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The work described in this article gives information on the effects of ageing on the hypothalamo-pituitary-testicular axis in rats. The hypothalami of young and old male rats contain similar amounts of luteinizing-hormone-releasing hormone (LHRH); when perifused in vitro they release comparable amounts of LHRH under basal conditions and in response to K+. The addition of an LHRH analogue to the perifusion medium blocks the release of LHRH induced by K+ from the hypothalami of young and old male rats, indicating that the ultrashort feedback mechanism controlling LHRH release functions normally in aged male rats. Ageing also exerts important effects on the density of mu- and kappa-opioid receptors in the brain. The number of hypothalamic mu-opioid receptors was significantly decreased in aged animals; a replacement treatment with testosterone does not reverse this decrease, indicating that the decline of hypothalamic mu receptors and of serum titres of testosterone in old rats are independent phenomena. The number of kappa-opioid receptors in the brain increases in the amygdala and in the thalamus with ageing. Apparently ageing does not influence the number of delta receptors in any of the brain areas investigated. The number of pituitary LHRH receptors decreases in old animals, which might explain the low serum concentration of gonadotrophins in aged rats caused by an inadequate response of the pituitary to hypothalamic LHRH. The impaired secretion of testosterone in aged male rats is accompanied by an increase in the number of testicular LHRH receptors, indicating that the intratesticular mechanisms controlling testosterone release also undergo significant alterations during ageing. The rate of conversion of testosterone to dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) is the same in the hypothalami of young and old rats. However, the yields of DHT obtained from the pituitaries of aged male rats are significantly lower than those recorded in the pituitaries of young animals. These results show that the enzymes necessary for metabolizing testosterone via the 5 alpha-reductase pathway are maintained both in the hypothalamus and in the anterior pituitary of aged male rats. However, the 5 alpha-reductase activity of the anterior pituitary of senescent animals appears to be lower than that in the younger controls.

The present experiments were performed to study the binding characteristics of delta opioid receptors in membrane preparations obtained from the brain of adult male rats, and to analyze whether aging modifies these binding parameters. The binding characteristics of delta opioid receptors were evaluated on membrane preparations derived from dissected brain regions (hypothalamus, amygdala, mesencephalon, corpus striatum, hippocampus, thalamus, frontal poles, anterior and posterior cortex) collected from male rats of 3 and 24 months of age; the highly selective ligand 3H-[D-Pen2-D-Pen5] enkephalin (3H-DPDPE) was used. The results obtained in young rats show that the distribution of delta opioid receptors is different in the various brain areas examined; these receptors appear to be maximally concentrated in the frontal poles, anterior and posterior cortex; lower concentrations were found in the other structures considered. Kd (dissociation constant) for the delta sites was found very similar in all areas. The distribution of delta opioid receptors in the brain of 24-month-old rats was similar to that observed in young animals; this result was surprising in view of the fact that aging modifies the number of other types of brain opioid receptors (mu and kappa).

Experiments have been designed in order to analyze whether the binding capability of mu opioid receptors in the brain of the male rat is modified by age. In a first experiment, the number of receptors (Bmax) and the constant of affinity (Ka) for the mu ligand 3H-dihydromorphine (3H-DHM) have been measured in the whole brain of male rats of 2, 15 and 22 months of age. In a second experiment the Bmax and the Ka for 3H-DHM have been evaluated in the hypothalamus of male rats of 2 and 22 months of age. In this experiment it was also investigated whether the administration of exogenous testosterone modifies the number and/or the affinity of the hypothalamic mu receptors. Serum levels of LH, FSH, prolactin and testosterone have been measured by specific RIAs. The results obtained show that: serum testosterone levels are significantly decreased in aged rats, while serum LH and FSH show only a small decline; serum prolactin is higher in old than in young animals; the number of mu receptors in the whole brain of 15 and 22 month old animals and in the hypothalamus of 22 month old rats is significantly lower than in the same tissues of young animals; the administration to old animals of testosterone, in doses able to bring back towards normal serum levels of testosterone, induces a decrease of LH and FSH, but has no effect on serum prolactin titers. Testosterone administration does not modify the number of hypothalamic mu opioid receptors, indicating that the decline of brain mu receptors in old animals is not the consequence of the physiological decline of testosterone secretion; in no instance the Ka for the mu ligand is significantly affected.

Several experiments have been performed in order to clarify the mechanisms through which aging in male rats brings about profound modifications of the neuroendocrine system (reduced pulsatile secretion of LH and FSH, decreased serum levels of gonadotropins and testosterone, etc.). (1) It has been found that the number of mu opioid receptors decreases significantly in the hypothalami of old male rats; the substitution therapy with testosterone is ineffective in increasing the number of mu opioid receptors. These data suggest that the decrease of hypothalamic mu opioid receptors is not due to a decline of serum testosterone levels, but appears to be an independent phenomenon. (2) K opioid receptors increase significantly in the amygdala and in the thalamus of old male rats. These results show that aging, in addition to mu receptors, affects also the number of K receptors in selected areas of the brain. The increase of the number of K receptors in the amygdala might have some bearing on the decrease of serum gonadotropins observed in aged rats, since the amygdala is involved in the nervous circuitry influencing the hypothalamo-pituitary-gonadal axis. (3) The study of the release of LHRH from the hypothalamus of old male rats with an in vitro perfusion system shows that the release of the hormone is comparable in young and old animals, both in basal and in K+ stimulated conditions. These results indicate that the hypothalamus of old male rats retains the capacity of releasing LHRH both in basal and in stimulated conditions. (4) It has been observed that the number of LHRH receptors at the level of the anterior pituitary is significantly reduced in old male rats. This finding might explain the low serum levels of gonadotropins and testosterone in aged rats, due to a lack of an adequate response of the pituitary to hypothalamic LHRH.

Aging exerts profound influences on the function of the hypothalamic-pituitary-testicular-axis. This work has been performed in order to verify whether, in male rats, the decreased secretion of LH and testosterone (T) occurring in old animals is reflected by modifications of luteinizing hormone-releasing hormone (LHRH) receptors at the level of the anterior pituitary and of the testes. To this purpose, the affinity constant (Ka) and the maximal binding capacity (Bmax) for the LHRH analog [D-Ser(tBu)6]des-Gly10-LHRH-N-ethylamide were evaluated, by means of a receptor binding assay, in membrane preparations derived from the anterior pituitary and testicular Leydig cells of male rats of 3 and 19 months of age. Serum levels of LH and T were measured by specific RIAs. The results obtained show that, in aged male rats, the concentration of pituitary LHRH receptors is significantly lower than that found in young animals. On the other hand, the concentration of LHRH binding sites is significantly increased on the membranes of Leydig cells of old rats. In no instance the Ka for the LHRH analog is significantly affected. Serum levels of LH and T are significantly lower in old than in young male rats. In conclusion, these results suggest that the reduced secretion of LH in old male rats may be linked, at least partially, to a decrease of the number of pituitary LHRH receptors. The impaired production of testosterone occurring in aged rats is accompanied by a significant increase of the number of testicular LHRH receptors, indicating that also the intratesticular mechanisms controlling testosterone release undergo significant alterations with aging.