Mechanism of LH release after peripheral administration of kisspeptin in cattle.

Kisspeptin modulates GnRH secretion in mammals and peripheral administration of 10-amino acid fragment of kisspeptin (Kp10) induces LH release and ovulation in cattle. Experiments were done to determine if iv administration of kisspeptin will activate GnRH neurons (i.e., after crossing the blood-brain barrier) and if pre-treatment with a GnRH receptor blocker will alter kisspeptin-induced LH release (from gonadotrophs) and ovulation. In Experiment 1, cows (n = 3 per group) were given human-Kisspeptin10 (hKp10; 3 x 15 mg iv at 60-min intervals) or normal saline and euthanized 150 min after treatment was initiated. Every 20th free-floating section (50 µm thickness) from the preoptic area to hypothalamus was double immunostained to colocalize GnRH- (DAB) and activated neurons (cFOS; Nickel-DAB). Kisspeptin induced plasma LH release from 15 to 150 min (P = 0.01) but the proportion of activated GnRH neurons did not differ between groups (5.8% and 3.5%, respectively; P = 0.11). Immunogold electron microscopy detected close contacts between kisspeptin fibers and GnRH terminals in the median eminence. In Experiment 2, pubertal heifers (n = 5 per group) were treated with 1) hKp10 iv, 2) Cetrorelix (GnRH antagonist; im) + hKp10 iv or 3) saline on Day 6 of the follicular wave under low-progesterone condition. A rise in plasma LH concentration was detected from 15 to 240 min in the hKp10 group but not in cetrorelix or control group (P<0.001). Ovulations were detected only in the hKp10 group (4/5; P = 0.02). Cetrorelix treatment was associated with regression of the preovulatory dominant follicle and emergence of a new follicular wave 3.4±0.75 days after the treatment in all five heifers. Results support the hypothesis that the effect of peripheral kisspeptin is mediated downstream of GnRH synthesis and does not involve GnRH-independent LH release from gonadotrophs. Peripheral kisspeptin may release pre-synthesized GnRH from the nerve terminals in areas outside the blood-brain barrier.

Hypothalamic involvement and the role of progesterone in the NGF-induced LH surge pathway.

To elucidate the mechanism by which nerve growth factor (NGF) influences the LH secretory pathway in camelids, a series of experiments were done to determine the involvement of the hypothalamus (Experiment 1), the role of GnRH neurons (Experiment 2), and the effect of progesterone (Experiment 3) on the NGF-induced LH surge and ovulation in llamas. In Experiment 1, the declining phase of the NGF-induced LH surge was used to determine if the decline is a result of pituitary depletion or hypothalamic unresponsiveness. Female llamas were treated with NGF and, 7 h later, assigned to three groups and given a second dose of NGF (n = 5), a dose of GnRH (n = 5), or saline (n = 6). The LH response was attenuated after the second dose of NGF vs GnRH. In Experiment 2, Fos expression (marker of neuronal activation) in GnRH neurons was examined in the hypothalamus of llamas after NGF or saline treatment (n = 3 per group). Despite an LH surge in the NGF group but not in the saline group, no differences were detected between groups in Fos/GnRH co-expression. In Experiment 3, llamas in low-, medium-, and high-plasma progesterone groups (n = 4 per group) were treated with NGF. The NGF-induced LH surge did not differ among treatment groups. Results from the present study show that the induction of a preovulatory LH surge by NGF may be controlled by a novel pathway involving GnRH neuro-terminals downstream of the hypothalamus and is independent of progesterone influence.

Kisspeptin induces LH release and ovulation in an induced ovulator†.

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus.

Kisspeptin induces ovulation in heifers under low plasma progesterone concentrations.

The objective of the present study was to compare the effect of a single versus multiple doses of a 10-amino acid fragment of human (hKp) or murine (mKp) kisspeptin on LH secretion and the fate of the dominant follicle. In all experiments, a new wave was induced (Day 0) by ultrasound-guided ablation of >5 mm follicles, a progesterone device (CIDR) was placed in the vagina, animals given prostaglandin F2α analog im on Day 3.5 and 4, and hKp or mKp treatment given on Day 6. The experimental design maintained growth and ovulatory potential of the dominant follicle for 12 days and allowed hypothesis testing during the low-progesterone period (plasma progesterone ≤1.8 ng/ml on Day 6) wherein spontaneous wave emergence and ovulation did not occur between Day 6 and Day 12. In Experiment 1, heifers (n = 10/group) were given single iv dose of 45 mg hKp, 45 mg mKp, or 2 ml normal saline (control). Post-treatment plasma LH concentrations from 15 to 90 min were higher (P < 0.01) in hKp group than in the mKp and control groups. Two heifers ovulated in hKp group versus none in other groups. In Experiment 2, heifers (n = 6/group) were given 45 mg hKp over a 2 h period divided into multiple iv doses treatments or 2 ml normal saline (control). Post-treatment plasma LH concentrations were higher (P < 0.01) in all hKp treatment groups than in the control group. The ovulation rate was higher (P = 0.06) after hKp treatments (11/18) than in the control group (0/6). In Experiment 3, heifers (n = 6/group) were given 45 mg mKp over a 2 h period divided into multiple iv doses treatments or a single iv dose of gonadorelin acetate (positive control). Plasma LH concentration was higher (P < 0.01) and the ovulation rate was greater (P = 0.01) in the GnRH group (5/6) than mKp groups (1/12). In summary, hKp was more effective to induce ovulation than mKp. Human kisspeptin-10 given over a 2 h period induced ovulations at a rate similar to that of GnRH treatment in heifers under a low plasma progesterone state.

Bilateral thyroid follicular compact-cellular carcinoma in a llama.

An 18-y-old female llama (Lama glama) in Saskatoon, Saskatchewan was examined during a routine herd check, and a mass was detected in the ventral cervical area just below the angle of the jaw. No clinical signs were evident except for the mass and chronic loss of body condition. Postmortem examination revealed bilateral enlargement of the thyroid gland with multiple cysts. Histopathology of the thyroid gland revealed follicular compact-cellular carcinoma lesions, with infiltration of neoplastic thyroid follicular cells in regional lymph nodes.

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

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.