Neuroendocrine involvement in aging: evidence from studies of reproductive aging and caloric restriction.

Neuroendocrine changes contribute to female reproductive aging, but changes in other tissues also play a role. In C57BL/6J mice, neuroendocrine changes contribute to estrous cycle lengthening and reduced plasma estradiol levels, but the midlife loss of cyclicity is mainly due to ovarian failure. Hypothalamic estrogen receptor dynamics and estrogenic modulation of gene expression are altered in middle-aged cycling mice. Although insufficient to arrest cyclicity, these neuroendocrine changes may contribute to other reproductive aging phenomena, such as altered gonadotropin secretion and lengthened estrous cycles. In women, the loss of ovarian oocytes, the cause of menopause, accelerates in the decade before menopause. Accelerated oocyte loss may in turn be caused by a selective elevation of plasma follicle stimulating hormone, and neuroendocrine involvement may thus be implicated in menopausal oocyte loss. Chronic calorie restriction retards both neural and ovarian reproductive aging processes, as well as age-related change in many other physiological systems. The diverse effects of food restriction raises the possibility of an underlying coordinated regulatory response of the organism to reduced caloric intake, possibly effected through alterations of neural and/or endocrine signalling. We are therefore attempting to identify neuroendocrine changes that may coordinate the life prolonging response of animals to food restriction. Our initial focus is on the glucocorticoid system. Food restricted rats exhibit daily periods of hyperadrenocorticism, manifest as elevated free corticosterone during the diurnal peak. We hypothesize that this hyperadrenocortical state potentiates cellular and organismic homeostasis throughout life in a manner similar to that achieved during acute stress, thereby retarding aging processes and extending life span.

Hyperadrenocorticism and food restriction-induced life extension in the rat: evidence for divergent regulation of pituitary proopiomelanocortin RNA and adrenocorticotropic hormone biosynthesis.

The increased diurnal elevation of plasma corticosterone (B) induced by food restriction (FR) may play a role in the life span extension of FR. We investigated whether FR alters adrenocorticotropic hormone (ACTH) and proopiomelanocortin (POMC) mRNA levels in plasma and anterior pituitary (AP), since these molecules both regulate and can be suppressed by B. Measurements were made in 3-month-old male Fischer 344 rats that had been ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. Plasma B was 2-fold higher in FR rats in the PM samples, but did not differ in AM samples. By contrast, plasma ACTH did not differ in the PM samples of FR and AL rats and was 20% lower in AM samples (p < .05) of FR rats. AP content of ACTH was 50% lower in FR rats in both AM and PM samples (p < .01). In contrast, AP contents of POMC and mRNA, primary transcript, and processing intermediate were not reduced in FR rats, and PM content of POMC primary transcript was elevated in FR rats (p < .05). The reduced pituitary and plasma ACTH of FR rats may be the consequence of their elevated plasma B levels. This study also suggests that factors other than elevated ACTH account for FR-induced hyperadrenocorticism. These results also indicate that POMC mRNA and ACTH biosyntheses are differentially regulated in FR rats.

Aging impairs estrogenic suppression of hypothalamic proopiomelanocortin messenger ribonucleic acid in the mouse.

Altered neuroendocrine sensitivity to estrogen is a characteristic of reproductive aging in female rodents, but its molecular basis is not well understood. The objective of this study was to determine whether altered modulation of hypothalamic proopiomelanocortin (POMC) mRNA by estradiol (E2) is a component of reduced neuroendocrine sensitivity to estrogen in the aging mouse. Young (4 month-old), middle-aged (13 month-old), and old (25 month-old) C57BL/6J mice were ovariectomized, implanted 2 weeks later with Silastic capsules containing E2 or cholesterol (CHOL), and sacrificed 3 days later. Hypothalamic POMC mRNA was measured by solution hybridization/RNase protection, using a RNA probe complementary to a fragment of mouse POMC mRNA. In the group with CHOL implants, POMC mRNA was 36% lower in middle-aged and old mice compared to young mice. E2 treatment reduced POMC mRNA levels by 44% in young mice but failed to lower POMC mRNA in middle-aged and old animals. These results confirm earlier evidence of reduced levels of POMC mRNA in hypothalami of aging rodents and indicate that the ability of E2 to reduce hypothalamic POMC mRNA is lost by middle age.

Up-regulation of the uterine estrogen receptor and its messenger ribonucleic acid during the mouse estrous cycle: the role of estradiol.

Uterine estrogen receptor (ER) and ER mRNA were measured in cycling and ovariectomized (OVX) estrogen-treated mice to probe the physiological regulation of the intracellular distribution and biosynthesis of ER. On proestrus, when plasma estradiol (E2) levels are highest, the cell nuclear ER concentration was 2.4-fold greater than on metestrus. This increase was primarily attributable to an increase in total cellular ER (cytosolic plus nuclear ER) and only secondarily to an activation of ER, as measured by its redistribution from the cytosolic (i.e. nuclear-extractable) to the nuclear (nonextractable) fraction. Total cellular ER concentration was 1.8-fold higher on proestrus than on metestrus, whereas the fraction of total ER in the nuclear compartment (i.e. the percentage activated) was only 1.3-fold higher. The concentration of cellular ER mRNA was 3-fold greater on proestrus than on the other days of the estrous cycle, suggesting that the increased concentration of ER on proestrus was a consequence of increased ER gene expression. In OVX mice, physiological and, to a lesser extent, supraphysiological levels of E2 increased cell nuclear ER. As in proestrous mice, the increased ER content contributed more than ER activation to the increased cell nuclear ER concentration. Physiological, but not supraphysiological, concentrations of E2 increased ER mRNA in OVX mice. Together, these results suggest that up-regulation by E2 of ER mRNA and ER accounts for most of the increased nuclear binding of ER on proestrus. E2-dependent activation and consequent DNA binding of ER presumably initiate this process, but quantitatively account for only a small fraction of the increased nuclear binding of ER.

Genetic influences on oestrous cyclicity in mice: evidence that cycle length and frequency are differentially regulated.

Studies in C57BL/6J, DBA/2J and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1) 2-5 months old revealed marked genotypic differences among inbred strains. C57 mice had three times as many regular (3-6 days) cycles as DBA and C3H mice, due largely to fewer pseudopregnant-like (7-14 day) cycles. C57 had longer regular cycles than DBA and C3H mice. Although the frequencies of regular cycles of DBA and C3H mice were similar, the cycles of C3H mice were shorter than those of DBA mice. The results indicated that the genetic determinants of the frequency of regular cycles differ from those specifying cycle length. Frequency of regular cycles of F1 hybrids was either intermediate between the parent strains (B6D2F1) or similar to the C57 strain (B6C3HF1), suggesting that regular cycle frequency shows additive genetic variation in the former crosses, but mostly dominant variance in the latter background. Regular cycles were either shorter than in both parent strains (B6D2F1) or similar to one of them (B6C3HF1), indicating heterosis and dominance for genes specifying short cycles. Although the lack of reciprocal crosses meant that maternal effects and possible genomic imprinting effects could not be assessed, these results reveal marked genetic influences on cycle length and frequency and suggest that some of the genes specifying these two traits differ.

Absence of estrogenic activity in a diet that promotes estrous cyclicity in C57BL/6J mice.

A breeder diet that shortens estrous cycles in mice has been reported to contain estrogenic substances, based on its ability to increase uterine weight of immature mice. However, the estrogenicity of the diet was inferred from uterine weight gain of immature mice that were intact. The increased uterine weight of mice on the breeder diet could thus have resulted from a precocious pubertal increase of endogenous estrogens induced by the diet rather than estrogenic substances in the diet. We therefore measured the estrogenicity of the breeder diet in ovariectomized animals. C57BL/6J mice were fed the breeder diet or a standard diet for 1 or 4 weeks. The breeder diet failed to increase uterine weights above control values for either treatment interval. Intact mice that were fed the breeder diet had twice the number of cycles of mice fed the standard diet, a confirmation of earlier studies. These results indicate that the breeder diet does not contain biologically significant estrogenic activity, and thus potentiates cyclicity by other means.

Age-related alterations in estrogen receptor dynamics are independent of cycling status in middle-aged C57BL/6J mice.

The objective of this study was to determine whether changes in estrogen receptor (ER) levels and dynamics that were previously observed in old acyclic mice were present in middle-aged mice and whether the cycling status of the mice influenced those changes. Young (3-6 months) regularly cycling and middle-aged (12-14 months) C57BL/6J mice that were either acyclic or still cycling regularly were injected with a dose of E2 (0.05 microgram/10 g body wt) sufficient to achieve maximal levels of nuclear ER (ERn) in all tissues examined: hypothalamus (HYPO), pituitary (PIT), and uterus (UT). The rise and fall of ERn and the replenishment of cytosolic ER (ERc) were measured 0, 1, 2, 4, 8, 12, and 24 h later. Cycling status did not affect ER binding profiles in middle-aged tissues. Therefore, data from cycling and acyclic subgroups were pooled for comparison with young mice. The increase in ERn following E2 injection, measured as the integrated area under the ERn profile, was reduced 33, 23, and 17%, respectively, in HYPO, PIT, and UT of middle-aged mice. In addition, the duration of elevated ERn was selectively reduced in middle-aged HYPO. ERc levels were reduced in middle-aged HYPO and UT, but replenishment rates were not altered. Reductions in total ER (ERn + ERc) were sufficient to account for the decline in ERn in middle-aged HYPO and UT, but factors in addition to ER loss appear to contribute to reduced ERn in middle-aged PIT. These results indicate that alterations in ER levels and dynamics occur prior to the transition to acyclicity, that these alterations are not secondary to hormonal or other changes associated with acyclicity, and that receptor loss appears to account for most of the age-related reduction in nuclear ER binding.

Effects of chronic exposure to estradiol on ovarian cyclicity in C57BL/6J mice: potentiation at low doses and only partial suppression at high doses.

Long-term exposure to ovarian hormones contributes to age-related changes in estrous cyclicity in rodents. Estrogens are implicated in this process, but the concentration of estrogen required to exert these effects is not well established. Also, although estrogens are presumed to alter vaginal cyclicity by affecting the hypothalamic-pituitary axis, they may also impair the ability of the vaginal epithelium to cornify. To address these issues, young and middle-aged ovariectomized (ovx) C57BL/6J mice were exposed for 7-10 wk to plasma levels of estradiol (E2) at one of three ranges (30-40, 50-80, or 120-160 pg/ml). Ovaries from young mice were then transplanted under the renal capsule, and vaginal cyclicity was monitored for 4 mo. Mice exposed to the lowest level of E2 not only failed to stop cycling, but had a higher monthly frequency of estrous cycles than did controls (nearly 1 extra cycle/mo). Mice exposed to the intermediate level of E2 showed no impairment in cyclicity. Although mice exposed to the highest concentrations of E2 showed no vaginal cyclicity, they continued to ovulate as evidenced by fresh, albeit reduced, numbers of corpora lutea. These results indicate that, in ovx mice, (1) chronic exposure to relatively low concentrations of E2 potentiates cyclicity, (2) very high levels of E2 are required to induce acyclicity, and (3) this acyclicity reflects vaginal as well as neuroendocrine alterations. The results also indicate that vaginal acylicity may be a poor indicator of ovulatory acyclicity in mice that have been chronically exposed to E2.

Genetic influences on the timing of puberty in mice.

Genetic influences on the timing of three pubertal events--vaginal opening, first vaginal cornification, and onset of cyclicity--were studied in C57BL/6J, DBA/2J, and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1). Marked genotypic differences were found. Among inbred strains, differences in the onset of vaginal opening and first vaginal cornification (C3H less than DBA less than C57) did not parallel those for the onset of cyclicity (C3H much greater than DBA = C57). Compared to parental strains, F1 hybrid strains were intermediate for times of vaginal opening and first vaginal cornification, consistent with the model in which the genetic effects on the timing of these events are additive. By contrast, onset of cyclicity occurred significantly earlier in the F1 hybrids than in their parent strains, indicating heterosis for one or more genes specifying this event. Body weights also differed among the genotypes from weaning onward, but these differences were only partially correlated with the differences in the timing of the pubertal events. Thus, genetic influences other than those affecting body weight contribute to the differential timing of pubertal events in these mouse strains. These results reveal marked genetic variation in the timing of puberty, and indicate that the set of genes specifying the timing of vaginal opening and first vaginal cornification differs from those specifying the onset of cyclicity.

Differential effects of aging on estrogen receptor dynamics in hypothalamus, pituitary and uterus of the C57BL/6J mouse.

Estrogen receptor (ER) dynamics and content were measured in the hypothalamus (HYPO), pituitary (PIT) and uterus (UT) of aging mice because of their potential importance to age-related changes in sensitivity to estrogen. Young (3-6 months), and old (22-24 months) C57BL/6J mice were injected with a dose of E2 (0.05 micrograms/10 g body wt) sufficient to achieve maximal levels of nuclear ER (ERn) in all tissues, and the rise and fall of ERn and the depletion and replenishment of cytosolic ER (ERc) were measured 0, 1, 2, 4, 8, 12 and 24 h later. Integrated areas under the ERn profiles in old HYPO, PIT and UT were reduced 34, 28 and 19%, respectively. These reductions were due to (1) lower levels of ERn throughout the profiles, (2) delays in attainment of peak ERn in UT and PIT, and (3) accelerated loss of peak ERn in HYPO. ERc levels were also reduced in old mice, and replenishment of ERc was delayed in old HYPO and PIT, but not in UT. Reductions in total ER (ERn + ERc) were sufficient to account for all reductions and altered dynamics of ERn, except for the delayed attainment of peak ERn in UT. These results indicate that levels and dynamics of nuclear ER are altered during aging, and that most of these changes are secondary to alterations in ER content and turnover rather than a reduced ability of ER to bind to nuclear sites.

Longitudinal studies of estrous cyclicity in C57BL/6J mice: III. Dietary modulation declines during aging.

Dietary modulation of estrous cyclicity was studied throughout the reproductive lifespan to assess the stability of age-related changes in cyclicity and to probe underlying mechanisms. Animals were fed a standard diet or an isocaloric breeder diet that differed in nutrient composition to promote fecundity. In young mice, the breeder diet more than doubled the frequency of short (4-day) cycles, and, as a result, increased the total number of cycles during the cycling lifespan by 10%. Dietary potentiation of short cycles disappeared between 7 and 9 months of age, and most subsequent age-related changes in cyclicity were resistant to dietary influence. The breeder diet had no effect on the transition from 4- to 5-day cycles, the onset of acyclicity, or on the incidence or duration of persistent vaginal cornification. It only delayed the increase of very long (greater than 5-day) cycles by 1 month. These results show that most age-related changes in cyclicity are not influenced by dietary differences that affect cyclicity in young mice, and that diminished responsiveness to dietary variation is among the earliest age-related changes in the reproductive system. In addition, the results suggest that differences in cycle frequency and, presumably, in cumulative exposure to pre-ovulatory elevations of ovarian steroids do not influence the cycling lifespan in this strain of mouse.

Tissue differences in estrogen receptor dynamics: nuclear retention, rate of replenishment, and transient receptor loss vary in hypothalamus, pituitary, and uterus of C57BL/6J mice.

Little is known about tissue differences in estrogen receptor (ER) dynamics, despite evidence that they could play a role in the tissue specificity of estrogen action. This study was designed to test the hypothesis that ER dynamics differ in uterus (UT), pituitary (PIT), and hypothalamus (HYPO), as measured by 1) duration of peak nuclear ER (ERn), 2) rate of replenishment of cytosolic ER (ERc), and 3) loss of total ER (ERt) after a bolus of estradiol (E2). Young adult mice were studied at two hormonally distinct stages of the cycle [days 2 or 3 (D2-3) and D5 (D1 = proestrus)]. Animals were injected with a dose of E2 (0.05 microgram/10 g BW) sufficient to achieve maximal ERn or with vehicle only, and ER was determined in nuclear and cytosolic fractions 1, 2, 4, 8, 12, and 24 h later. ERn peaked concomitantly with plasma E2 at 1 h in all tissues, but the duration of peak ERn varied among tissues: 4 h in HYPO compared to 1-2 h in UT and PIT. ERc replenishment was complete by 12 h in HYPO, but not until 24 h or more in PIT; replenishment in UT was intermediate (12-24 h). The transient loss of ERt after E2 injection was pronounced in UT and PIT, but was undetectable in HYPO. These tissue differences were maintained across cycle state, despite effects of cycle state on ER dynamics. The effects of cycle state on ER dynamics were also tissue specific; they were greatest in UT and absent in HYPO. On D2-3 in UT, ERn and ERt were lower, and replenishment of ERc was slower than on D5. Parallel effects of cycle state were seen in PIT, with the exception of ERn, which was unaffected. Because altered ER dynamics similar to those observed on D2-3 can be produced by progesterone pretreatment, the altered ER dynamics on D2-3 may be a consequence of recent exposure on D1 to the ovulatory surge of progesterone. Taken together, these results indicate that the mechanisms governing intracellular ER dynamics vary markedly among tissues and provide an impetus for further examination of their role in the tissue specificity of estrogen action.

Aging of the hypothalamo-pituitary-ovarian axis: hormonal influences and cellular mechanisms.

Longitudinal studies employing heterochronic ovarian grafts and long-term ovariectomy indicate that there is no single pacemaker of reproductive aging. Neuroendocrine dysfunction, the declining follicular reserve, and ovarian secretions all contribute to reproductive decline, and their relative importance to the different stages of reproductive aging varies markedly. Moreover, although ovarian secretions during adulthood potentiate certain aspects of the reproductive aging process, their behavior does not fit a simple model of cumulative steroidal damage incurred over the lifespan. Current data are more consistent with temporally distinct windows of steroidal vulnerability for the events affected: cycle lengthening is affected by ovarian secretions during the period of cyclicity, and post-cyclic neuroendocrine failure is potentiated by ovarian secretions during the peri- and post-cyclic period of the lifespan. Recent examination of estradiol receptor dynamics reveals multiple, albeit selective, changes during aging that may contribute to the age-related impairments of tissue sensitivity to estrogen. These changes vary qualitatively and quantitatively among target tissues. Thus, aging of the hypothalamo-pituitary-ovarian axis at the cellular level mirrors, in its multifactorial nature, aging at the organismic level.