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

Abstract

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 LHbeta-subunit, FSHbeta-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 LHbeta, although only 53% of LHbeta-positive cells expressed PRL-R. A small proportion (2%) of gonadotrophs expressing PRL-R immunoreactivity were negative for FSHbeta, 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.

Expression and regulation of osteopontin and connective tissue growth factor transcripts in rat anterior pituitary

“…Again, this might be explained by the fact that in the rat, the PRL serum levels are not increased despite elevated 17 -estradiol levels (Escalada et al 1996). In this context it may be interesting to note that Opn is expressed in gonadotropic cells, a cell type that is surrounded by clusters of lactotropic cells (Horvath & Kovacs 1988, Allaerts et al 1991 and equipped with PRL receptors (Morel et al 1994, Jin et al 1997, Tortonese et al 1998.…”

Differential expression and seasonal modulation of VGF peptides in sheep pituitary

“…In sheep, lactotrope cells are increasingly stimulated from winter to summer, serum prolactin being higher during long days (from spring to summer) and lower during short days (from autumn to winter; Pelletier 1973, Ravault 1976). In the same species, the pituitary pars tuberalis is probably involved in stimulating prolactin secretion (Graham et al 2002), while prolactin may be involved in seasonal inhibition of gonadotropin release via a paracrine intrapituitary mechanism (Tortonese et al 1998), probably also involving dopamine (Gregory et al 2004). Conversely, thyrotropes can be stimulated in response to reduced ambient luminosity, in apparent connection with a higher need for heat production via increased thyroid hormone release during the winter months (Hassi et al 2001).…”

Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle

“…These antibodies had been previously tested to specifically recognize long and short sheep PRLrs by immunohistochemical and immunoblotting techniques (Tortonese et al 1998, Bispham et al 1999, Budge et al 2000, 2003.…”