Age-related changes in cytoplasmic estradiol receptor concentrations in microdissected brain nuclei: correlations with changes in steroid-induced sexual behavior.

Abstract The purpose of this study was to: (1) determine at what age changes in cytoplasmic estradiol receptors are evident in specific microdissected brain areas of the female rat; (2) assess whether alterations parallel previous changes observed when large brain areas were used for determination of receptor concentrations; and (3) assess whether changes in cytoplasmic estradiol receptors are correlated with changes in steroid-mediated physiological functions. To assess the effects of age on cytoplasmic estradiol receptor concentrations, we used virgin female Sprague-Dawley rats at 3–4 months, 7–8 months and 10–11 months of age. They were ovariectomized 7–14 days prior to use to allow maximal translocation of receptors to the cytoplasm. The animals were anesthetized and perfused with a 10% (v/v) solution of dimethylsulfoxide to protect the receptor proteins from the effects of freezing. Brains were removed and frozen. This procedure of freezing the brains caused a minimal (15–18%) loss in the number of receptors and no change in the dissociation constant. Consecutive 300 μm sections were sliced and the following nuclei and brain areas were microdissected: bed nucleus of the stria terminalis, suprachiasmatic-preoptic area, medial preoptic nucleus, periventricular preoptic nucleus, periventricular anterior hypothalamic area, paraventricular nucleus, dorsomedial nucleus, ventromedial nucleus, arcuate-median eminence, medial amygdala, and cortical amygdala. The pituitary gland was also removed and analyzed. The cytoplasmic fraction from a tissue pool from 3 animals was prepared and aliquots were incubated with [ 3 H]estradiol at a final concentration of 1.5 nM in the presence or abssence of 100-fold excess moxestrol. Receptor-bound [ 3 H]estradiol was separated from free hormone by gel filtration. There was no difference in cytoplasmic estradiol receptor concentrations in any brain area in 7–8-month-old rats compared to 3–4-month-old rats. In marked contrast, by 10–11 months of age, there was a significant decrease in the number of cytoplasmic estradiol receptors in the suprachiasmatic-preoptic area and medial preoptic nucleus with a similar trend in the bed nucleus of the stria terminalis and periventricular preoptic nucleus. These brain areas are included in the grossly dissected preoptic area used in previous studies. We observed an unexpected decrease in the ventromedial nucleus, but no change in the dorsomedial nucleus, paraventricular nucleus, periventricular anterior hypothalamic area or arcuate-median eminence, areas included in the grossly dissected medial basal hypothalamus. Since the ventromedial nucleus is involved in steroid-induced reproductive behavior we examined whether or not the decrease in cytoplasmic estradiol receptors in this area is correlated with behavioral changes. Young (3–4 month) and middle-aged (10–11 month) rats were ovariectomized and 1–3 weeks later, they received Silastic capsules which produced physiological levels of plasma estradiol. Two days later, 4 h before testing, rats received subcutaneous injections of progesterone. Proceptive behavior, lordosis quotient and lordosis quality score were assessed. Middle-aged rats showed significant deficits in all aspects of mating behavior. The data demonstrate that by 10–11 months of age, rats exhibit decreased cytoplasmic estradiol receptor levels in components of the preoptic area. The use of microdissection methods allowed us to detect changes in a particular component of the medial basal hypothalamus, the ventromedial nucleus. This was correlated with deficits in estradiol-induced behavior. The data suggest that changes in estradiol receptors may affect the ability of aging rats to respond to estradiol and may be a contributing factor to the age-related transition to acyclicity and infertility.