Hypothalamic LH-RH content in male rats is lowered after castration. The s.c. implantation of testosterone or testosterone propionate-packed Silastic tubing (from 0.5 to 6 cm in length) in a range which encompassed the normal circulating plasma testosterone concentration, prevented this lowered LH-RH content 21 days following castration and simultaneous implantation. The temporal response to implanted testosterone was then studied: rats were killed 1, 2, 4, 8, 14 and 21 days after castration and simultaneous implantation of 3 cm testosterone-packed Silastic tubing. The hypothalamic LH-RH content began to decrease in the castrated group after 4 days and fell progressively thereafter. However, the hypothalamic LH-RH content of the castrated group maintained with constant physiological levels of testosterone showed no such reduction at any time following castration. These experiments indicate that circulating testosterone in physiological concentrations can maintain a normal hypothalamic LH-RH content and demonstrate an action of testosterone, in physiological concentrations, in the feedback regulation of LH-RH secretion.

No abstract

Luteinizing hormone-releasinghormone (LH-RH), a water soluble decapeptide, was found to be so firmly bound to a subcellular particulate structure in rat hypothalamus that only negligible amounts were solubilized during the homogenization and centrifugation processes. The structures binding LH-RH were localized in the 1.2 Msucrose portion of a discontinuous sucrose gradient. Another neurohormone, vasopressin, was more dispersed and located in the heavier portion of the gradient. Cholinesterase activity was also widely distributed throughout the gradient and its distribution was not closely related to the LH-RH section of the gradient.

Several aqueous solvent systems were tested for their efficiency in extracting luteinizing hormone releasing hormone (LH-RH) from rat hypothalamus. Although LH-RH is a water-soluble decapeptide, neutral distilled water extracted only 10% of the LH-RH obtained using acid extraction methods. The efficiency of the acid extraction procedure suggests that in the hypothalamus the releasing hormone is bound to a relatively large molecular weight compound. Using the acidic extraction procedure, we found that hypothalamic LH-RH content is significantly lower in the castrated animal than in the normal rat.

LH-RH in rat hypothalamic extract (HE) emerges in 2 peaks after gel filtration on Sephadex G-25 with pyridine acetate buffer (PAB), pH 5.8,1 peak in the unretarded protein fraction and the other near the salt region. A synthetic LH-RH marker was found only in the peak near the salt region by elution with acid. Using a dialysis technique, a protein fraction, from which LH-RH and other small molecular weight material were dissociated by gel filtration on Sephadex G-25 with acid, showed affinity for synthetic LH-RH. Synthetic LH-RH in the hypothalamic protein solution was retained in the dialysis bags more effectively than other types of protein solutions. This binding protein is of relatively small molecular size according to gel filtration behavior on Sephadex G-75. Since the dialysis experiment showed extremely poor recovery of total LH-RH, we determined the rate of loss of LH-RH under several conditions. The rate of disappearance was significantly less in solutions containing protein derived from cerebellum and hypothalamus, compared to bovine serum albumin (BSA) solution or salt solution. The ability of the hypothalamic protein to retard the rate of disappearance of LH-RH is further evidence for a special affinity between LH-RH and the protein factor. It is concluded that the protein LH-RH complex is dissociated by lowering the pH and that the dissociated protein and synthetic LH-RH can be re-associated to form a protein-hormone (PrH) complex at neutral pH.