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Endocrinology Vol. 139, No. 12 5215-5223
Copyright © 1998 by The Endocrine Society

ARTICLES

Detection of Prolactin Receptor Gene Expression in the Sheep Pituitary Gland and Visualization of the Specific Translation of the Signal in Gonadotrophs

Domingo J. Tortonese1, Julie Brooks, Patricia M. Ingleton and Alan S. McNeilly

Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, (D.J.T., J.B., A.S.M.), Edinburgh EH3 9EW, Scotland, United Kingdom; and the Institute of Cancer Studies, University of Sheffield Medical School (P.M.I.), Sheffield S10 2RX, England, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In sheep, as in other mammalian species, the pronounced reduction in GnRH and gonadotropin secretion that characterizes stages of infertility is normally associated with a conspicuous increase in the secretion of PRL. A possible role of PRL in modulating gonadotropin release implies the presence and activation of specific receptors in target tissues (i.e. pituitary, hypothalamus). In this study, we investigated the expression of PRL receptor (PRL-R) messenger RNA (mRNA) in the sheep pituitary and the distribution of the translated product in specific pituitary cell types. Using primers designed to flank different regions of the extracellular and cytoplasmic domains of the PRL-R, two complementary DNA (cDNA) fragments, one of which was specific for the long-form PRL-R, were amplified by reverse transcriptase-PCR. Sequencing revealed more than 95% identity with nucleotides 267-1272 of the bovine PRL-R cDNA. When these cDNA fragments were used as probes for the detection of PRL-R mRNA expression by Northern analysis, three major transcripts of approximately 13, 10, and 3.5 kb were identified in the pituitary. Both probes detected identical transcripts, suggesting that primarily the long form of PRL-R is expressed in the sheep pituitary gland. No difference in the abundance of pituitary PRL-R mRNA transcripts was observed between anestrous and breeding season ewes (P > 0.05). Additional RT-PCR studies revealed the existence of a cDNA variant bearing a 39-bp insert with a premature stop codon. Translation of the PRL-R mRNA was confirmed by Western blot analysis. The identification of PRL-R in specific pituitary cell types was carried out by immunocytochemistry. Double immunofluorescent staining, using antibodies to the rat liver PRL-R and specific monoclonal antibodies to the LHß-subunit, FSHß-subunit, free {alpha}-subunit, PRL, or GH, revealed that in both the pars distalis and pars tuberalis, all pituitary cells expressing PRL-R immunoreactivity were positive for LHß, although only 53% of LHß-positive cells expressed PRL-R. A small proportion (2%) of gonadotrophs expressing PRL-R immunoreactivity were negative for FSHß, indicating the specific localization of PRL-R in LH (or LH/FSH) secreting cells. Further, a selective cytological association was detected in the pars distalis where LH gonadotrophs appeared surrounded by lactotrophs. In contrast to these observations, PRL-R immunoreactivity was completely absent in lactotrophs and in the vast majority (>98%) of somatotrophs. In conclusion, here we show the expression of PRL-R mRNA in the sheep pituitary and the specific translation of the signal in LH (or LH/FSH) gonadotrophs. These results support the hypothesis that PRL may be involved in the regulation of gonadotropin secretion through a paracrine mechanism within the pituitary gland and that this action does not seem to be mediated by changes in PRL-R mRNA expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN SEVERAL mammalian species, an interaction between the gonadotropic and PRL axes has long been recognized (1). A conspicuous increase in the secretion of PRL is normally associated with the pronounced reduction in gonadotropin secretion that results in stages of infertility (i.e. seasonal anestrus in sheep, lactational amenorrhea in primates, pseudopregnancy in rodents). Even though these inversed endocrine changes can be regarded merely as temporal associations, experimental observations in rodents provide evidence for a cause-effect. The administration of exogenous PRL to rats in vivo reduced the proportion of pituitary cells secreting LH in vitro (2), whereas experimentally induced hyperprolactinemia inhibited gonadotropin secretion (1, 3, 4) and blocked the postcastrational rise in LH release (5). Moreover, in vitro treatments with PRL resulted in suppression of both basal and GnRH-stimulated LH release from cultured pituitary fragments (6). Similarly, lactational hyperprolactinemia was shown to reduce the LH response to exogenous GnRH in vivo and in vitro (7, 8).

In sheep, a TRH-stimulated increase in the secretion of PRL impaired the estradiol-induced surge of gonadotropins (9). Furthermore, the acute administration of PRL into the lateral cerebral ventricles of ewes provoked a significant reduction of endogenous PRL secretion (10), an effect that most likely involved the activation of dopaminergic networks within the hypothalamus. Because, in this species, dopamine is also a potent inhibitor of GnRH/gonadotropin release (11, 12, 13, 14), this system may provide the neural link for the interaction between the secretions of gonadotropins and PRL. This inference is supported by observations in the rat, showing that PRL augments the concentrations of dopamine in hypophysial portal blood (15) and stimulates dopamine output from perfused hypothalamic fragments (16). However, in a recent preliminary study (17), we found substantial evidence for a specific gonadotroph-lactotroph association, suggesting that, in sheep, another mechanism of control may be operating locally within the pituitary to regulate gonadotropin release.

The referred effects of PRL on the GnRH/gonadotropic axis might consequently be exerted at a hypothalamic and/or pituitary level and require the presence and activation of specific receptors within those tissues. Indeed, after the cloning of the complementary DNA (cDNA) encoding the rat PRL receptor (PRL-R) (18), PRL-R messenger RNA (mRNA) expression was reported in the rat pituitary gland (19), as well as in the hypothalamus (19, 20). In sheep, preliminary data from our group have provided evidence for PRL-R gene expression within those tissues (17, 21). In view of the physiological relevance and the clinical implications of the dramatic temporal changes in fertility shown by this species, we have extended our studies to further investigate the expression of PRL-Rs within the sheep pituitary as a possible key component of the proposed mechanism of control. Here we show the expression of PRL-R mRNA in the sheep pituitary gland, the changes throughout the seasonal reproductive cycle, and the specific translation of the PRL-R signal in pituitary gonadotrophs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissues
Sheep tissues (pituitary, liver, adrenal) for the detection of PRL-R mRNA and PRL-R protein expression by RT-PCR, Northern, and Western analysis, were obtained from ewes of known photoperiodic history selected from our flock (n = 8). Animals were killed with an overdose of pentobarbitone, and tissues were dissected out immediately post mortem and frozen in liquid nitrogen within 5 min of death. For immunocytochemical identification of pituitary PRL-R, sheep pituitary glands were collected immediately after death, halved either transversally or sagittally (to include the pars tuberalis), and then immersed in Bouin’s solution for 16 h at room temperature. After subsequent immersion in 70% ethanol for 24 h, tissues were processed and embedded in paraffin using an automatic processor (Shandon 2LE, Life Sciences International, Basingstoke, Hampshire, UK). All experimental procedures were conducted in accordance with the Home Office Animals (Scientific Procedures) Act 1986 of the United Kingdom.

RT-PCR
Total RNA was extracted and purified following the method described by Chomczynski and Sacchi (22). Two sets of oligonucleotide primers were designed from the bovine PRL-R cDNA sequence and were synthesized using an PE Applied Biosystems 391 DNA synthesizer. Primers 1 (sense, 5'-GCAGATGGAGGACTTCCTACCAATTA-3') and 2 (antisense, 5'-GCAGGTCACCATGCTATAGCCCTT-3') were predicted to generate a 645-bp product common to both the long and short forms of the PRL-R expanding from nucleotide 240 (extracellular domain) to nucleotide 884 (intracellular domain) of the bovine PRL-R cDNA (23). Primers 3 (sense, 5'-CTGGTTGGTTCATTATCCAGTACG-3') and 4 (antisense, 5'-CCTTCCACGCTTGTGCTCTGAG-3') were predicted to generate a 727-bp product specific to the long form of the PRL-R expanding from nucleotide 568 (extracellular domain) to nucleotide 1294 (intracellular domain) of the bovine PRL-R cDNA. cDNA was synthesized using a first-strand cDNA synthesis kit (Advantage RT-for-PCR Kit, CLONTECH, Cambridge, UK). Briefly, 1 µg total pituitary RNA was incubated with 20 pmol oligo (dT)18 primer in a total vol of 13.5 µl for 2 min at 70 C and then placed on ice. A reaction mix (6.5 µl), comprising buffer (50 mM Tris-HCL, pH 8.3, 75 mM KCL, 3 mM MgCl2), 0.5 mM each deoxynucleotide triphosphate, 1 U ribonuclease inhibitor, and 200 U Moloney murine leukemia virus RT, was added to each tube and the tubes then incubated at 42 C for 1 h. The reaction was stopped, by heating the tubes to 94 C for 5 min, and subsequently diluted to a final vol of 100 µl with H2O.

The cDNA was then amplified by incubating 5 µl RT product with 1.25 U Taq DNA polymerase (Gibco BRL, Paisley, UK) in buffer (50 mM KCl, 1.5 mM MgCl2, 20 mM Tris-HCl, pH 8.4, 0.1% Triton-X-100) with 0.2 mM each deoxynucleotide triphosphate, and 50 pmol each of primers 1 and 2, or 3 and 4, as appropriate, in a total vol of 50 µl. A control tube was run in parallel in which water replaced the total RNA. The PCR amplification conditions were: an initial denaturation step at 95 C for 3 min, followed by 30 cycles of denaturation at 95 C for 30 sec, annealing at 58 C for 1 min, and extension at 70 C for 3 min; a final extension period at 70 C for 10 min completed the amplification.

After amplification, 15 µl of each PCR reaction were electrophoresed through a 2% agarose gel containing 0.5 µg/ml ethidium bromide and the RT-PCR products visualized under UV light. These RT-PCR products were then subcloned into the TA cloning vector, pCRII (Invitrogen, Leek, The Netherlands), and the resulting clones were sequenced using an PE Applied Biosystems 373A automated sequencer. The sequences of our cDNA products and those of known PRL-R cDNAs were compared using the Genejockey II sequence analysis software (Biosoft, Cambridge, UK).

Northern blot analysis
PRL-R cDNA for Northern analysis was generated from plasmid DNA by PCR amplification using SP6 and T7 oligonucleotide primers. The resulting PRL-R cDNA probes (both common and long-form) were purified through spin columns (Chromaspin TE-100, CLONTECH) and labeled to high specific activities (1 x 109 cpm/µg) with [{alpha}-32P] deoxycycine triphosphate using a random primed DNA labeling kit (Rediprime, Amersham, Buckinghamshire, UK).

Total RNA (20 µg) or poly(A)+ RNA (10–20 µg) was denatured at 60 C for 5 min in 50% formamide, 17.4% formaldehyde, 20 mM 3-(N-morpholino)propanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA. RNAs were then fractionated by electrophoresis through 1.5% agarose/0.66 M formaldehyde gels and transferred to Hybond-N membranes (Amersham). After air drying and UV cross-linking, the membranes were prehybridized at 65 C for 6 h in prehybridization buffer: 0.5 M sodium phosphate, pH 7.2, 1 mM EDTA, pH 8.0, 7% (wt/vol) SDS, 1% (wt/vol) BSA, and 15% (vol/vol) deionized formamide. Hybridization was carried out in fresh prehybridization buffer containing 1.5 x 106 cpm/ml 32P-radiolabeled cDNA probe overnight at 65 C. Membranes were subsequently washed in 2 x saline-sodium citrate (1 x saline-sodium citrate being 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at 65 C (1 x 5 min, 1 x 10 min, and 1 x 20 min washes) and then exposed to a phosphorimaging system screen for analysis (PhosphorImager 425, Molecular Dynamics, Inc., Chesham, Buckinghamshire, UK). Efficiency of loading was determined by reprobing the membranes with a rat 18S ribosomal RNA oligonucleotide probe.

Immunoprecipitation and Western blot analysis
Tissue samples (sheep liver and pituitary) were homogenized (100 mg/ml) in ice-cold 0.1% SDS, 1 mM phenylmethylsulfonylfluoride, and 10 ng/ml aprotinin for 30 sec (Kinematica polytron, Lucerne, Switzerland). Homogenates were then centrifuged at 2500 rpm for 10 min, and the supernatant was assayed for protein concentration (protein assay kit, Bio-Rad Laboratories Ltd., Hemel Hempstead, Hertfordshire, UK). This crude cell lysate was analyzed by immunoprecipitation and Western analysis by two different methods. In the first method, 300 µg protein extract was incubated overnight at 4 C in 1 ml immunoprecipitation buffer (1% Triton-X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonylfluoride, 0.5% NP-40) and 10 µl (1:100 final dilution) of PRL-R antibody, either R120 (common domain) or R118 (long form) (24). These antibodies were raised in rabbits (25) using oligopeptides designed from the proposed sequences of the rat liver PRL-R (18, 26) with residues 53–68 (R120) of the external domain common to both long and short forms of the receptor and residues 309–325 (R118) specific to the intracellular region of the long-form PRL-R. After incubation with primary antibodies, protein A sepharose (50 µl 10% solution; Sigma Chemical Co., Poole, Dorset, UK) was added and the mixture incubated for a further 1 h at 4 C. The sepharose complex was collected by centrifugation and washed three times in immunoprecipitation buffer. The pellets were resuspended in 30 µl gel loading buffer (125 mM Tris-HCl, pH 8.0 containing 4% SDS, 2% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue), denatured by boiling for 5 min, and centrifuged for 5 min. Samples (10 µl) were electrophoresed through a 12% SDS-PAGE minigel system and electrotransferred to polyvinylidene diflouride (PVDF) membrane (Millipore Corp., Bedford, MA). The membranes were incubated sequentially at room temperature, with 4 x 5-min washes in Tris-buffered saline (TBS, pH 7.4) containing 0.05% Tween (TBS-T) between steps, in: 1) 5% milk powder in TBS-T for 1 h; 2) PRL-R R118 or PRL-R R120 antibody, as appropriate, both 1:500 dilution, for 1 h; 3) biotinylated swine antirabbit immunoglobulins (1:2500, Dako Ltd., High Wycombe, Buckinghamshire, UK) for 1 h; 4) avidin-biotin complex conjugated with horseradish peroxidase (Dako Ltd.) for 1 h; and 5) enhanced chemiluminescence detection solution (ECL, Amersham) for 1 min, followed by 15 sec exposure to Hyperfilm-ECL (Amersham). Control incubations were performed by replacing the primary antibodies with normal rabbit serum (1:500, Dako Ltd.). In the second method, 100 µg protein extract was incubated overnight at 4 C in 1 ml immunoprecipitation buffer with 5 µl PRL-R R120 antibody (1:200 final dilution). Sheep antirabbit IgG Dynabeads (50 µl; Dynal, Bromborough, Merseyside, UK) were then added, and the mixture was incubated for a further 2 h at 4 C. The magnetic beads were collected on a magnet and washed three times in 0.01 M PBS (pH 7.4) containing 0.1% BSA, resuspended in 20 µl gel loading buffer, and denatured by boiling for 5 min. The beads were collected on a magnet, and the remaining supernatant was electrophoresed through a 7.5% SDS-PAGE 16 cm x 16 cm gel and electrotransferred to PVDF membrane, as before. The membranes were incubated sequentially at room temperature, with 3 x 15 min washes in TBS-T between steps, in: 1) 5% donkey serum (SAPU, Carluke, Lanarkshire, UK) in TBS-T overnight at 4 C; 2) PRL-R R120 antibody (1:500 dilution in 5% donkey serum) alone, or R120 antibody preadsorbed for 24 h at room temperature with 100 µg/ml PRL-R peptide, for 2 h at room temperature; 3) donkey antirabbit IgG linked to horseradish peroxidase (1:2000 in TBS-T, Amersham) for 1 h at room temperature; and 4) ECL detection solution (Amersham) for 1 min, followed by 15 sec exposure to Hyperfilm-ECL. The PRL-R peptide used for the preadsorption control was newly synthesized using the same sequence originally used to raise the R120 antibody.

Immunocytochemistry
Pituitary sections were cut at 6 µm, mounted onto TESPA-coated slides, and dried overnight at 60 C. Sections were subsequently dewaxed, rehydrated, and washed in 0.05 M TBS. Nonspecific binding sites were blocked with normal donkey serum and/or normal goat serum (1:5 dilution in TBS) for 30 min at room temperature, and sections were then incubated overnight at 4 C in a humidity chamber with specific primary antibodies (diluted in 1:5 blocking serum). Each antibody was used first in single staining to determine the optimal working dilutions. For double immunostaining, the PRL-R polyclonal antibody R120 (1:50 dilution) was used in combination with one of the following mouse monoclonal antibodies: 1) bovine LHß-subunit (518 B7; 1:100; gift from Dr. J. F. Roser, University of California-Davis); 2) ovine FSHß-subunit (1:50; gift from Dr. K. Henderson, AgResearch, Wallaceville, New Zealand); 3) human free {alpha}-subunit (AHT 20; 1:100; gift from Dr. J.-M. Bidart, Institut Gustave-Roussy, Villejuf, France); 4) ovine PRL (K32; 1:100; DRG International Inc., Mountainside NJ); or 5) bovine GH (GH1/D4; 1:100; gift from Dr. M. Wallis, University of Sussex, UK). In addition, a double staining for LH and PRL was conducted using the same monoclonal antibody to the LHß-subunit (518 B7; 1:100) and a rabbit polyclonal antibody raised in house against ovine PRL (ASMcN R50; 1:200). After exposure to primary antibodies, sections were rinsed (2 x 5 min) in 0.1 M PBS containing 0.1% BSA (PBS/BSA, pH 7.6) and incubated sequentially (double stainings) in secondary antibodies, i.e. donkey antirabbit serum conjugated to fluorescein (SAPU) and goat antimouse serum conjugated to rhodamine (Sigma Chemical Co.), both diluted 1:20 in PBS/BSA, for 60 min (each) at room temperature. For single stainings, only one of the two secondary antibodies was used. Sections were then rinsed (4 x 5 min) in PBS/BSA and coverslip mounted with Citifluor (University of Kent Chemical Laboratory, Canterbury, Kent, UK). Controls included: 1) omission of primary antibodies; 2) replacement of primary antibodies by normal rabbit serum and/or normal mouse serum; 3) preadsorption of primary antibodies with the specific antigen (i.e. LH, FSH, PRL, GH or PRL-R peptide newly-synthesized with the same sequence as the one used for immunization); 4) preadsorption of primary antibodies with nonspecific antigens (positive control); and 5) use of nonspecific secondary antibody in single stainings.

Statistical analysis
The effects of reproductive status (i.e. anestrus vs. breeding season) on the expression of pituitary PRL-R mRNA transcripts determined by Northern analysis were examined by ANOVA for a completely randomized design.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PRL-R mRNA in the sheep pituitary gland
RT-PCR, using primer sets predicted to amplify either the coding region common to both short and long forms of the PRL-R or, specifically, the long form (Fig. 1AGo), revealed the existence of PRL-R mRNA in the sheep pituitary gland. Primers 1 and 2 amplified a 645-bp sequence, as expected (Fig. 1BGo). Primers 3 and 4 amplified the predicted 727-bp sequence and, in addition, a 766-bp product of similar intensity to the latter (Fig. 1BGo). These products were then subcloned and fully sequenced, which revealed that they all had homology to the PRL-R. The sequences of these 645- and 727-bp products overlapped, producing a composite sequence of 1006 bp that had more than 95% identity with the bovine PRL-R cDNA sequence (23). The 766-bp product was found to contain a 39-bp insert, in the cytoplasmic domain, identical to that reported by Anthony et al. (27). Our nucleotide sequence data (not shown) has been deposited in the EMBL, GenBank, and DDBJ Nucleotide Sequence Database under accession numbers Y10578 and Y10808.



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  		Figure 1. A, Schematic representation of PRL-R mRNA. The 5' and 3' untranslated regions are shown by straight lines and the coding region by boxes. Open boxes show extracellular and cytoplasmic domains common to both the long and short forms of the PRL-R, and the black box indicates the position of the common transmembrane domain. The hatched box shows the cytoplasmic domain specific to the long form of the receptor. Two sets of primers were used to generate either common form (i.e. will amplify both long and short PRL-R; primers 1 and 2) or long-form (primers 3 and 4) RT-PCR products. The positions of these primers are shown, as are the sizes of the expected products. Primers 3 and 4 were found to amplify an additional RT-PCR product with a 39-bp insert (see Results). The location of this insert in the sheep PRL-R is depicted by the triangle. B, RT-PCR analysis of the PRL-R cDNA in the sheep pituitary gland. RT-PCR reactions were separated on a 2% agarose gel containing 0.5 µg ethidium bromide/ml, visualized under UV light and photographed. Lane A, Negative control (no RT template); lane B, primers 1 and 2 with 5 µl sheep pituitary RT template; lane C, primers 3 and 4 with 5 µl sheep pituitary RT template. The numbers show the size of the PCR products (in bp).

 
Northern analysis for the PRL-R mRNA, using both the common and long-form cDNA probes, revealed identical transcript sizes. The results for the common form cDNA probe only are shown in Fig. 2Go. In the pituitary, three major transcripts of approximately 13, 10, and 3.5 kb were identified. The 3.5-kb transcript was more abundant in liver and adrenal tissue than in the pituitary. The larger-size mRNA transcripts seemed to be slightly larger in the liver than in the pituitary or adrenal. An additional mRNA transcript of approximately 12 kb was also present in liver and adrenal. However, because no large-size markers are available, it is difficult to determine the exact size of the large mRNA transcripts detected.



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  		Figure 2. Northern blot analysis of poly A+ mRNA from sheep pituitary (20 µg), liver (10 µg), and adrenal (10 µg), hybridized with 32P-labeled sheep pituitary common-form PRL-R cDNA probe. The positions of RNA size markers are shown.

 
A comparison of the abundance of the 13- and 10-kb PRL-R mRNA transcripts in the pituitaries of anestrous and breeding season (follicular phase) ewes is shown in Fig. 3Go. There was no significant effect of season on the expression of either of the two transcript sizes measured.



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  		Figure 3. Quantification of 13- and 10-kb pituitary PRL-R mRNA transcripts in anestrous- and breeding-season (late follicular phase) ewes. Total RNA (20 µg) from individual sheep pituitary samples was run on Northern blots and quantified for PRL-R mRNA transcripts using a phosphorimaging system. Values are means ± SEM, n = 6.

 
Western analysis
After immunoprecipitation, the polyclonal antibodies R120 and R118 both detected bands of approximately 95, 50, and 27 kDa under reducing conditions in both liver and pituitary gland (Fig. 4AGo). These three immunoreactive species were also detected with a different method using a larger gel for more accurate protein separation (Fig. 4BGo); however, not only were additional bands running at 220 and 120 kDa observed with this method, but the 50-kDa band shown in Fig. 4AGo was found to be composed of two distinct species running at about 46 and 62 kDa. The 95-kDa band, detected with the two antibodies and by the two methods, was blocked in control samples of both tissues by preadsorption of the R120 antibody with the specific peptide (Fig. 4BGo); this band is thus specific and corresponds to the expected size of the long-form PRL-R. In contrast, the strong band of about 46 kDa was nonspecific, because it was present in control samples after preadsorption (Fig. 4BGo) and also when the antibodies were replaced by normal rabbit serum (data not shown). Interestingly, the better protein separation obtained in the larger gel revealed the presence of a specific immunoreactive signal of approximately 62 kDa; this band may represent the partial degradation of the long-form PRL-R, despite the addition of protease inhibitors to buffers, or perhaps a dimer of the truncated form of the receptor (because it was detected by the R120 antibody). Similarly, the specific bands of 27 kDa (detected by both methods) and 220 and 120 kDa (observed only in the larger gel) may represent modified PRL-R-related species.



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  		Figure 4. Western analysis of PRL-R in sheep pituitary (P) and liver (L). A, Protein lysates were immunoprecipitated with either PRL-R R118 antibody (which specifically recognizes long-form PRL-R) or PRL-R R120 (which recognizes both long and short forms) and protein-A sepharose, run on 12% SDS-PAGE minigels and transferred to PVDF membranes. The blots were probed with PRL-R R118 or PRL-R R120, as appropriate. B, Protein lysates were immunoprecipitated using PRL-R R120 antibody and sheep antirabbit IgG dynabeads, run on large format 7.5% SDS-PAGE gels, and transferred to PVDF membrane. The blots were probed with PRL-R R120 antibody alone or R120 antibody that had been preadsorbed with synthetic PRL-R peptide. The Mr of protein markers are shown.

 
Expression of PRL-R proteins in specific pituitary cell types
The expression and distribution of PRL-R in specific pituitary cell types was studied by immunocytochemistry using double immunofluorescent staining with PRL-R R120 antibody and monoclonal antibodies specific to the LHß-subunit, FSHß-subunit, common {alpha}-subunit, PRL, and GH. The R120 antibody is a polyclonal antibody to the rat liver PRL-R that recognizes both the long and short PRL-R forms in the rat (24). When, in a preliminary study, this antibody was used on its own in single staining, PRL-R immunoreactivity was observed throughout the pituitary but particularly localized in the dorsal and ventral regions and laterally to the pars intermedia. Identical distribution was detected with the PRL-R antibody R118, which selectively recognizes the long form of the receptor (data not shown). Because the intensity of the staining reaction was higher with the antibody R120, this antibody was used in the double-staining studies. Figure 5Go shows the expression of PRL-R in specific pituitary cell types. The two upper panels (A and E) reveal that all cells expressing PRL-R immunoreactivity are gonadotrophs positive for LHß. However, not all LHß-positive cells express PRL-R (arrows). In fact, out of 5,173 LH gonadotrophs counted, only 53% (i.e. 2,723) showed PRL-R immunostaining. Similarly, the majority of cells expressing PRL-R immunoreactivity were positive for FSHß (Fig. 5Go, B and F). However, a small proportion (2%) of PRL-R positive cells were negative for FSHß (arrow). The double staining for the common {alpha}-subunit and PRL-R produced results similar to those for LH gonadotrophs (data not shown). In contrast to these observations, PRL-R immunoreactivity was completely absent in lactotrophs (Fig. 5Go, C and G) and in almost all somatotrophs (Fig. 5Go, D and H). Indeed, less than 2% of GH positive cells expressed PRL-R immunoreactivity.



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  		Figure 5. Double immunofluorescent staining for LHß (A), FSHß (B), PRL (C), or GH (D), and PRL-R (E–H) in the ovine pituitary pars distalis, for LHß (I), and PRL (J) in the pars distalis, and for LHß (K) and PRL-R (L) in the pars tuberalis. Paraffin-embedded sections were incubated with PRL-R polyclonal antibody R120, which recognizes both long and short PRL-Rs, and mouse monoclonal antibodies specific to the LHß-subunit, FSHß-subunit, PRL, or GH. A polyclonal antibody raised against ovine PRL (ASMcN R50) was also used in combination with the LHß monoclonal antibody. Subsequently, sections were incubated with donkey antirabbit serum conjugated to fluorescein and goat antimouse serum conjugated to rhodamine. Note that: 1) all cells expressing PRL-R immunoreactivity, in both the pars distalis and the pars tuberalis, are positive for LHß, but not all LHß-positive cells express PRL-R (arrows, A and K); 2) a small proportion of cells expressing PRL-R (arrow, F) are negative for FSHß, indicative of the specific expression of the receptor in LH (or LH/FSH gonadotrophs); 3) lactotrophs and the vast majority of somatotrophs do not express PRL-R immunoreactivity; and 4) gonadotrophs are completely surrounded by lactotrophs (examples indicated by arrows and arrowheads in I and J, respectively). No staining was observed in control sections where the first antibodies were omitted or replaced by normal rabbit serum or normal mouse serum (e.g. inset in A: monoclonal LHß antibody replaced by normal mouse serum), nor was it detected in control sections incubated with nonspecific secondary antibodies; preadsorption of primary antibodies with specific antigens blocked immunoreactive staining (e.g. inset in E: polyclonal PRL-R antibody preadsorbed with specific peptide; see Materials and Methods). Magnification, x500 (except for D, H, K, and L, x400; insets in K and L, x100).

 
The distribution of gonadotrophs and lactotrophs throughout the pituitary was studied by immunocytochemistry using double immunofluorescence staining with the same monoclonal antibody for LHß as above and a polyclonal antibody to ovine PRL (ASMcN R50). The study revealed that, in the pars distalis, the gonadotrophs (Fig. 5IGo) are completely surrounded by lactotrophs (Fig. 5JGo). Indeed, lactotrophs appear always in clusters of cells, whereas gonadotrophs were shown to be isolated, i.e. not in contact with each other. No staining for PRL was observed in the pars tuberalis (data not shown). However, the specific localization of PRL-R immunoreactivity in gonadotrophs of the pars distalis was corroborated in the pars tuberalis. All cells expressing PRL-R immunoreactivity were positive for LHß (Fig. 5Go, K and L). No staining was detected in control sections where the first antibodies were omitted or replaced by normal rabbit serum or normal mouse serum (Fig. 5Go, insert in A), nor was it observed in control sections incubated with nonspecific secondary antibodies; furthermore, preadsorption of primary antibodies with specific antigens blocked immunoreactive staining (Fig. 5Go, insert in E), whereas preadsorption with nonspecific antigens did not affect it. These five methods of control corroborate the high specificity of immunoreactivity; in addition, the blockade of PRL-R immunostaining with the specific peptide (Fig. 5Go, insert in E) confirm the results from Western analysis for the expression of PRL-R protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated the expression of PRL-R mRNA in the ovine pituitary gland. This finding is in agreement with a previous report in the rat where PRL-R gene expression was observed in this tissue (19). The cDNAs amplified by RT-PCR in the current study were shown to be similar to those reported previously for the rat (18), cow (23), and sheep liver and corpus luteum (27); indeed, comparison of the nucleotide sequences revealed high identity (95.6%) with the bovine PRL-R cDNA sequence (23). In the rat, two forms of the PRL-R, short (18) and long (26) differ in the sequence and length of their cytoplasmic domains and were proposed to result from alternative splicing of a common primary transcript (28). Both forms were detected in the rat pituitary, but in general, the expression of these two variants is tissue specific; whereas the short form is more abundant in the liver, the long form is highly expressed in the gonads (26). Although it is thought that these forms may mediate different actions of PRL, a role for the short form is still unknown. In contrast, the long form was reported to mediate PRL-induced signal transduction (29, 30) and is responsive to posttranscriptional regulation by PRL (24, 31). In this study, using RT-PCR with primers designed to amplify specifically the long form of the PRL-R, we isolated two cDNAs, one of which had a 39-bp insert in the cytoplasmic domain. This insertion is identical to that reported by Anthony et al. (27) in ovine fetal liver and adult luteal tissue; the insertion site corresponds to amino acid 263 of the bovine PRL-R. It is predicted that the mRNA containing these additional 39 nucleotides should encode for a truncated form of PRL-R, because the 11 amino acids resulting from the 39-bp insert are followed by two in frame stop codons. The point of insertion in the cytoplasmic domain is the same in which the sequences of the two forms of the receptor in the rat begin to differ (18, 26). However, in sheep, the nucleotide sequences of the two cDNAs are identical also after the insertion. Therefore, it seems that, in this species, the long form of PRL-R mRNA is expressed with two variants, one of which has a cytoplasmic insert that will result in a truncated form of the receptor. In a recent publication, the full-length coding sequence for the ovine PRL-R cDNA was reported, corroborating the existence of a second cDNA bearing the 39-bp insert (32). Genomic analysis revealed that the presence or absence of this insert was the result of alternative splicing of a common transcript, rather than of the existence of different genes (32).

The expression of PRL-R mRNA in the sheep pituitary gland was corroborated by Northern analysis. The two cDNA probes used, i.e. one that does not distinguish between short and long forms of the receptor and the other one specific to the long form, gave identical results supporting the specific expression of the long form of PRL-R mRNA in this tissue. The three major-sized transcripts detected in the pituitary were also observed in liver and adrenal. The 3.5-kb band, however, was less abundant in the pituitary than in the other two tissues and, therefore, could not be quantified to compare the effects of reproductive status (breeding season vs. anestrus) on the expression of this mRNA. Interestingly, this transcript was similar to a major transcript of approximately 3.8 kb detected in bovine liver, endometrium, and corpus luteum (23). In addition, the three transcripts described in this study were of similar size to three out of the five reported for the human decidua PRL-R mRNA (33, 34). The reason for and physiological significance of different-sized transcripts are, as yet, to be elucidated. The heterogeneity may arise from the existence of multiple polyadenylation or transcription start sites or linked to alternative splicing. An important finding of this study is that when the abundance of the two largest-sized transcripts (approximately 10 and 13 kb) in the pituitary was analyzed throughout the annual reproductive cycle, no differences were apparent between sexually active animals in the breeding season and anestrous ewes. This implies that possible effects of PRL on the annual reproductive cycle are not mediated through changes in pituitary PRL-R mRNA expression.

Translation of pituitary PRL-R mRNA was confirmed, in the first instance, by Western analysis. In both pituitary and liver (positive control tissue), a specific band of approximately 95 kDa was identified using the PRL-R R120 antibody that recognizes both variants of the receptor; this band was blocked by preadsorption with synthetic peptide. The same-sized band was observed with the PRL-R R118 antibody specific to the long form. The intensity of the signal, however, was slightly stronger with the R120 antibody, suggesting that the amino acid sequence resulting from the 39-bp insertion may not be recognized by the R118 antibody. In addition, the antibody R120 matches 11 out of 17 targeted amino acids, according to the bovine or ovine sequence, whereas the antibody R118 matches 11 out of 16. In agreement with these results, PRL-R protein bands of similar size were reported for the long form of PRL-R in bovine endometrium (31) and for the human PRL-R in decidua (34). The major 46-kDa band observed in this study was nonspecific and is likely to represent Ig heavy chain, because it was also present when normal rabbit serum was used instead of primary antibodies and, in addition, was not blocked by preadsorption controls. The remaining immunoreactive signals of approximately 120, 62, and 27 kDa are of similar size to PRL-R proteins shown previously for the rat prostate (24), bovine liver and endometrium (31), and human decidua (34). Indeed, considerable size heterogeneity has been reported for the PRL-R protein in several species (31, 33, 34, 35). As indicated previously, the 220- and 120-kDa bands may represent modified types of the long-form PRL-R. The 62- and 27-kDa bands, however, may reflect proteolytic degradation (despite the addition of protease inhibitors to samples), although it is not possible to distinguish between proteolytic artifacts and specific posttranscriptional proteolytic cleavage. Alternatively, the 62-kDa immunoreactive species may represent a dimer of the truncated form of the receptor, which is predicted to migrate at approximately 33 kDa. Differences in molecular size might also be related to different degrees of glycosylation, which may be responsible for differences of up to 10 kDa in the mobility of PRL-R proteins during electrophoresis on SDS-PAGE (31). Nevertheless, our observation that the same-sized band of 95 kDa was found with the two antibodies is consistent with the results for gene expression, and with previous work in the cow and human, and provides evidence that in the sheep pituitary the long form of PRL-R is predominantly expressed.

An important finding of this study is that translation of the PRL-R signal is cell specific, i.e. the PRL-R protein was found to be expressed only in the gonadotroph. This observation strengthens the long-term proposed association between PRL and gonadotropin secretion. Moreover, the specific cytological organization of the sheep pituitary observed in this study, which revealed that gonadotrophs are surrounded by lactotrophs, is supportive of a functional role of the PRL-R expressed in gonadotropin-secreting cells. This implies that, in addition to the reported central effects of PRL on the inhibitory regulation of GnRH/gonadotropin secretion (9, 36, 37, 38, 39), PRL is likely to participate in the control of gonadotropin release also at the pituitary level. Indeed, studies in the rat have shown that PRL is capable of suppressing LH release from pituitary fragments (6, 16) and of reducing the percentage of pituitary cells secreting LH in vitro (2). Similarly, the in vivo and in vitro LH response to exogenous GnRH administration was shown to be reduced in hyperprolactinemic rats, compared with controls (7, 8). The possible direct effects of PRL on gonadotropin secretion in the rat are further supported by cytological studies showing functional contacts between lactotrophs and gonadotrophs (40) and paracrine interactions between these two cell types in pituitary cell aggregates (41). In addition, PRL binding sites have been identified in rat primary pituitary cell cultures (42). However, in contrast to the results of the current study, a recent report indicated that all pituitary cell types seemed to express PRL-R immunoreactivity in this species (43). Similarly, in the human, a recent publication has provided evidence for the presence of PRL-R in several pituitary cell types, including not only gonadotrophs but also lactotrophs (44). PRL-R in human lactotrophs had been previously identified by radioreceptor assay (45) and were suggested to mediate the regulation of PRL on its own secretion at the pituitary level, a mechanism of autoregulation originally proposed for the rat (46, 47, 48). In sheep, the specific expression of PRL-R in gonadotrophs observed in this study implies that the aforementioned role of PRL on gonadotropin secretion directly at the pituitary level is perhaps of more physiological significance in a species in which dramatic changes in fertility occur throughout the annual reproductive cycle (49). To our knowledge, this is the first report showing a selective association between gonadotrophs and lactotrophs in the sheep pituitary gland and the specific expression of PRL-R in gonadotropin-secreting cells. These findings are suggestive of a paracrine role of PRL on gonadotroph function in this species. The specific nature of that role, however, remains to be elucidated. Although possible, it is unlikely that the proposed effects of PRL involve dramatic changes in gonadotroph responsiveness to GnRH. In this species, the in vivo LH response to GnRH in ovariectomized ewes was not affected by the level of prolactinemia (9). Moreover, pulsatile exogenous administration of GnRH to seasonal anestrous ewes, where PRL release is at maximum, induced reciprocal pulsatile secretion of LH similar to that observed during the follicular phase of the estrous cycle, when PRL concentrations are low (50). The lack of difference in PRL-R mRNA expression between breeding season and anestrous ewes reported here is in agreement with that observation. Because PRL is capable of inducing the expression of its own receptor (24, 51), our finding may merely reflect constant high concentrations of PRL within the pituitary throughout the seasonal reproductive cycle. Nevertheless, in view of the fact that only 53% of the total pituitary gonadotroph population express PRL-R, it remains plausible that PRL participates in recruiting the proportion of gonadotrophs that will polarize in response to a GnRH surge (52). Alternatively, PRL may be involved in the pituitary control of gonadotropin secretion by modulating the feedback actions of gonadal steroids (53), because it is well established that estrogens induce PRL secretion in rodents (54, 55), and that estradiol can stimulate PRL release by acting directly on the lactotrophs (56, 57). Thus, through specific receptors in the surrounded gonadotroph, PRL may mediate steroid hormone effects on gonadotropin secretion locally within the pituitary.

In conclusion, in this study, we have shown: 1) the expression of PRL-R mRNA in the sheep pituitary gland; 2) the occurrence of two variants of the long-form PRL-R mRNA in this tissue, one of which bears a 39-bp insert with a premature stop codon; 3) the specific expression of the translated PRL-R protein in gonadotrophs of the pars distalis and pars tuberalis; and 4) a selective association between gonadotrophs and lactotrophs in the pars distalis, where gonadotrophs appear surrounded by PRL secreting cells. These findings are supportive of a role of PRL in the regulation of gonadotropin secretion in sheep and suggest the existence of a paracrine mechanism within the pituitary gland.


   Acknowledgments
 
We would like to thank Dr. J. F. Roser (University of California-Davis) and Quidel Corporation (San Diego, CA) for the gift of LHß antibody; Dr. K. M. Henderson (AgResearch, New Zealand) for the gift of FSHß antibody; Dr. J.-M. Bidart (Departement de Biologie Clinic, Institut Gustave-Roussy, France) for providing the free {alpha}-subunit antibody; Dr. M. Wallis (University of Sussex, UK) for GH antibody; and SAPU (Carluke) for supplying donkey antirabbit fluorescein isothiocyanate antibody. We thank Miss N. Anderson for the care of the animals, Mr. M. Millar for his assistance in immunocytochemical studies, Dr. H. Jabbour for helpful discussions, and Mr. T. McFetters and Mr. E. Pinner for the art work. The nucleotide sequence data has been deposited in the EMBL, GenBank, and DDBJ Nucleotide Sequence Database under accession numbers Y10578 and Y10808.


   Footnotes
 
1 Address all correspondence and requests for reprints to the following current address: Dr. Domingo J. Tortonese, Department of Anatomy, University of Bristol, Southwell Street, Bristol, BS2 8EJ, United Kingdom. Back

Received February 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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