Fig 1.
Experimental model used in Experiment 2.
Emergence of a new follicular wave (Day 0) was induced by follicular ablation (FA) three days after ovulation, and an intravaginal progesterone-releasing device (CIDR) was inserted immediately after follicle ablation and left in place for 13 days. Luteolytic doses of a prostaglandin F2α analog (cloprostenol) were given on Day 3.5 and 4. Ovarian ultrasonography was performed daily during the experiment. Blood collection and treatment administration was done on Day 6.
Fig 2.
Plasma LH concentration in the experiment 1.
A) Plasma LH plasma concentrations in lactating cows treated with three iv injections of 15mg human Kisspeptin-10 (kisspeptin; n = 3) or normal saline (control; n = 3) at 1h intervals on Day 7 (Day 0 = day of wave emergence) in Experiment 1. B) Plasma LH profiles of individual cows from the kisspeptin group illustrating similar pattern in two cows but varying magnitude of response among the three animals. Black arrows along the x-axes indicate the time of treatments.
Table 1.
Ovarian and endocrine responses (mean±SEM) of lactating cows in proestrus treated with three iv injections of 15mg human Kisspeptin-10 or normal saline (control) at 1h intervals on Day 7 (Day 0 = day of wave emergence) in Experiment 1.
Data were compared by t-test.
Fig 3.
Distribution of GnRH and c-FOS immunoreactive cells in the preoptic area and hypothalamus after administration of 3 doses of 15 mg human Kisspeptin-10 intravenously (at 1 hr intervals) during proestrous phase of estrous cycle in cows.
A) GnRH immunoreactive neurons (arrow) in the medial preoptic area (mPOA) nucleus showing cytoplasmic DAB (brown) staining. B) GnRH immunoreactive neurons that co-express c-FOS immunoreactive reaction (Nickel-DAB black color). Note that c-FOS is localized in the nucleus (arrow). C-D) GnRH (brown) and c-FOS (black) immunoreaction cells and nerve fibers in the arcuate nucleus and median eminence.
Table 2.
Number of neurons expressing immunoreactivity for c-FOS positive perikarya (c-FOS), GnRH positive perikarya (GnRH) and GnRH neurons bodies that co-expressed cFOS protein (c-FOS and GnRH in the preoptic and hypothalamic area of cows 150 minutes after initiation of treatment with kisspeptin or saline (Time 0 = first administration).
Fig 4.
Representative immuno-electron microscopy at the border of median eminence region of medio-basal hypothalamus in a proestrous cow after intravenous kisspeptin administration.
A) black arrows indicate synaptic terminals kisspeptin fiber projection, and white arrows indicate neurosecretory GnRH granules in axon terminals; B) Higher magnification from Fig A (black box) showing kisspeptin immunoreactive axon fiber projection is identified with gold particles (small black dots); C) Higher magnification from Fig A (dotted white rectangle) showing GnRH immunoreactive axon nerve terminal is identified with GnRH neurosecretory granules (DAB staining) surrounded by kisspeptin immunoreactivity (gold particles).
Fig 5.
Ovarian follicular dynamic and LH profile in heifers treated in the Experiment 2.
A) Growth profile of dominant follicle in pubertal heifers treated at Day 6 of follicular wave (day of wave emergence = Day 0) with human Kisspeptin-10 given as three injections of 15 mg at 60 minutes interval over a 2-hour period (Kp10 group, n = 5). Cetrorelix group (n = 5) was injected GnRH antagonist (Cetrorelix, 20 μg/Kg body weight, intramuscular) 3 hours before kisspeptin treatment while the Control group (n = 5) was given three intravenous injections of normal saline. B) Plasma LH concentrations in Kp10, Cetrorelix and Control group heifers.
Table 3.
Ovarian and endocrine responses (mean±SEM) of heifers treated with human Kisspeptin-10 (Kp10 group), pre-treatment with cetrorelix before Kisspeptin-10 (Cetrorelix group) or normal saline (Control group) intravenously under subluteal levels of plasma progesterone in Experiment 2.