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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
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Abstract |
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-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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 [-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 -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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
revealed the existence of PRL-R mRNA in the sheep pituitary gland.
Primers 1 and 2 amplified a 645-bp sequence, as expected (Fig. 1B).
Primers 3 and 4 amplified the predicted 727-bp sequence and, in
addition, a 766-bp product of similar intensity to the latter (Fig. 1B). 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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. 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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. There was no significant effect of
season on the expression of either of the two transcript sizes
measured.
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). These three immunoreactive species
were also detected with a different method using a larger gel for more
accurate protein separation (Fig. 4B); however, not only were
additional bands running at 220 and 120 kDa observed with this method,
but the 50-kDa band shown in Fig. 4A 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. 4B); 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. 4B) 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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-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 5 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. 5, B and F). However, a
small proportion (2%) of PRL-R positive cells were negative for FSHß
(arrow). The double staining for the common -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. 5, C and G) and in almost all
somatotrophs (Fig. 5, D and H). Indeed, less than 2% of GH positive
cells expressed PRL-R immunoreactivity.
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) are
completely surrounded by lactotrophs (Fig. 5J). 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. 5, 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. 5, 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. 5, 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. 5, insert in E) confirm the results from Western analysis for
the expression of PRL-R protein. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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-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 |
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Received February 20, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 R. Flores, M. L. Looper, R. W. Rorie, D. M. Hallford, and C. F. Rosenkrans Jr Endocrine factors and ovarian follicles are influenced by body condition and somatotropin in postpartum beef cows J Anim Sci, June 1, 2008; 86(6): 1335 - 1344. [Abstract] [Full Text] [PDF]
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 H. L Henderson, J. Townsend, and D. J Tortonese Direct effects of prolactin and dopamine on the gonadotroph response to GnRH J. Endocrinol., May 1, 2008; 197(2): 343 - 350. [Abstract] [Full Text] [PDF]
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 K. Janssens, O. Krylyshkina, N. Hersmus, H. Vankelecom, and C. Denef {beta}1-Adrenoceptor Expression in Rat Anterior Pituitary Gonadotrophs and in Mouse {alpha}T3-1 and L{beta}T2 Gonadotrophic Cell Lines Endocrinology, May 1, 2008; 149(5): 2313 - 2324. [Abstract] [Full Text] [PDF]
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G. M. Anderson, D. C. Kieser, F. J. Steyn, and D. R. Grattan Hypothalamic Prolactin Receptor Messenger Ribonucleic Acid Levels, Prolactin Signaling, and Hyperprolactinemic Inhibition of Pulsatile Luteinizing Hormone Secretion Are Dependent on Estradiol Endocrinology, April 1, 2008; 149(4): 1562 - 1570. [Abstract] [Full Text] [PDF]
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 H. L Henderson, D. J Hodson, S. J Gregory, J. Townsend, and D. J Tortonese Gonadotropin-Releasing Hormone Stimulates Prolactin Release from Lactotrophs in Photoperiodic Species Through a Gonadotropin-Independent Mechanism Biol Reprod, February 1, 2008; 78(2): 370 - 377. [Abstract] [Full Text] [PDF]
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D. R. Grattan, C. L. Jasoni, X. Liu, G. M. Anderson, and A. E. Herbison Prolactin Regulation of Gonadotropin-Releasing Hormone Neurons to Suppress Luteinizing Hormone Secretion in Mice Endocrinology, September 1, 2007; 148(9): 4344 - 4351. [Abstract] [Full Text] [PDF]
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C. Brancia, P. Nicolussi, P. Cappai, G. La Corte, R. Possenti, and G.-L. Ferri Differential expression and seasonal modulation of VGF peptides in sheep pituitary J. Endocrinol., July 1, 2005; 186(1): 97 - 107. [Abstract] [Full Text] [PDF]
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R A Picazo, J P Garcia Ruiz, J Santiago Moreno, A Gonzalez de Bulnes, J Munoz, G Silvan, P L Lorenzo, and J C Illera Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle Reproduction, November 1, 2004; 128(5): 545 - 553. [Abstract] [Full Text] [PDF]
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S. J. Gregory, J. Townsend, A. S. McNeilly, and D. J. Tortonese Effects of Prolactin on the Luteinizing Hormone Response to Gonadotropin- Releasing Hormone in Primary Pituitary Cell Cultures During the Ovine Annual Reproductive Cycle Biol Reprod, May 1, 2004; 70(5): 1299 - 1305. [Abstract] [Full Text] [PDF]
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 D. Meeran, H. F. Urbanski, S. J. Gregory, J. Townsend, and D. J. Tortonese Developmental Changes in the Hormonal Identity of Gonadotroph Cells in the Rhesus Monkey Pituitary Gland J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2934 - 2942. [Abstract] [Full Text] [PDF]
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G. A. Lincoln, J. Townsend, and H. N. Jabbour Prolactin Actions in the Sheep Testis: A Test of the Priming Hypothesis Biol Reprod, September 1, 2001; 65(3): 936 - 943. [Abstract] [Full Text] [PDF]
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J. R. E. Davis, J. McVerry, G. A. Lincoln, S. Windeatt, P. R. Lowenstein, M. G. Castro, and A. S. McNeilly Cell Type-Specific Adenoviral Transgene Expression in the Intact Ovine Pituitary Gland after Stereotaxic Delivery: An in VivoSystem for Long-Term Multiple Parameter Evaluation of Human Pituitary Gene Therapy Endocrinology, February 1, 2001; 142(2): 795 - 801. [Abstract] [Full Text] [PDF]
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R. C. Eagle and D. J. Tortonese Characterization and Distribution of Gonadotrophs in the Pars Distalis and Pars Tuberalis of the Equine Pituitary Gland During the Estrous Cycle and Seasonal Anestrus Biol Reprod, September 1, 2000; 63(3): 826 - 832. [Abstract] [Full Text]
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D. Coss, C. B. Kuo, L. Yang, P. Ingleton, R. Luben, and A. M. Walker Dissociation of Janus Kinase 2 and Signal Transducer and Activator of Transcription 5 Activation after Treatment of Nb2 Cells with a Molecular Mimic of Phosphorylated Prolactin Endocrinology, November 1, 1999; 140(11): 5087 - 5094. [Abstract] [Full Text]
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