In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

The effects of kisspeptin agonist canine KP-10 and kisspeptin antagonist p271 on plasma LH concentrations during different stages of the estrous cycle and anestrus in the bitch.

Kisspeptin (KP) plays a key role in the regulation of the hypothalamic-pituitary-gonadal axis via the release of GnRH. As normal KP signaling is essential for reproductive function, it could be an interesting new target for therapeutic interventions, e.g., nonsurgical contraception in dogs. The aims of the present study were to investigate the effect of KP-10 administration on plasma LH concentration in different stages of the reproductive cycle and to investigate the suitability of p271 as KP antagonist in the bitch. Two groups of six adult Beagle bitches were used. In one group, plasma LH concentration was determined before (40 and 0 minutes) and 10, 20, 40, and 60 minutes after the intravenous administration of 0.5-µg/kg body weight (BW) canine KP-10. In the other group, the bitches received a continuous intravenous infusion with p271 (50 µg/kg BW/h) for 3 hours, and 0.5-µg/kg BW canine KP-10 was administered intravenously 2 hours after the start of the p271 infusion. Their plasma LH concentration was determined before (-40 and 0 minutes) and 30, 60, 90, 120, 130, 140, 160, and 180 minutes after the start of the p271 infusion. In both groups, the experiments were performed during the follicular phase, the first and second half of the luteal phase, and during anestrus. Canine KP-10 induced an increase of plasma LH concentration during all estrous cycle stages and anestrus. There was no difference in LH response between the two groups. The lowest LH response was seen during the follicular phase and the highest response during anestrus. The area under the curve (AUC) for LH and LH increment in the follicular phase were lower than those in anestrus. The AUC LH and LH increment in the first half of the luteal phase were lower than those in the second half of the luteal phase and anestrus. The AUC LH and LH increment in the second half of the luteal phase were not different from those in anestrus. Continuous administration of the antagonist p271 did not alter basal plasma LH concentration and could not prevent or lower the LH response to KP-10 in any of the cycle stages and anestrus. It can be concluded that the LH response to KP-10 is dependent on estrous cycle stage and that peripheral administrated p271 cannot be used as KP antagonist in the dog. This provides new insight in reproductive endocrinology of the bitch, which is important when KP signaling is considered for therapeutic interventions, such as for estrus induction or nonsurgical contraception in the bitch.

Successful treatment for subinvolution of placental sites in the bitch with low oral doses of progestagen.

Subinvolution of placental sites (SIPS) is the major cause of persistent sanguineous vaginal discharge after parturition in the bitch. Spontaneous remission is common but may take several months, and hence, medical therapy to end the discharge is often requested. In this retrospective study, we evaluated the effect of treatment for SIPS with low oral doses of a progestagen. Nine bitches with SIPS, but otherwise clinically healthy, were found in the computer database of the Department of Clinical Sciences of Companion Animals. Seven of these bitches were treated with low oral doses of a progestagen (megestrol acetate, 0.1 mg/kg body weight (bw) once daily for the 1st week, then 0.05 mg/kg bw once daily for the 2nd week). The other two bitches were untreated. Treatment results were evaluated by a telephone questionnaire. Progestagen treatment was successful in all of the treated dogs; sanguineous vaginal discharge stopped within the treatment period. One of the two untreated dogs remained symptomatic until the next oestrus, approximately 120 days after parturition, and the other remained symptomatic until 6 weeks before the start of the next pro-oestrus, 270 days after parturition. No side effects of the progestagen treatment were observed. Subsequent gestations, parturitions and puerperal periods of 5 mated bitches were uneventful. One bitch did not become pregnant after mating. In conclusion, the results of this study indicate that oral administration of low doses of progestagen for 2 weeks is effective in stopping persistent sanguineous vaginal discharge in bitches with SIPS, with neither side effects nor reduced subsequent fertility.

Effects of gonadotropin-releasing hormone administration on the pituitary-gonadal axis in male and female dogs before and after gonadectomy.

GnRH-stimulation tests were performed in 14 female and 14 male client-owned dogs of several breeds, before and 4 to 5 mo after gonadectomy. The aim of the study was to obtain more insight into the pituitary-gonadal axis in intact and neutered dogs and to establish reference values. Basal plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were increased significantly after gonadectomy in both bitches and male dogs. In both males and females ranges of the basal plasma FSH concentrations, before and after gonadectomy, did not overlap as opposed to the overlap in ranges of the basal plasma LH concentrations. Before gonadectomy basal plasma LH concentrations were lower and basal plasma FSH concentrations were higher in bitches than in male dogs. After gonadectomy these basal values did not differ significantly. GnRH administration before gonadectomy resulted in an increase in plasma LH and FSH concentrations in both genders. GnRH administration after gonadectomy produced an increase only in plasma LH concentrations in both genders, and a just significant increase in plasma FSH in castrated male dogs. GnRH administration before gonadectomy resulted in a significant increase in plasma testosterone concentration in both genders. In males ranges of basal and GnRH-stimulated plasma testosterone concentrations before and after gonadectomy did not overlap. Basal plasma estradiol concentrations were significantly higher in intact males than in castrated males and their ranges did not overlap. The basal estradiol concentrations in bitches before and after ovariectomy were not significantly different. At 120 min after GnRH administration, ranges of plasma estradiol concentration of intact and ovariectomized bitches no longer overlapped. In conclusion, basal plasma FSH concentration appears to be more reliable than basal plasma LH concentration for verification of neuter status in both male and female dogs. The basal plasma testosterone concentration appears to be reliable for verification of neuter status in male dogs. The plasma estradiol concentration at 120 min after GnRH administration can be used to discriminate between bitches with and without functional ovarian tissue.