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 [20]. 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.