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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
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
-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.
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