Tissue collections
Animals were sacrificed by decapitation, with prior CO2sedation, 17 ±1 hours after lights OFF at 75 days old. During brain
dissection, special care was taken to include the intact pituitary stalk
containing the pars tuberalis by cutting the stalk half way between
hypothalamus and pituitary gland residing in the hypophysial fossa. To
further dissect the hypothalamus, the cerebellum, bulbus olfactorius and
frontal cortex were removed by coronal cuts. A sagittal hypothalamic
tissue block was obtained by lateral sagittal cuts at the hypothalamic
sulci and a horizontal cut at 4 mm distance from the ventral border of
the hypothalamus. The posterior hypothalamus (containing pars tuberalis,
DMH, VMH and Arcuate nucleus) and anterior hypothalamus (containing POA,
PVN, PVz and SCN) were separated by a coronal cut at posterior border of
the optic chiasm and the mammillary bodies. After dissection, it was
checked that the remainder of the pituitary gland, containing the pars
nervosa, pars intermedialis and pars distalis, was still intact in the
sella turcica covered by dura mater. The extracted tissues were flash
frozen within 2-4 minutes after death in liquid N2 and
stored at -80°C until RNA extraction. Reproductive organs were dissected
and cleaned of fat, and wet masses of testes, paired ovary and uterus
were measured (±0.0001 g).