In Vitro Kisspeptin Effects on Steroid Production

Kisspeptin-10 (rat) and kisspeptin antagonist (p234) were purchased from Tocris Bioscience (Abingdon, Oxon, UK). Kisspeptin-10 corresponds to the C-terminal region of the translated kisspeptin 54 peptide (residues 112–121), and its sequence is identical in rat and bovine. Kisspeptin-10 and p234 (antagonist) were dissolved in water and 20% (w/v) acetonitrile in water, respectively, to give a stock concentration of 10^–3 M. These were further diluted in sterile medium to give final concentrations of 10^–6, 10^–7, 10^–8, 10^–9 and 10^–10 M. In each culture model, the effects of kisspeptin-10 and kisspeptin antagonist on steroid production and viable cell number were evaluated under both basal and gonadotrophin or forskolin (FSK)-stimulated conditions as explained below. Highly purified ovine FSH (oFSH 19SIAPP) and LH (oLH-S-16) were provided by the NHPP (Torrance, CA, USA). In NLTC cultures, LH was used at a final concentration of 150 pg/mL, shown previously to elicit maximal A4 secretion (Glister et al. 2005 (link)); A4 and P4 secretion were evaluated. In NLGC cultures, FSH was used at a final concentration (0.3 ng/mL) shown previously to elicit optimal E2 secretion (Glister et al. 2001 (link), 2004 (link)); E2 and P4 secretion were evaluated. As LGC and LTC are largely unresponsive to gonadotrophin stimulation in this serum-supplemented culture model (Kayani et al. 2009 (link)), the adenylate cyclase activator, forskolin (FSK; 10 μM), was used as an alternative secretagogue; only P4 secretion was evaluated for these cells since A4 and E2 secretion are both extremely low.