Heifers express G-protein coupled receptor 61 in anterior pituitary gonadotrophs in stage-dependent manner

Gonadotropin-releasing hormone receptors (GnRHRs) colocalize with insulin and glucocorticoid receptors in lipid rafts of the gonadotroph plasma membrane, where they facilitate downstream signaling. We recently found that orphan G-protein-coupled receptor (GPR)61 is expressed in the anterior pituitary (AP) of heifers, leading us to speculate that GPR61 colocalizes with GnRHR in the plasma membrane of gonadotroph and is expressed at specific times of the reproductive cycle. To test this hypothesis, we examined the coexpression of GnRHR, GPR61, and either luteinizing hormone (LH) β subunit or follicle-stimulating hormone (FSH) β subunit in AP tissue and cultured AP cells by immunofluorescence microscopy. GPR61 was detected in gonadotrophs, with a majority of them being colocalized with GnRHR and the remainder present at other parts of the cell surface or in the cytoplasm. We obtained a strong positive overlap coefficient (0.71±0.01) between GPR61 and GnRHR on the cell-surface of cultured GnRHR-positive AP cells. Real-time PCR and western blot analyses found that expression was lower (P<0.05) in AP tissues during early luteal phase as compared to pre-ovulation or mid- or late luteal phases. Additionally, the 5ꞌ-flanking region of the GPR61 gene contained several sites with response elements similar to those of estrogen or progesterone. These data suggested that GPR61 colocalizes with GnRHR in the plasma membrane of gonadotrophs, and its expression changes stage-dependently in the bovine anterior pituitary gland.