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

• Published in: Journal of Veterinary Medical Science Vol. 78; no. 11; pp. 1699 - 1702
• Main Authors: PANDEY, Kiran, NAHAR, Ashrafun, KADOKAWA, Hiroya
• Format: Journal Article
• Language: English
• Published: Japan JAPANESE SOCIETY OF VETERINARY SCIENCE 2016; Japan Science and Technology Agency; The Japanese Society of Veterinary Science
• Subjects: Animals; Antibodies; Cattle; Cells, Cultured; Confocal microscopy; Dextran; Easysep; Female; gonadotroph; Gonadotrophs - cytology; Gonadotropin-releasing hormone; Gonadotropins; Immunomagnetic Separation - methods; Luteinizing hormone; Luteinizing Hormone - metabolism; Magnetite Nanoparticles; Nanoparticles; Pituitary (anterior); Pituitary Gland, Anterior - cytology; Receptors, LHRH - immunology; Receptors, LHRH - metabolism; ruminant; Theriogenology
• ISSN: 0916-7250; 1347-7439; 1347-7439
• DOI: 10.1292/jvms.16-0157

No methods are currently available for rapidly isolating gonadotrophs from the anterior pituitary (AP) in any species. We developed a method for preparing pure bovine gonadotrophs from a heterogeneous AP cell mixture by magnetic separation and our original antibody against the N terminus of bovine gonadotropin-releasing hormone receptor (GnRHR). A bovine AP cell mixture was incubated with the anti-GnRHR antibody, anti-dextran antibody-conjugated secondary antibody and dextran-coated magnetic nanoparticles for magnetic isolation. Approximately 5.2 × 106 cells were isolated per AP of Japanese Black heifers (26 months of age) and cultured, and confocal microscopy confirmed to be GnRHR- and luteinizing hormone-positive, corresponding to a purity of 100%. Approximately 44.5 µg of total protein was extracted from the pure gonadotrophs per AP.