27 Neurokinin B, Kisspeptin, and Gnrh Hormone Analogs Fail to Stimulate Secretion of Luteinizing Hormone (LH) in KISS1 Knockout Boars

Abstract

Abstract We generated kisspeptin knockout (KISS1-/- ) pigs exhibiting hypogonadotropic hypogonadism and pubertal failure. Humans and rodents with inactivating KISS1 mutations exhibit increased gonadotropin secretion in response to GnRH and kisspeptin analogs, but it is unknown if KISS1-/- pigs respond similarly. Our goal was to define endocrine responses of KISS1-/- boars to hormone agonists that target different levels of the hypothalamic-pituitary-gonadal axis. Boars (302.6 ± 0.9 d of age) were fitted with indwelling jugular catheters to collect serial blood samples. Serum concentrations of LH and testosterone were quantified with radioimmunoassay. Data were analyzed as repeated measures with genotype (KISS1+/+ ; KISS1+/- ; KISS1-/- ) and time as fixed effects. Blood samples were collected beginning 60 to 120 min before a bolus injection (i.v.) of the agonist and continued for 5 to 48 h after treatment, depending on experiment, with at least 1 wk between studies. Boars received an agonist of either: 1) neurokinin B (senktide; 10 mg/kg BW; Exp. 1); 2) kisspeptin (C6; 15 nMol/kg BW; Exp. 2); 3) GnRH (cystorelin, 150 ng/kg BW; Exp. 3); or 4) LH (hCG; 20 IU/kg BW; Exp. 4). There was a genotype x time interaction for all experiments (P < 0.09). In the 60 min following senktide treatment (Exp. 1), mean LH increased 2.5-fold in KISS1+/+ and KISS1+/- boars, but was unchanged in KISS1-/- boars (P < 0.04). In Exp. 2, C6 increased LH concentrations in KISS1+/+ and KISS1+/- boars within 10 min (P < 0.05) and LH did not return to baseline until 28 h after treatment. However, secretion of LH in KISS1-/- boars was unaffected by C6. Next, cystorelin treatment (Exp. 3) increased circulating LH (P < 0.05) in KISS1+/+ and KISS1+/- males (2.1 ± 0.2 ng/mL), but not in KISS1-/- boars (0.5 ± 0.2 ng/mL). In Exp. 4, hCG increased mean testosterone concentrations for 48 h in KISS1+/+ (32.9 ± 6.7 ng/mL) and KISS1+/- (26.7 ± 4.7 ng/mL) boars, but testosterone in KISS1-/- animals remained at near undetectable levels after hCG administration. Therefore, we have shown for the first time that both neurokinin B and kisspeptin agonists (senktide and C6, respectively) stimulate robust secretion of LH in pigs. In addition, these agonists stimulate similar secretory patterns of LH in KISS1+/+ and KISS1+/- boars, confirming that a single functional copy of KISS1 is sufficient to confer normal reproductive endocrine function in pigs. Failure of a GnRH agonist to stimulate LH secretion in KISS1-/- boars likely reflects insufficient pituitary stores of LH, resulting from a lack of GnRH priming, and underlies the inability of either senktide or C6 to stimulate LH secretion in these animals. Lack of hCG-stimulated testosterone secretion in KISS1-/- boars may indicate a gonadotropin-independent deficit in testicular Leydig cells.