The subcellular distribution and accumulation of LHRH in hypothalamic neurons of female rats were studied as a function of age (1-24 months). Hypothalami were homogenized in isoosmotic sucrose solution, and a 900 × g supernatant fluid was prepared. Free granules and synaptosomes containing LHRH were separated by means of continuous sucrose density gradient centrifugation. Homogenization and subcellular fractionation were carried out at 4 or 25 C. In the homogenates, LHRH was found to be associated with free granules and with two types of synaptosome-like particles: rigid synaptosomes (the presence of which was evident after homogenization and fractionation were carried out at 4 as well as 25 C) and fragile synaptosomes (the presence of which was evident onlyafter homogenization and fractionation at 25 C). The amount of LHRH recovered in rigid synaptosomes prepared from hypothalami of 1.5-month-old rats was significantly (P < 0.01) greater than that in rigid synaptosomes prepared from 1-month-old animals. However, the amount of LHRH in rigid synaptosomes changed little after the animals reached 1.5 months of age, remaining at about 0.9 ng/hypothalamus. Fragile synaptosomes prepared from immature (1-month-old) rat hypothalami did not contain significant amounts of LHRH, whereas those from adult or old rats contained about 0.3 ng LHRH/hypothalamus. The amount of LHRH recovered in free granules (at 25 C) increased until the donor animals were 10 months of age but remained constant thereafter at about 1.1 ng/hypothalamus. In the mature rat hypothalamus, the subneuronal structures that give rise to the rigid synaptosomes are the major storage sites of LHRH. The increase in the LHRH content of the hypothalamus in the aging female rat occurs predominantly in subneuronal structures, which give rise to fragile synaptosomes and free granules. Based on these results, it is suggested that the physicochemical properties of the LHRH neurons as well as the subneuronal distribution of LHRH change with aging. Such changes may be related to the alteration in the mode of function of the hypothalamic- pituitary-gonadal axis in the aging female rat.
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