The introduction of a novel male stimulates the hypothalamic-pituitary-gonadal axis of female sheep during seasonal
anestrus, leading to the resumption of follicle maturation and ovulation. How this pheromone cue activates pulsatile
secretion of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) is unknown. We hypothesised that
pheromones activate kisspeptin neurons, the product of which is critical for the stimulation of GnRH neurons and fertility.
During the non-breeding season, female sheep were exposed to novel males and blood samples collected for analysis of
plasma LH profiles. Females without exposure to males served as controls. In addition, one hour before male exposure, a
kisspeptin antagonist (P-271) or vehicle was infused into the lateral ventricle and continued for the entire period of male
exposure. Introduction of a male led to elevated mean LH levels, due to increased LH pulse amplitude and pulse frequency
in females, when compared to females not exposed to a male. Infusion of P-271 abolished this effect of male exposure.
Brains were collected after the male effect stimulus and we observed an increase in the percentage of kisspeptin neurons
co-expressing Fos, by immunohistochemistry. In addition, the per-cell expression of Kiss1 mRNA was increased in the rostral
and mid (but not the caudal) arcuate nucleus (ARC) after male exposure in both aCSF and P-271 treated ewes, but the percell
content of neurokinin B mRNA was decreased. There was also a generalized increase in Fos positive cells in the rostral
and mid ARC as well as the ventromedial hypothalamus of females exposed to males. We conclude that introduction of
male sheep to seasonally anestrous female sheep activates kisspeptin neurons and other cells in the hypothalamus, leading
to increased GnRH/LH secretion.
Conceived and designed the experiments: JTS IJC. Performed the
experiments: JPD QL JTS. Analyzed the data: JPD QL JTS. Contributed
reagents/materials/analysis tools: RPM. Wrote the paper: JPD JTS IJC.
Figure S1 Male exposure in anestrous ewes increased
the number of kisspeptin cells in the rostral ARC.
Sections representing the rostral, middle, and caudal regions of the
ARC (as above) were chosen from each ewe and mounted on
SuperFrost slides. Fluorescent immunocytochemistry was performed
as previously described . The primary kisspeptin
antibody (AC566) was used at a concentration of 1:2000 and was
visualized with a goat anti-rabbit secondary antibody (Alexa 448,
1:400; Molecular Probes Inc., Eugene, OR). Kisspeptin-ir cells
were identified under fluorescent illumination, with a single
observer counting the total number of cells. For each ewe, the
number of kisspeptin-ir cells per section in each region was
averaged to produce a mean (6SEM). A, Representative
photomicrographs of the rostral ARC showing kisspeptin immunoreactive
neurons (green). 3V, Third ventricle. Scale bar, 200 mm.
B-C, The number of detectable kisspeptin neurons in the rostral
ARC (B) was higher (P,0.05) in ewes exposed to males compared
to control aCSF treated ewes. The number of kisspeptin neurons
did not differ in the Mid (C), or Caudal (D) ARC. Data are the
mean 6 SEM, n= 4 per group.