction, primers specific to the ubiquitous L19 ribosomal protein were included as internal control. We first examined and compared the mRNA expression of PRL-RL and PRL-RS in corpora lutea and follicles of rats at the same stage of pregnancy (day 14). As shown in Fig. 1, both PRL-RL and PRL-RS were detected in both ovarian tissues with PRL-RL being the most abundant. However, levels of both receptor mRNAs were much higher in the corpus luteum than in the follicle (P , 0.05). Developmental studies shown in Fig. 2A indicate that both forms of the PRL-R mRNA are expressed in the corpus luteum throughout pregnancy. The amount of the RT-PCR product corresponding to the long form of the PRL-R mRNA was consistently higher than that obtained for the short form of the PRL-R mRNA (Fig. 2B). Throughout gestation and until day 20 of pregnancy, there were no significant changes in mRNA levels of either form of the PRL-R in the corpus luteum. However, most interestingly, between days 20 and 22, just before parturition, an abrupt decline in mRNA for both forms of the receptor occurred. Using a polyclonal antiserum that recognizes both forms of the receptor, we examined by Western analysis (Fig. 3) the expression of PRL-R protein in corpora lutea of early (day 4) and late (day 22) pregnancy. PRL-RL and PRL-RS proteins were detected in the rat corpus luteum at 66 and 45 kDa, respectively. Similarly to PRL-RL and PRL-RS mRNA expression, the PRL-RL protein was expressed at higher levels than the PRL-RS, and lower levels of both receptor forms were found on day 22 when compared with day 4 of pregnancy. Expression of PRL-R and 20a-HSD at the end of pregnancy

A possible consequence of the decline in PRL-R levels in late gestation is an alteration in PRL-regulated gene expression in the corpus luteum. The 20a-HSD gene is negatively regulated by PRL, and therefore we would predict an increase in the level of the 20a-HSD mRNA at day 22 and after parturition. As depicted in Fig. 4 (A and B), PRL-RL and PRL-RS mRNA levels decreased markedly on day 21 and

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FIG. 6. Effect of LH antiserum (LH-As) on PRL-R mRNA levels in rat corpora lutea. Day 21 pregnant rats were injected with either normal horse serum (2) or with LH-As (1). On day 22, corpora lutea were removed from the ovaries, and RNA was isolated and subjected to RT-PCR as described in Materials and Methods. Data were quantified by densitometry and corrected for L19. Normalized mRNA levels are graphically represented as the mean 6 SEM (n 5 3).

FIG. 7. Effect of PRL on PRL-RL and PRL-RS mRNA expression in rat corpora lutea. Pregnant rats were hypophysectomized on day 3 and treated with either PRL (1) or vehicle (2) for 4 days as described in Materials and Methods. Corpora lutea from these treatments were isolated, and RNA was prepared and subjected to RT-PCR. Results were quantified by densitometry and corrected using L19. Normalized mRNA levels are graphically depicted as the mean 6 SEM (n 5 3).

remained at low levels until a day after parturition; at these times, 20a-HSD mRNA levels increased dramatically (Fig. 4C). 20a-HSD could not be detected before day 21, but became abruptly and highly expressed from day 21 until the day after parturition. The rapid rise in luteal 20a-HSD mRNA was accompanied (Fig. 5, A and B) by a sharp increase in 20a-dihydroprogesterone and a corresponding fall in progesterone in the circulation of the same animals. The decline in PRL-R expression appears to render the corpus luteum less responsive to the inhibitory action of PRL and allows for the increase in 20a-HSD mRNA levels and the catabolism of progesterone to 20a-dihydroprogesterone. Hormonal regulation of PRL-R mRNA expression in vivo

Because LH concentrations rise in the circulation at the end of pregnancy (26, 27), we examined whether LH was responsible for the down-regulation seen in the levels of both forms of the PRL-R. In this set of experiments, day 21 pregnant rats were injected with a dose of LH antiserum (LH-As), known to neutralize circulating LH (16, 17). Corpora lutea were isolated from the rats 24 h later, and total RNA was subjected to RT-PCR. Results shown in Fig. 6 indicate that neutralization of LH at the end of pregnancy caused a marked decrease (P , 0.01) in PRL-RL and PRL-RS mRNA levels indicating that LH may have an up-regulatory rather than a down-regulatory role in PRL-R expression. Since the corpus luteum is subjected to high levels of pituitary PRL in early pregnancy and to even higher levels of placental PRL-related hormones later on in pregnancy (1),

we examined the role of PRL on PRL-RL and PRL-RS mRNA. Treatment of day 3 hypophysectomized rats with PRL for 4 days caused a substantial increase in PRL-R mRNA (Fig. 7). Interestingly, the up-regulation was restricted to the long form of the receptor (P , 0.05). Hormonal regulation of PRL-R mRNA expression in culture

To examine further the hormonal regulation of the PRL-R mRNA expression, we used highly luteinized granulosa cells in culture. As shown in Fig. 8 (A and C), PRL caused a significant increase in PRL-RL mRNA levels, but not in the short form of the PRL-R (Fig. 8, B and C). Because the PRL-R is known to transduce many of PRL’s actions through the JAK/Stat pathway involving Stat phosphorylation on tyrosine, we examined the effect of the tyrosine kinase inhibitor, genistein, on the induction of PRL-RL mRNA by PRL. Genistein was shown previously to inhibit PRL stimulation of a2-macroglobulin in the corpus luteum (28). As shown in Fig. 9, PRL induced within 12 h a significant increase (P , 0.05) in PRL-RL mRNA (panel A); however, when genistein was added along with PRL (panel B), a sharp decrease (P , 0.05) in PRL-R expression was observed, suggesting that PRL stimulation of PRL-R gene expression involves tyrosine kinase (s). Similar to the effect of PRL, 8-bromo-cAMP (Fig. 10) also caused the up-regulation of the long but not the short form of the PRL-R, suggesting that both protein kinase A and a tyrosine kinase pathway are involved in the regulation of PRL-R mRNA expression in ovarian cells.


FIG. 8. Effect of PRL on PRL-RL and PRL-RS mRNA expression in luteinized granulosa cells. Granulosa cells were isolated from preovulatory follicles and cultured in DMEM/F12 1:1 containing 1% FBS. After a 72-h incubation, the medium was changed and cells were incubated for an additional 12 h in the presence of different doses of oPRL in the absence of serum. Total RNA was prepared and subjected to RT-PCR analysis as described in Materials and Methods. RT-PCR products were visualized by autoradiography and normalized to the amount of the L19 mRNA internal control. The autoradiograms from one experiment are shown for the PRL-RL (panel A) and PRL-RS (panel B) mRNA. Panel C depicts the densitometric analysis from three independent experiments (mean 6 SEM of values expressed as percentage of the control, which was considered 100%). PRL-RL mRNA values for three groups treated with PRL (0.1, 1, and 10 mg/ml) were significantly different (P , 0.05) from vehicle-treated controls.

Discussion

During pregnancy in the rat, both the survival of the corpus luteum and its capacity to produce progesterone are highly dependent on the action of PRL and PRL-like hormones of decidual and placental origin (1, 29). These hormones are produced sequentially during pregnancy and are able to bind to the same receptor on luteal membranes (30). PRL-R mRNA and protein expression has previously been

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FIG. 9. Effect of the tyrosine kinase inhibitor, genistein, on PRL-RL mRNA expression in luteinized granulosa cells. Granulosa cells were isolated from preovulatory follicles and cultured in DMEM/F12 1:1 containing 1% FBS. After 72 h, the medium was changed and cells were incubated in serum-free conditions for an additional 12 h with 1 mg/ml of PRL and different doses of genistein. Total RNA was prepared and subjected to RT-PCR analysis as described in Materials and Methods. RT-PCR products were visualized by autoradiography and normalized to the amount of the L19 mRNA internal control. Panel C depicts the densitometric analysis from three independent experiments (mean 6 SEM of values expressed as percentage of the vehicle-treated control, which was considered 100%).

detected in the ovary of the cycling rat (21) and in extracts of whole ovaries of hormonally induced pseudopregnant rats (9). This report demonstrates the differential expression of both forms of the PRL-R in the corpora lutea of pregnant rats and provides the first analysis of the changes in both mRNA and protein expression for both PRL-RL and PRL-RS during pregnancy. This investigation also examines the hormonal regulation of each form of the PRL-R and the correlation between the luteal PRL-R and 20a-HSD gene expression at the end of pregnancy. Using RT-PCR and Western analysis, both forms of the PRL-R were found to be expressed in the rat corpus luteum with the PRL-RL being the most abundant at all stages of

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FIG. 10. Effect of cAMP on PRL-RL and PRL-RS mRNA expression in luteinized granulosa cells. Granulosa cells were isolated from preovulatory follicles and cultured in DMEM/F12 1:1 containing 1% FBS. After a 72-h incubation, the medium was changed and cells were incubated for an additional 12 h in the presence of different doses of 8-bromo-cAMP without serum. Total RNA was prepared and subjected to RT-PCR analysis as described in Materials and Methods. RT-PCR products were visualized by electrophoresis and autoradiography and normalized to the amount of the L19 mRNA internal control. Panel C depicts the densitometric analysis from three independent experiments (mean 6 SEM of values expressed as percentage of the control, which was considered 100%). Values obtained with the dose of 250 mM cAMP were statistically different (P , 0.05) when compared with controls.

pregnancy. A similar pattern was observed in the whole ovary of pregnant mice which express, however, three different forms of the PRL-RS (31). In the rat, the short form of the PRL-R is encoded by a transcript of 1.8 kb, whereas the long form is encoded by three larger transcripts (8, 32). Al-

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though the heterogeneity of PRL-R mRNA species appears to arise from alternative splicing (8, 32), it is evident from recent data (33, 34) that differential transcriptional initiation and alternative polyadenylation also contribute to the mRNA heterogeneity. Coexpression of both PRL-R forms has been reported in several tissues of pregnant rats (35). However, in contrast to rat liver and mammary gland where PRL-RS is the major receptor (8, 36), it is the PRL-RL that predominates in the corpus luteum. The developmental expression during pregnancy of the PRL-R also differs in these three major PRL target tissues. Whereas in both mammary gland and liver PRL-R mRNA levels remain low during pregnancy but increase just before parturition (36), the receptor mRNA levels in the corpus luteum are elevated throughout pregnancy and drop just before parturition. Thus, in spite of the similar hormonal environment during pregnancy, a marked difference in the developmental expression of the PRL-R takes place in these three major PRL target tissues. Differential regulation may be due not only to particular sets of transcription factors present and active at a given time, but also to different promoters that may be regulated differently. Indeed the recent finding of three alternative promoters in the 59-untranslated region of the PRL-R gene may provide the molecular basis of this tissue specificity in the developmental regulation of the PRL-R expression (33). Whereas the rise in PRL-R in the mammary gland just before parturition helps in preparing this gland to become highly responsive to PRL, a hormone crucial for lactation, the drop in PRL-R mRNA and protein at the end of pregnancy found in this investigation appears to play a crucial role in terminating pregnancy. Indeed for years it was known that despite very high levels of placental lactogen in the circulation, progesterone secretion by the corpus luteum declines abruptly at the end of pregnancy. Recently we have cloned 20a-HSD, the enzyme responsible for the catabolism of progesterone (22), and have shown that the expression of this gene is silenced by PRL, rat placental lactogen-1, and rat placental lactogen-2 (14). However, despite high levels of rat placental lactogen-2 in the circulation and the rise of pituitary PRL (26, 27), 20a-HSD mRNA and protein become abruptly expressed just before parturition (Ref. 4 and present study). We originally thought that 20a-HSD mRNA may be induced, despite high levels of PRL, by the rise in LH at the end of pregnancy. Indeed, a recent report indicates the ability of ovine LH to stimulate 20a-HSD activity in corpora lutea of late pregnancy (37). However, based on a preliminary study in which we examined LH and cAMP stimulation of 20a-HSD expression in cultured cells that express the PRL-R, it became apparent that PRL totally blocks the stimulatory effect of both LH and cAMP on 20a-HSD expression (38). The more detailed study reported in this investigation clearly indicates that 20a-HSD mRNA, which is undetectable on day 20 of pregnancy, becomes highly expressed within 24 h and remains elevated the day of parturition (day 22) and the day following parturition exactly at a time when a profound decline in the expression of both forms of the PRL-R occurs. This decrease in PRL-R expression most probably renders the


corpus luteum less responsive to the circulating PRL and PRL-related hormones and diminishes the PRL-mediated inhibition of 20a-HSD expression. What induces the drop in both forms of the PRL-R at the end of pregnancy remains unclear. We originally thought that the rise in LH may be responsible for such an event; however, neutralization of LH with a dose of LH antiserum known to block LH action (16, 17) caused a further decline in PRL-R mRNA expression, suggesting that LH is more likely to be involved in the up-regulation of this receptor. PRL also can up-regulate the expression of the PRL-R in the rat corpus luteum. Our finding that PRL causes a selective increase in the expression of PRL-RL with little, if any, effect on that of PRL-RS mRNA suggests that PRL and rat placental lactogen may be responsible for the higher expression of the long form of the receptor in the rat corpus luteum during pregnancy. Both receptor types appear to be derived from a single primary transcript and whether PRL’s effect is at the level of RNA splicing or mRNA stability remains to be investigated. Since the increase in PRL-RL mediated by PRL was blocked by the tyrosine kinase inhibitor genistein, and also since cAMP up-regulates the expression of PRL-RL, it appears that the up-regulation of PRL-RL mRNA may involve both protein kinase A and tyrosine kinase pathways. In summary, results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and the long form of the PRL-R with the long form being much more abundant; 2) the PRL-R mRNA remains elevated throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20a-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and 5) both A kinase- and tyrosine kinase-mediated pathways appear to participate in the up-regulatory mechanism of PRL-R mRNA expression. Acknowledgments We are very grateful to Dr. Quadri for the 20a-dihydroprogesterone antibody and to the NIDDK and National Hormonal and Pituitary Program (NIH) for the oPRL, R. Clepper for animal care, L. Alaniz-Avila for photography, and V. Rogala for the preparation of the manuscript. We especially wish to thank Dr. J. Ou for his expert assistance.

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Conference on the Diagnosis and Treatment of the Unborn Child The Buttes, Tempe, Arizona Saturday, April 18 –Monday, April 20, 1998 Participants: Maria I. New (Chairman), New York, NY; Arleen Auerbach, New York, NY; Ted Brown, New York, NY; Frank Chervenak, New York, NY; Jessica Davis, New York, NY; Robert Desnick, New York, NY; Miroslav Dumic, Zagreb, Croatia; Mark I. Evans, Detroit, MI; Alan W. Flake, Philadelphia, PA; Maguelone G. Forest, Lyon, France; Judith Hall, Vancouver, BC; Margaret Hilgartner, New York, NY; Eric Hoffman, Pittsburg, PA; Sheila Innis, Vancouver, BC; Y. W. Kan, San Francisco, CA; Joshua Lederberg, New York, NY; Lucio Luzzatto, New York, NY; Arno Motulsky, Seattle, WA; John Opitz, Salt Lake City, UT; Darwin J. Prockop, Philadelphia, PA; David L. Rimoin, Los Angeles, CA; Joe Leigh Simpson, Houston, TX; Jean Wilson, Dallas, TX; Don Wolf, Beaverton, OR. The Conference will include: Genetic Disorders in Ashkenazim; Hemophilia Problems in Carrier Identification and Fetal Diagnosis; Fragile X Syndrome; Fanconi Anemia; Inherited Variation in Susceptibility to Cancer in the Unborn Child; Smith Lemli-Opitz Syndrome, Marfan Syndrome, Androgen Receptor Defects, Skeletal Dysplasias, Muscular Dystrophies, Congenital Adrenal Hyperplasia, Uniparental Disomy, Diagnosis and Treatment of Fetal and Neonatal Malnutrition, Fetal Cells in Maternal Blood to Diagnose Aneuploidy; Fetal Cells in Maternal Blood to Diagnose Hemoglobinopathy; Open Surgery; Percutaneous Approaches, Stem Cell Therapy, Stem Cell Techniques, Preimplantation Genetic Diagnosis, Ethical Fetal Therapy; New Obligations of Physicians in Genetics, Cloning and Ethics. The deadline for abstracts of poster presentations is December 15, 1997. For further information, please contact Maria I. New, MD, Prenatal Diagnosis Conference—Arizona ’98, Ross Professional Services, 40 Old Ridgebury Road, Ste. 309, Danbury, CT 06810; telephone 203-730-8724; fax 203-778-1121.