DETECTION OF PROLACTIN RECEPTOR GENE-EXPRESSION IN THE SHEEP PITUITARY-GLAND AND VISUALIZATION OF THE SPECIFIC TRANSLATION OF THE SIGNAL INGONADOTROPHS

In sheep, as in other mammalian species, the pronounced reduction in G nRH and gonadotropin secretion that characterizes stages of infertilit y is normally associated with a conspicuous increase in the secretion of PRL. A possible role of PRL in modulating gonadotropin release impl ies the presence and activation of specific receptors in target tissue s (i.e. pituitary, hypothalamus). In this study, we investigated the e xpression of PRL receptor (PRL-R) messenger RNA (mRNA) in the sheep pi tuitary and the distribution of the translated product in specific pit uitary cell types. Using primers designed to flank different regions o f the extracellular and cytoplasmic domains of the PRL-R, two compleme ntary DNA (cDNA) fragments, one of which was specific for the long-for m PRL-R, were amplified by reverse transcriptase-PCR. Sequencing revea led more than 95% identity with nucleotides 267-1272 of the bovine PRL -R cDNA. When these cDNA fragments were used as probes for the detecti on of PRL-R mRNA expression by Northern analysis, three major transcri pts of approximately 13, 10, and 3.5 kb were identified in the pituita ry. Both probes detected identical transcripts, suggesting that primar ily 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). Addit ional 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 m RNA was confirmed by Western blot analysis. The identification of PRL- R in specific pituitary cell types was carried out by immunocytochemis try. Double immunofluorescent staining, using antibodies to the rat li ver PRL-R and specific monoclonal antibodies to the LH beta-subunit, F SH beta-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 LH beta, although only 53% of LH beta-positive cells expressed PRL-R. A small proportion (2%) of go nadotrophs expressing PRL-R immunoreactivity were negative for FSH bet a, indicating the specific localization of PRL-R in LH (or LH/FSH) sec reting cells. Further, a selective cytological association was detecte d in the pars distalis where LH gonadotrophs appeared surrounded by la ctotrophs. In contrast to these observations, PRL-R immunoreactivity w as completely absent in lactotrophs and in the vast majority (>98%) of somatotrophs. In conclusion, here we show the expression of PRL-R mRN A in the sheep pituitary and the specific translation of the signal in LH (or LH/FSH) gonadotrophs. These results support the hypothesis tha t PRL may be involved in the regulation of gonadotropin secretion thro ugh a paracrine mechanism within the pituitary gland and that this act ion does not seem to be mediated by changes in PRL-R mRNA expression.