4 Induction of ovulation by kisspeptin in llamas

Abstract

After mating, female camelids ovulate in response to nerve growth factor (NGF) present in semen (formerly referred to as ovulation-inducing factor). The ovulatory effect appears to be induced by stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) neuronal activity and LH secretion. Recent studies have identified kisspeptin as an important mediator of GnRH secretion in several species. In the present study, we tested the hypothesis that kisspeptin is involved in the ovulatory pathway in llamas and investigated the mechanism of this effect. In Experiment 1, ovarian function in non-pregnant, non-lactating adult female llamas was synchronized by intramuscular administration of a GnRH analogue (50 µg of gonadorelin acetate; Fertiline, Vetoquinol, Quebec, QC, Canada). When a growing dominant follicle ≥8 mm in diameter was detected, llamas were assigned randomly to 3 treatment groups and given an intravenous dose of purified seminal NGF (1 mg, single dose; n = 5), kisspeptin (0.1 mg kg⁻¹ of body weight, 2 doses 1 h apart; n = 5), or PBS (n = 4). The bioactive 10 amino acid fragment of murine kisspeptin was used. Ovulation and corpus luteum development were assessed by transrectal ultrasonography every other day from the day of treatment (Day 0) to Day 8. In Experiment 2, ovarian function among female llamas was synchronized, as in Experiment 1. When a growing dominant follicle ≥8 mm in diameter was detected, llamas were given kisspeptin (0.1 mg kg⁻¹ of body weight IV, 2 doses 1 h apart) beginning 2 h after pretreatment with either a GnRH receptor blocker (cetrorelix acetate, 1.5 mg per llama IV; Sigma, Oakville, ON, Canada; n = 6) or saline (n = 6). Llamas were examined 48 h later by transrectal ultrasonography to detect ovulation and 8 days later to determine the presence of a corpus luteum. Chi-square tests were used to compare ovulation rates, and ANOVA for repeated measures was used to compare diameter profiles of the corpus luteum. In Experiment 1, ovulation rate did not differ between the NGF and kisspeptin groups (5/5 in each; 100%) and was greater than in the control group (0/4; 0% P < 0.05). Corpus luteum diameter did not differ between llamas that ovulated in response to treatment with NGF or kisspeptin (13.2 ± 0.8 and 14.0 ± 1.2 mm on Day 8, respectively). In Experiment 2, none of the llamas pretreated with cetrorelix ovulated in response to kisspeptin treatment (0/6; 0%), whereas all of the llamas pretreated with saline ovulated in response to kisspeptin treatment and had a corpus luteum at Day 8 (6/6; 100%; P < 0.05). Results supported the hypothesis that kisspeptin induces ovulation in llamas. Because a GnRH receptor antagonist blocked ovulation, our interpretation is that the site of action of kisspeptin is upstream of the pituitary gland and involves control of GnRH release from the hypothalamus. These findings raise the possibility that kisspeptin mediates the ovulation-inducing effect of NGF.

Research was supported by the Natural Sciences and Engineering Research Council of Canada.