An estradiol-induced protein synthesized in the ventral medial hypothalamus and transported to the midbrain central gray

Abstract

Estradiol (E2) facilitates the lordosis reflex that occurs in response to flank stimulation in female rats. Lordosis appears to be regulated in part by the synthesis of proteins in the ventral medial hypothalamus (VMH) that are transported to the midbrain central gray (MCG). We developed a stategy involving microinfusion of radioactive amino acids, followed by 2-dimensional gel electrophoresis, to identify proteins that may be regulated by E2, in the VMH and transported to the MCG. A mixture of 35S-methionine and 35S-cysteine (2:1, total 500-1000 μCi), suspended in 1 μl PBS, was infused bilaterally into the VMH over a period of 2 hr into matched pairs of ovariectomized female rats, one of which was given a Silastic implant containing E2 at the beginning of infusion or 1 week earlier. The rats were sacrificed 12 hr after the end of infusion, and several brain regions were obtained by microdissection. Samples were analyzed by 2-dimensional gel electrophoresis, entailing isoelectric focusing in the first dimension and SDS-PAGE (molecular-weight separation) in the second dimension, followed by fluorography. We could routinely separate at least 250 spots. We consistently found a protein spot with an apparent molecular weight of 70 kDa, pl of about 5.9, which almost always appeared in the VMH and MCG of rats given E2 replacement but very rarely in samples from ovariectomized rats given no E2 replacement. A spot immediately acidic to this protein (70 kDa, pl about 5.8) appeared to vary inversely with this E2-induced protein. Three lines of evidence suggest that the labeled protein in the MCG was due primarily to transport from the VMH: (1) Radioactivity after infusion was much higher in regions with hypothalamic projections than in other regions, and in the MCG tended to be found over axonal tracts rather than cell bodies; (2) colchicine reduced the radioactivity in the MCG by about 90%; and (3) despite variation between individuals, the relative densities of the 70 kDa proteins were identical in the VMH and MCG in the same individual. It is not clear if the 70 kDa, 5.9 pl protein was synthesized de novo in response to E2 or was the result of a modification of the 5.8 pl protein. In any case, these studies suggest that E2 causes the appearance of this protein in the VMH, from whence it is transported to the MCG. The function of this protein remains unknown; however, the molecular weight and isoelectric point of this protein are similar to those of an isoform of the proteins that remove clathrin from coated secretory vesicles, themselves part of the family of stress-induced proteins.