Ovarian and steroidal influences on neuroendocrine aging processes in female rodents.

Some Mammalian aging processes involve effects of steroids on the brain and pituitary. An ovary-dependent, neuroendocrine aging syndrome of laboratory rats and mice is described in this article. This syndrome can be attenuated during aging by chronic ovariectomy and can be prematurely induced in young rodents by sustained exposure to estradiol (E2). The limited follicular stock in the ovary is proposed to be a major pacemaker of aging in this neuroendocrine syndrome; ovarian aging may interact with neuroendocrine aging. Ovary-independent neuroendocrine changes occur as well. We also discuss developmental influences on adult aging in rodents and other examples in which adult lower mammals are sensitive to long lasting effects of steroids on the brain and pituitary. Possible molecular mechanisms are considered. In view of the long lasting effects of E2 and other steroids on lower mammals, the potential for long term effects of ovarian steroids on the human brain and pituitary warrants continued evaluation.

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.

Differential contributions of ovarian and extraovarian factors to age-related reductions in plasma estradiol and progesterone during the estrous cycle of C57BL/6J mice.

The relative contributions of ovarian and extra-ovarian factors to the altered ovarian steroidal profiles of middle-aged mice were assessed by reciprocal, heterochronic ovarian grafting. Ovaries from cycling, young (2 months), and middle-aged (12 months) mice were exchanged by grafting under the renal capsules. Blood samples were obtained daily at midday throughout the estrous cycle for measurement of estradiol (E2) and 3-4 h after lights-out on proestrus to measure the preovulatory elevation of progesterone (P4). Middle-aged intact mice had lower mean concentrations of E2 during the cycle, no detectable midday preovulatory elevation of E2, and an attenuated preovulatory increase of P4 compared to young mice. Ovarian grafts from young donors failed to increase mean E2 levels of middle-aged mice, but did restore the preovulatory elevation of E2 and preovulatory P4 to levels of young controls. Reciprocal grafting confirmed these findings: ovaries from middle-aged donors in young hosts produced mean E2 levels equivalent to those of young mice but were unable to support a preovulatory increase of E2 or a preovulatory P4 level equivalent to that of young controls. These results reveal differential contributions of ovarian and extra-ovarian factors to age changes in E2 and P4. They indicate that ovarian aging plays an important role in attenuating the preovulatory increase of E2 and P4, but extra-ovarian, presumably neuroendocrine, age changes underlie the mean reduction of E2 levels across the estrous cycle.

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.

Spontaneous tumors in aging female mice are more prevalent in the lateral pituitary zones.

The localization of gross and microscopic spontaneous pituitary tumors was examined in aging female C57BL/6J mice. These tumors were lactotroph adenomas, by morphological and immunocytochemical criteria. Each lobe of the pars distalis was divided into three zones of approximately equal size and the number of tumors in each zone was counted. Twelve out of 30 tumors were located entirely within the most lateral zone. An additional 14 tumors occurred in both the most lateral and the interjacent zones. Thus, almost 90% of the observed tumors were localized in more lateral zones of the pars distalis (Chi-squared test, p less than 0.01). These findings support a hypothesis that lower portal blood dopamine levels reaching lateral portions of the pars distalis are a factor in the higher incidence of lactotroph adenomas in these zones.

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.

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.

Dietary modulation of estrous cyclicity in singly and multiply housed C57BL/6J mice.

Two commercial diets differed markedly in their ability to support estrous cyclicity in both singly and multiply housed, 3- to 4-month-old virgin C57BL/6J mice. When fed a breeder diet developed to promote reproduction, mice had shorter cycles and a decreased number of acyclic intervals than when fed a standard diet. In addition, the breeder diet counteracted the inhibitory influence of multiple housing on cyclicity. Although the breeder diet had a three-fold higher fat content and a two-fold lower fiber content, it was not possible to ascribe a causal role to these differences since the diets differed in the relative amounts and sources of many other components.

Radical ovarian resection advances the onset of persistent vaginal cornification but only transiently disrupts hypothalamic-pituitary regulation of cyclicity in C57BL/6J mice.

In aging laboratory rodents, neuroendocrine failure to support estrous cyclicity is in part the consequence of exposure to ovarian secretions during adulthood. Moreover, some evidence suggests that those secretions associated with the predominant postcyclic state, persistent vaginal cornification (PVC), are more deleterious than those associated with cyclicity. However, it is not clear whether postcyclic hormonal secretions are intrinsically more deleterious or whether vulnerability to ovarian secretions, regardless of their nature, increases during aging. Using relatively young, age-matched mice, this study was designed to control for age and to determine if the hormonal milieu associated with PVC would be more deleterious to neuroendocrine function than that associated with regular cyclicity. Onset of PVC was advanced about 5 mo by resecting 90-95% of the ovarian tissue from 5-mo-old mice. The resultant PVC was similar in duration, vaginal cytology and ovarian histology to that seen in normally aging mice. At age 13 mo, when mean duration of PVC was 3 mo in resected mice but only 1 mo in sham-operated controls, the ability of mice to support cyclicity upon receipt of ovarian grafts from 4-mo-old donors was tested as an index of neuroendocrine function. The response of resected mice was slightly impaired, but only during the first month after grafting. This transient disruption of neuroendocrine function in mice prematurely exposed to PVC stands in contrast to the irreversible loss of cycling potential in older animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Longitudinal studies of estrous cyclicity in aging C57BL/6J mice: II. Cessation of cyclicity and the duration of persistent vaginal cornification.

The of onset of acyclicity and the duration of persistent vaginal cornification (PVC), the predominant postcyclic anovulatory condition, were measured in five cohorts of virgin mice staggered across a 6-yr interval. The average age of cessation of cyclicity varied significantly among cohorts, ranging from 11 to 16 mo of age. Seventy-five percent of the mice entered PVC after ceasing to cycle, the remainder entered directly into a persistent diestrous condition. The duration of PVC, which averaged between 2 and 4 mo, varied markedly among individuals and was inversely related to the age of onset of acyclicity. Consequently, the variance associated with the cessation of cyclicity was about 4-fold greater than that associated with the cessation of PVC. These differing variances suggest that the factors governing ovulatory failure and PVC cessation may differ.

Effect of dietary restriction on estrous cyclicity and follicular reserves in aging C57BL/6J mice.

Restricting the food intake of female mice by alternating days of feeding and fasting delayed the age-related loss of estrous cycling potential and retarded the rate of follicular depletion, as determined after reinstatement of ad libitum (AL) feeding. During the period of food restriction (FR; 3.5-10.5 mo), food intake and body weight were about 80% of AL values. Mice were acyclic and predominantly in a state of diestrus during FR, but after reinstatement of an AL diet at 10.5 mo all FR mice resumed cycling regularly. By contrast, 80% of AL controls had become acyclic by this age, and the cycles of the remaining mice were significantly longer than those of the reinstated FR mice. Follicular reserves of 12.5-mo-old FR mice were twice those of age-matched AL controls. Cycling performance of reinstated FR mice, measured by cycle length and the proportion of mice still cycling, was equivalent to that of AL mice when the latter were 2-5 mo younger. Ovarian age, measured by the size of the follicular reserve, was similarly retarded in FR mice. Based on these data and previous evidence that follicular depletion plays a major role in the cessation of cyclicity in this strain, we hypothesize that the delayed loss of estrous cyclicity in aging FR mice is mediated at least in part by the retarding effect of dietary restriction on the rate of follicular depletion.

Prolongation and cessation of estrous cycles in aging C57BL/6J mice are differentially regulated events.

The relative contributions of ovarian failure and hypothalamic-pituitary dysfunction to the prolongation and cessation of estrous cycles were assessed by measuring the ability of acutely ovariectomized (OVX) middle-aged (12 mo) mice to cycle after receiving grafts (under the renal capsule) of ovaries from young (2 mo) mice. The potentially disruptive effect of the acyclic state on the cycling response to grafted, young ovaries was avoided restricting grafting to middle-aged hosts that were still cycling. The effect of chronic exposure to ovarian secretions before the cessation of cyclicity on age-related hypothalamic-pituitary dysfunction was also assessed. The cycling ability of long-term OVX middle-aged mice (i.e., OVX at 3 mo) bearing grafts of young ovaries was compared to that of age-matched acutely OVX controls. Grafted young ovaries extended the cycling lifespan of acutely OVX middle-aged hosts by 60%. The length of this extended cycling lifespan, however, was only 80% of that achieved by young hosts bearing grafts of young ovaries. Young ovaries in middle-aged mice markedly lowered the incidence of long cycles (greater than 5 days), shifting the modal cycle length to 5 days. However, young ovaries in middle-aged mice failed to increase the incidence of 4-day cycles, the modal cycle of young controls. Middle-aged ovaries grafted into young hosts lengthened their cycles and shortened their cycling lifespan to middle-aged values. Long-term ovariectomy failed to increase the cycling lifespan of middle-aged hosts bearing grafts of young ovaries beyond that achieved in acutely OVX mice. Long-term ovariectomy did shorten the modal cycle length of middle-aged mice to 4 days, although the duration of 4-day cycling was only one-third (2 mo) that of young controls. These results indicate that the relative contributions of ovarian and neuroendocrine factors to three major events of reproductive aging vary with each event. Whereas the hypothalamic-pituitary unit appears to play an important role in the initial shift from 4- to 5-day cycles, the aging ovary plays the major role in the subsequent shift to longer cycles and in the ultimate cessation of cyclicity. Although chronic exposure to ovarian secretions during the period of cyclicity does not play a major role in the cessation of cyclicity, it appears to contribute to the hypothalamic-pituitary changes responsible for the initial shift from 4- to 5-day cycles.

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.

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.

Pituitary tumorigenesis in aging female C57BL/6J mice: a light and electron microscopic study.

Spontaneous pituitary tumors have been studied by light and electron microscopy in female C57BL/6J mice at 2 1/2, 11, 15, 22, 23, 24 and 30 months. Tumors were evident macroscopically in greater than 50% of mice 22 months or older, and greater than 80% on microscopic evaluation. Active and hypertrophied mammotrophs were the predominant cell type within the tumors at 22-30 months, often totally filling large portions of the tumor mass. Exocytosis of secretory granules was extensive from the mammotrophs, but much less abundant from other parenchymal cells. Somatotrophs and gonadotrophs were also present, and appeared active and often strikingly hypertrophied. The tumors were characterized by disruptions of parenchymal and capillary integrity which resulted in the formation of large vascular lakes lined solely by tumor cells, generally mammotrophs. Apparent metastasis of tumor cells into the vascular lakes was also observed. In 11- and 15-month mice small tumors or pretumor foci were evident in some mice on microscopic evaluation, although they were not visible macroscopically. Their degree of development was somewhat variable, but they had essentially the same features as more advanced tumors in older mice. pretumor foci were characterized by more moderate disruptions of parenchymal cell and capillary integrity; cellular hypertrophy, particularly of somatotrophs and gonadotrophs; and the presence of small vascular lakes. In 2 1/2-month mice tumors could not be localized macroscopically or microscopically, and the pituitary was composed of well-defined cell cords and an intact capillary bed. However, small focal zones of capillary and tissue disruption were apparent occasionally in 2 1/2-month mice. These findings indicate that the process of pituitary tumorigenesis in female C57BL/6J mice is initiated by midlife, with subsequent progressive development into large, mammotroph-dominated tumors.

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.

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.

A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology.

Cycle frequency, length, and vaginal cytology were measured longitudinally in three cohorts of singly housed virgin mice staggered across a 3-year interval. The age profiles of these parameters were qualitatively similar, but quantitatively different, among cohorts. Cycle frequency was initially low (Phase I), due to prolonged cycles and late-starting cycles, and did not peak (Phase II) until mice were 3-5 months old. Phase II lasted for 7-10 months, depending on the cohort. Thereafter cycle frequency declined steadily (Phase III). The average age of cessation of cyclicity varied among cohorts, occurring between 13 and 16 months of age. Age changes in cycle length paralleled those of cycle frequency. During Phase II, median cycle length was less than 5 days and variance was lowest. During Phases I and III, variance was about twofold greater and median cycle length was greater than 5 days. Although median cycle length remained stable for several months during Phase II, the peak period of 4-day cycles was much shorter. In all cohorts, 4-day cycles did not peak until 7-8 months of age and began to decline by 9 months. The decrease in 4-day cycles was associated with a progressive lengthening of cycles-first from 4 to 5 days, then to longer cycles. The fraction of cycles with extended cornification (greater than 2 days) increased with advancing age from less than 0.35 during the initial period of cycle lengthening to a maximum of 0.60. The observation that the initial phase o cycle prolongation was not usually associated with extended cornification is consistent with earlier evidence that this period is characterized by a delayed, rather than prolonged, preovulatory rise of estradiol. However, the increased fraction of prolonged cycles with extended cornification at later ages suggests that the preovulatory elevation of estradiol may ultimately be prolonged.