Effect of Kisspeptin-10 on plasma luteinizing hormone concentrations and follicular dynamics during the luteal phase in cattle

Highlights

• Intravenous route of administration of Kisspeptin-10 was more effective in increasing plasma LH levels than the intramuscular injection.

• Kisspeptin-10 given as single intravenous injection increased plasma concentrations of LH during the luteal phase in a dose dependent manner.

• The treatments resulted in increased size of the dominant follicle.

• However, the treatments failed to induce ovulations.

Abstract

Two experiments were carried out to determine the effect of Kisspeptin-10 on plasma LH concentrations and follicular dynamics during the luteal phase in cattle. We tested the hypothesis that a single treatment of Kisspeptin-10 will increase plasma LH concentration and the diameter of the dominant follicle, and induce ovulation during the luteal phase of the estrous cycle in cattle. In the Experiment 1, Hereford-cross heifers (n = 28, 14–16 mo) were given PGF2α im to induce luteolysis and ovulation. On Day 5 (Day 0 = ovulation), a new follicular wave was induced by ultrasound-guided follicular ablation. Heifers were treated on Day 10 (4 days after wave emergence) with 100 μg GnRH im (n = 9), 2 mL saline im (n = 7), 1 mg Kisspeptin-10 im (Kp im, n = 6) or 1 mg Kisspeptin-10 iv (Kp iv; n = 6). Blood samples were collected at −60, −15, 0, 5, 15min (0 min = time of injection) and every 15 min thereafter until 3 h. Transrectal ovarian ultrasonography was performed at 12 h intervals from Day 10–14. In Experiment 2, non-lactating beef cows on Day 5 were treated with 100 μg GnRH im (n = 9), saline im (n = 5), 10 mg of Kisspeptin-10 iv (Kp 10 mg; n = 5) or 15 mg of Kisspeptin-10 iv (Kp 15 mg; n = 5). Blood samples were collected at −15, 0, 15, 30, 60, 120, 180 min and twice daily ovarian ultrasonography was done from Day 5–10. In Expt 1, plasma LH concentrations increased for 1 h following Kp iv administration. The peak concentration occurred at 15 min and was higher in the Kp iv group than in the Kp im group (P = 0.01). The LH peak was 3.5-folds higher in the GnRH group than the Kp iv group (P < 0.0001). In Expt 2, GnRH induced higher (P < 0.001) plasma LH concentrations for all time-points than other groups. Kp 15 mg at peak (15min), 30 and 60 min induced higher (P < 0.0001) plasma LH concentrations than Kp 10 mg and saline. Kisspeptin-treated animals did not ovulate in either experiment while GnRH induced ovulation (n = 5/9 in Expt 1; 9/9 in Expt 2). The diameter of the dominant follicle was greater (P = 0.02) at 12–48 h after kisspeptin treatment (Kp groups combined) than the Saline group in Expt 2. In conclusion, Kisspeptin-10 increased plasma LH concentrations and follicle size, and although plasma LH concentrations were higher after iv than im administration, but at the doses used, Kisspeptin-10 did not induce ovulation during the luteal phase in cattle.

Introduction

Kisspeptin is a neuropeptide that is produced by the hypothalamic neurons. Initially synthesized 145-aminoacid peptide is proteolytically cleaved in a 54-amino acid product (Kisspeptin-54) and further degraded in one of the three shorter peptide forms (Kisspeptin-14, Kisspeptin-13 or Kisspeptin-10). All forms of kisspeptin share the same C terminal amino acid sequence that is able to activate G protein-coupled receptor 54 (GPR-54) [1,2]. GPR-54, also known as Kiss 1 receptor, is highly expressed in GnRH neurons in rodents and sheep [3,4]. The mutations in human Kisspeptin receptor and the deletion of GPR-54 in murine GnRH neurons were associated with idiopathic hypothalamic hypogonadism, a condition that leads to delayed puberty and subsequently infertility [[5], [6], [7]]. Kisspeptin protein stimulates GnRH secretion [8] in mouse. Likewise, GnRH concentration in the cerebrospinal fluid increases after kisspeptin injection in ewe and monkeys [9,10]. Additionally, kisspeptin antagonist reduces GnRH secretion and inhibits kisspeptin effect on LH releasing [10,11]. GnRH is the pivotal hormone in control of LH/FSH secretion [[12], [13], [14]]. Resulting LH and FSH surges are responsible to control follicle development control in the ovary [15,16]; therefore, treatments of kisspeptin were able to induce cyclicity and ovulation in sheep and rat [9,17,18].

The shortest form of kisspeptin (i.e, 10-amino acid fragment; Kisspetin-10) is able to increase concentrations of LH and FSH in sheep, goat, mouse and cattle [9,17,19,20]. Furthermore, single intravenous administration of human and murine Kisspeptin-10 in sheep increased LH plasma concentration, and in calves single injections of human Kisspeptin-10 induced the release of gonadotrophin hormones when given by intravenous and intramuscular injections [9,21]. In ovariectomized cows, human Kisspeptin-10 increased concentrations of LH with or without sex steroids hormones influence [20] and a peak in plasma LH appear to occur around 20 min in prepubertal Holstein heifers [22]. In both male and female calves, concentrations of LH after intramuscular injections were lower than the intravenous administration [21]. Full-length bovine Kisspeptin (Kisspeptin-53) was able to induced ovulation in 1 out of 5 pubertal heifers treated [23]; however, we do not fully understand the dynamics of LH release and fate of the dominant follicle after a single iv and im injection of Kisspeptin-10 under luteal phase, such as high progesterone concentration in non-pregnant adult cattle. For example, can a single injection of Kisspeptin-10 induce ovulation under high-progesterone environment and what is the effect of Kisspeptin-10 on dominant follicle growth, if the Kisspeptin-10 fails to induce ovulation. It is known that after selection of the dominant follicle (around 8.5 mm diameter), high concentrations of LH are necessary to keep the dominant follicle growing, while increasing progesterone concentrations during early- and mid-luteal phase suppress endogenous plasma LH concentrations and dominant follicle size [16]. We expect that exogenous administration of Kisspeptin-10 will cross the blood brain barrier, activate GPR-54 receptors on hypothalamic neurons inducing GnRH release, which will cause LH release from adenohypophysis. If kisspeptin is able to sufficiently increase plasma LH during luteal phase, downstream effect would be an increase in size of the dominant follicle and perhaps also ovulation during the luteal phase.

The objective of the study was to determine the effect of Kisspeptin-10 on plasma LH concentrations, dominant follicle growth, and ovulation in cattle during the luteal phase. We tested the hypotheses that administration of Kisspeptin 10 during the high-progesterone phase of the bovine estrous cycle will 1) increase plasma LH, 2) increase the diameter of the dominant follicle, and 3) induce ovulation. The experimental design permitted assessment of the route of administration of kisspeptin (Experiment 1), and the effect of dose (Experiment 2).