Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations

Method for isolating pure bovine gonadotrophs from anterior pituitary using magnetic nanoparticles and anti-gonadotropin-releasing hormone receptor antibody

“…The FITC-positive cells observed in this study were larger than those that were negative for FITC, and their shape and size were very similar to those of bovine gonadotrophs that were immunoreactive to anti-LH and anti-GnRHR antibodies in previous studies [1, 7]. The western blot analysis indicated that the isolated cells contained LH.…”

“…We recently developed a highly specific antibody that recognizes the extracellular region at the N terminus of bovine gonadotropin-releasing hormone receptor (GnRHR) [7]. We could obtain pure bovine gonadotrophs utilizing the antibody for fluorescence-activated cell sorting analysis of bovine AP cell mixture [7].…”

“…We could obtain pure bovine gonadotrophs utilizing the antibody for fluorescence-activated cell sorting analysis of bovine AP cell mixture [7]. However, the slow speed of this method was unsatisfactory, since the system could only process one sample at a time.…”

“…The total amount of protein in 3 µl of each sample was measured with a bicinchoninic acid kit (Thermo Fisher Scientific), and 2.5 µ g of total protein was analyzed alongside bovine luteinizing hormone (LH) [AFP11118B; National Hormone and Pituitary Program (NHPP), Bethesda, CA, U.S.A.] and protein extracted from the AP tissue of a heifer for size comparison. Western blotting was carried out using a previously described protocol [7] using a mouse monoclonal anti-LH antibody (1: 40,000 dilution) [9] that recognizes the bovine LHβ subunit and does not cross-react with other bovine pituitary hormones [5]. Protein bands were visualized using a chemiluminescence kit (ECL Prime, GE Healthcare, Amersham, U.K.) and CCD system (LAS-3000 Mini; Fujifilm, Tokyo, Japan).…”

“…The cultured cells in the µ -Slide VI microscopy chamber were treated with 4% paraformaldehyde in PBS for 3 min and then with 0.1% Triton X-100 in PBS for 3 min. Immunocytochemistry was carried out as previously described [7] using the same primary antibodies for GnRHR and LH as well as Alexa Fluor 546 goat anti-mouse IgG (Thermo Fisher Scientific), Alexa Fluor 647 goat anti-guinea pig IgG (Thermo Fisher Scientific) and 4′,6′-diamino-2-phenylindole (DAPI; Wako). Cells were visualized by confocal microscopy.…”