Menopause: the aging of multiple pacemakers.

Menopause signals the permanent end of menstrual cyclicity in a woman's life. Its impact reaches far beyond just the reproductive system. An understanding of the factors that interact and govern the process of aging in the reproductive system will help us to develop strategies for alleviating the negative aspects of menopause and may help us to better comprehend the process of biological aging.

Neuroendocrine influences and repercussions of the menopause.

In summary, the evidence that both the ovary and the brain are key pacemakers in the menopause is compelling. Our appreciation that estrogens are important neurotrophic and neuroprotective factors has grown rapidly. Future studies will allow us to better understand the ensemble of factors that interact to maintain regular reproductive cyclicity and how this precise dynamic balance changes with age. Furthermore, understanding how estrogen exerts trophic and protective actions should lead to its use as an important therapeutic agent in the maintenance of normal neural function during aging and after injury.

Estrogen therapy: does it help or hurt the adult and aging brain? Insights derived from animal models.

Hormone therapy and estrogen therapy in postmenopausal women have been thought to ameliorate cognitive dysfunction and decrease the risk and/or progress of neurodegenerative conditions such as Alzheimer's disease and stroke. Furthermore, estrogens have been shown to exert neuroprotective actions in a variety of in vitro and in vivo models of brain injury. However, the findings of the Women's Health Initiative have made us re-evaluate these assumptions. Our laboratory has shown that physiological levels of estradiol attenuate ischemic brain injury in young and middle-aged female rats. We have begun to probe the cellular and molecular mechanisms that underlie these novel non-reproductive actions of this steroid. Our findings demonstrate that in both young and aging rats, treatment with physiological concentrations of estradiol decreases ischemic injury by almost 50%, compared with oil-treated controls. Additionally, our data suggest that estradiol acts by altering the expression of genes that suppress apoptosis and enhance survival in the penumbral region of the infarct. These observations demonstrate that estrogen therapy protects against stroke-related injury in young and aging female rats and strongly suggest that middle-aged animals remain responsive to the protective actions of estradiol. Furthermore, they suggest that estrogen therapy protects against cell death by influencing the expression of genes that suppress apoptotic cell death pathways.

Stroke injury in rats causes an increase in activin A gene expression which is unaffected by oestradiol treatment.

Activins are members of the transforming growth factor-beta superfamily that exert neurotrophic and neuroprotective effects on various neuronal populations. To determine the possible function of activin in stroke injury, we assessed which components of the activin signalling pathway were modulated in response to middle cerebral artery occlusion (MCAO). Furthermore, because oestradiol replacement protects against MCAO-induced cell death, we explored whether oestradiol replacement influences activin gene expression. Female Sprague-Dawley rats underwent permanent MCAO and the expression of activins and their corresponding receptors was determined by semiquantitative reverse transcriptase-polymerase chain reaction at 24 h after onset of ischaemia. We observed up-regulation of activin betaA and activin type I receptor A mRNA in response to injury. Dual-label immunocytochemistry followed by confocal z-stack analysis showed that the activin A expressing cells comprised neurones. Next, we monitored the time course of activin betaA mRNA expression in oestradiol- or vehicle-treated rats at 4, 8, 16 and 24 h after MCAO via in situ hybridisation. Starting at 4 h after injury, activin betaA mRNA was up-regulated in cortical and striatal areas in the ipsilateral hemisphere. Activin betaA mRNA levels in the cortex increased dramatically with time and were highest at 24 h after the insult, and oestradiol replacement did not influence this increase.

Diurnal pattern of proopiomelanocortin gene expression in the arcuate nucleus of proestrous, ovariectomized, and steroid-treated rats: a possible role in cyclic luteinizing hormone secretion.

Opiate peptides are thought to modulate the pattern of LH release in female rats. We tested the hypothesis that changes in proopiomelanocortin (POMC) gene expression occur in proestrous (PRO) and ovariectomized (OVX) steroid-treated rats which may explain their unique patterns of LH secretion. Using in situ hybridization, we examined whether diurnal changes in POMC gene expression occur in the arcuate nucleus. Four groups of rats were used in this study. 1) PRO rats were used after exhibiting at least two consecutive 4-day estrous cycles; 2) OVX rats were killed 9 days after ovariectomy; 3) estradiol (E2)-treated rats were OVX for 7 days and then treated for 2 days; and 4) E2-progesterone (P4)-treated rats were treated with E2 as described above, and on day 9 at 1030 h, P4 was administered. Rats were killed at 2300, 0300, 1000, 1300, 1500, 1800, or 2300 h, beginning on the evening of diestrous day 2 or day 8 after ovariectomy. POMC gene expression exhibited a diurnal rhythm on PRO. Levels of mRNA rose during the morning, peaked between 0300-1000 h, and decreased by 2300 h. In E2-treated rats, which exhibited a LH surge similar in timing to the PRO surge, POMC mRNA levels exhibited a diurnal rhythm strikingly similar to that observed in PRO animals. OVX abolished the rhythm; however, average POMC mRNA levels across the 24-h period were not significantly different from those in PRO or E2-treated rats. P4 treatment increased POMC mRNA levels by 2300 h compared to those in all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of peptide secretion and gene expression in the same cell.

A combined reverse hemolytic plaque-in situ hybridization assay was developed to allow analysis of the relationship between peptide secretion and gene expression within individual cells. We used the pituitary lactotroph as a model system, but this strategy should be widely applicable. It can be used to test hypotheses regarding if and when peptide secretion and gene expression are coupled in any system in which antibodies to the secreted peptide and probes complementary to the mRNA are available. Using the mRNA hybridization signal to identify certain cell types, this method may also be useful in further studies on the biochemical mechanism of peptide secretion. In addition, questions regarding whether a cell known to secrete a given peptide contains other specific mRNAs and the relationship between these mRNAs and the secretion of the peptide can be studied using this strategy. We found striking heterogeneity among lactotrophs in both gene expression and PRL secretion and a lack of correlation of these parameters within individual lactotrophs under every treatment examined. We also present the first direct visualization and quantitation of the percentage of nonsecreting PRL mRNA-containing cells after estradiol treatment and in the presence or absence of the PRL secretagogue, TRH. Finally, we found that in ovariectomized rats, nonsecreting lactotrophs exhibited significantly higher levels of PRL mRNA than lactotrophs that were actively secreting PRL during the assay.

Age-related changes in light-induced Jun-B and Jun-D expression: effects of transplantation of fetal tissue containing the suprachiasmatic nucleus.

Fos and Jun mRNA and peptide exhibit a daily light-induced rhythm in the suprachiasmatic nucleus (SCN). The authors previously have reported that Fos expression in the SCN is elevated prematurely during the dark, light-induced Fos expression is attenuated in middle-aged rats, and transplantation of fetal SCN tissue into the third ventricle of rats of this age restores the daily pattern of Fos expression to that of the young. Using immunocytochemistry, the authors performed the present study to determine whether Jun-B and Jun-D expression in the SCN is altered at the same stage during aging and, if so, whether transplantation of fetal tissue containing the SCN can restore the light-induced rhythms of these two immediate early genes. All groups of rats were transcardially perfused 90 min prior to and after light onset. In young rats, light induced a robust increase in the number of Jun-B positive cells in the SCN, whereas very few cells were labeled before light onset. In middle-aged rats, the light-induced increase in the number of Jun-B positive cells was significantly attenuated. Transplantation of fetal SCN tissue into the middle-aged rats successfully restored light-induced Jun-B expression to the levels of young rats. By contrast, Jun-D exhibited a constitutively high level of expression in the SCN both before and after light onset, and light induced only a slight but significant increase. No age-related changes were detected in the expression of Jun-D either before or after light onset. Transplantation of fetal SCN tissue did not alter the daily pattern of Jun-D expression in the middle-aged rats. These data suggest that (1) light-induced activation of SCN neural activity is blunted during aging, (2) fetal SCN tissue can provide the critical support to allow the host to respond properly to light cues, and (3) the age-related change in Jun-B expression in the middle-aged host SCN can be rescued by fetal SCN transplants.

Effects of hyperprolactinemia on estrous cyclicity, serum luteinizing hormone, prolactin, estradiol, and progesterone concentrations, and catecholamine activity in microdissected brain areas.

The purpose of the present study was to determine the duration of PRL treatment required to suppress estrous cyclicity and preovulatory LH surges and to ascertain whether such treatment affects cyclicity by altering catecholamine activity and/or ovarian steroid secretion. Rats exhibiting 4-day estrous cycles were treated with ovine PRL (oPRL) or vehicle beginning on diestrous day 1 at 0900 h, diestrous day 2 at 2400 h, proestrus at 0600 h, or proestrus at 1200 h. Jugular veins of rats were cannulated to the level of the right atrium on diestrous day 2. Unrestrained rats were bled on proestrus. Preovulatory LH surges and ovulation were completely blocked, and vaginal cytology remained leukocytic on the expected day of proestrus and estrus when oPRL treatment was begun on diestrous day 1. Such treatment elevated progesterone levels beginning on diestrous day 2 and suppressed the preovulatory rise in estradiol observed on proestrous morning and afternoon in control rats. To determine the effect of oPRL on catecholamine activity, alpha-methylparatyrosine was administered to groups of oPRL- or vehicle-treated rats at 0900 or 1500 h on diestrous day 1, diestrous day 2, or proestrus. Animals were killed 0, 45, or 90 min later. Norepinephrine and dopamine concentrations were measured in the median eminence, suprachiasmatic nucleus, medial preoptic nucleus, striatum, ventral aspect of the stria terminalis, and posterior pituitary gland by radioenzymatic assay. Controls exhibited increased norepinephrine turnover rates in the median eminence, suprachiasmatic nucleus, and medial preoptic nucleus on proestrous afternoon concomitant with preovulatory LH surges. In contrast, oPRL-treated rats showed no such increase. In addition, median eminence dopamine turnover rates were elevated beginning on the afternoon of diestrous day 1 in oPRL-treated rats compared to control values. No other differences in norepinephrine and dopamine turnover rates were observed in oPRL-treated rats compared with controls in any other brain area on any day examined. Thus, the data indicate that elevated PRL concentrations have profound effects on reproductive cyclicity by disrupting ovarian steroid secretion and essential preovulatory neurochemical events in selected brain areas involved in the regulation of LHRH.

Estradiol-induced daily luteinizing hormone and prolactin surges in young and middle-aged rats: correlations with age-related changes in pituitary responsiveness and catecholamine turnover rates in microdissected brain areas.

These studies assessed the effects of age on the ability of estradiol-17 beta (E2) to induce LH and PRL surges in ovariectomized young and middle-aged rats that previously had normal estrous cycles. We determined whether any changes in the timing or amplitude of these surges could be correlated with changes in pituitary responsiveness to GnRH or with changes in norepinephrine (NE) and dopamine (DA) turnover rates in microdissected brain regions involved in cyclic gonadotropin release. Young (3-4 months old) and middle-aged (9-12 months old) rats were ovariectomized. One week later (day 0), they received Silastic capsules containing E2 which produced physiological serum concentrations of E2. Groups of rats were bled sequentially via indwelling right atrial cannulae 1-4 days after capsule implantation (days 1-4). All young rats displayed maximal LH surges by day 2 and exhibited equivalent surges on days 3 and 4. Middle-aged rats required the presence of E2 for at least 3 days before a maximal positive feedback response was achieved. Even at these times the timing of the LH rise was delayed by 1 h and peak concentrations were lower in middle-aged rats. E2-induced PRL surges did not exhibit any age-related differences. Pituitary responsiveness to GnRH was tested by administering two injections of GnRH to pentobarbital-blocked young and middle-aged rats on days 2 and 4. Pituitary responsiveness to the first injection on day 2 was blunted in middle-aged rats; however, the LH response at all other times was normal. Catecholamine turnover rates were examined on days 2 and 4 by giving alpha-methyl-para-tyrosine at 1000 or 1500 h and killing rats 45 or 90 min later. Resting initial catecholamine concentrations were assessed in untreated rats killed at 1000 h or 1500 h. The medial preoptic nucleus, suprachiasmatic nucleus, and median eminence were microdissected and assayed for NE and DA by radioenzymatic assay. In young rats, NE turnover rates increased during the afternoon in all brain areas on both days. In contrast, in middle-aged rats, no increase in NE turnover rates was observed during the afternoon of day 2. By day 4, the delayed and attenuated LH surge was accompanied by increased turnover rates in the median eminence only; no change occurred in the suprachiasmatic nucleus or medial preoptic nucleus. No age-related differences were observed in DA turnover rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen alters the diurnal rhythm of alpha 1-adrenergic receptor densities in selected brain regions.

Norepinephrine regulates the proestrous and estradiol-induced LH surge by binding to alpha 1-adrenergic receptors. The density of alpha 1-receptors may be regulated by estradiol, photoperiod, and noradrenergic neuronal activity. We wished to determine whether alpha 1-receptors exhibit a diurnal rhythm in ovariectomized and/or estradiol-treated female rats, whether estradiol regulates alpha 1-receptors in those areas of brain involved with LH secretion and/or sexual behavior, and whether the concentrations of alpha-receptors vary inversely relative to previously reported norepinephrine turnover patterns. Young female rats, maintained on a 14:10 light-dark cycle were ovariectomized. One week later, half of them were outfitted sc with Silastic capsules containing estradiol. Groups of animals were decapitated 2 days later at 0300, 1000, 1300, 1500, 1800, and 2300 h. Brains were removed, frozen, and sectioned at 20 micron. Sections were incubated with [3H]prazosin in Tris-HCl buffer, washed, dried, and exposed to LKB Ultrofilm. The densities of alpha 1-receptors were quantitated using a computerized image analysis system. In ovariectomized rats, the density of alpha 1-receptors exhibited a diurnal rhythm in the suprachiasmatic nucleus (SCN), medial preoptic nucleus (MPN), and pineal gland. In SCN and MPN, receptor concentrations were lowest during the middle of the day and rose to peak levels at 1800 h. In the pineal gland, the density of alpha 1-receptors was lowest at middark phase, rose to peak levels before lights on, and remained elevated during the day. Estradiol suppressed the density of alpha 1 binding sites in the SCN, MPN, median eminence, ventromedial nucleus, and the pineal gland but had no effect on the lateral septum. Estrogen treatment altered the rhythm of receptor densities in MPN, median eminence, and the pineal gland. In MPN, a transient suppression in the density of receptors occurred at 1500 h, a time associated with increased norepinephrine turnover and the onset of the LH surge. Therefore, we conclude that alpha 1-receptor densities undergo a diurnal rhythm in brain regions associated with entrainment to the photoperiod. Estrogen alters the rhythm of alpha 1-receptors in areas involved with the regulation of LH secretion and decreases the density in other estrogen-responsive regions.

Age-related changes in pulsatile luteinizing hormone release precede the transition to estrous acyclicity and depend upon estrous cycle history.

Aging of the female reproductive system in rats is marked by discrete stages in the disappearance of regular estrous cyclicity. We determined at which point in the transition to acyclicity changes in the LH pulse generator could be detected by comparing pulsatile LH release in ovariectomized young rats to three groups of middle-aged rats that were chronologically matched, yet exhibited different stages of reproductive senescence. Since changes in LH pulse amplitudes can result from hypothalamic and/or pituitary factors, we also assessed pituitary responsiveness to exogenous GnRH. Young rats (2-3.5 months) that had shown regular 4- or 5-day estrous cycles and middle-aged rats (9.5-12 months) that exhibited either regular cycles, irregular cycles, or persistent estrus were bilaterally ovariectomized and used 4 weeks later. Rats were implanted with right atrial cannulae and were bled 2 days later at 5-min intervals for 3 h. To test pituitary responsiveness to GnRH, rats were bled at 10-min intervals for 3 h and received GnRH (25 ng/100 g BW, iv) after the first and second hours. The mean inter-peak interval increased in middle-aged irregularly cycling and persistent estrous rats. The frequency distribution of inter-peak intervals was already significantly different in middle-aged regularly cycling compared to that in young regularly cycling rats. Middle-aged rats displayed fewer short inter-peak intervals and a greater frequency of longer intervals between LH pulses. LH pulse duration increased gradually in parallel with increasing reproductive senescence. Pulse amplitude decreased in all groups of middle-aged rats regardless of their prior reproductive status. Mean LH concentrations were significantly lower in middle-aged than in young regularly cycling rats, and a further significant decline was detected in the middle-aged irregularly cycling and persistent estrous groups. Middle-aged regularly and irregularly cycling rats showed decreased pituitary responsiveness to GnRH compared to young regularly cycling rats, whereas middle-aged persistent estrous rats displayed an intermediate level of pituitary responsiveness to GnRH. These data are the first evidence of changing pulse generator function in middle-aged rats tha had previously exhibited no change in the regularity of their estrous cycles. We suggest that such changes may play a role in the age-related transition to acyclicity.

Neurotensin gene expression increases during proestrus in the rostral medial preoptic nucleus: potential for direct communication with gonadotropin-releasing hormone neurons.

Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.

Neuroprotective effects of estradiol in middle-aged female rats.

Estrogen replacement therapy in postmenopausal women ameliorates cognitive dysfunction and decreases the risk and/or severity of neurodegenerative conditions such as Alzheimer's disease and stroke. Furthermore, estradiol exerts neuroprotective effects in a variety of in vitro and in vivo models of brain injury. We have previously shown that physiological levels of estradiol attenuate ischemic brain injury in young female rats. However, neurodegenerative events occur more frequently in elderly women who are chronically hypoestrogenic. Therefore, we investigated whether aging rats remain responsive to the neuroprotective actions of estradiol. Young (3-4 months) and middle-aged (9-12 months) rats were ovariectomized and treated for 1 week with estradiol before middle cerebral artery occlusion (MCAO). Regional cerebral blood flow was monitored in some animals at the time of injury. Brains were collected 24 h after MCAO and infarct volume was analyzed. Our data demonstrate that in both young and aging rats, low and high physiological doses of estradiol decrease ischemic injury by almost 50%, compared with oil-treated controls. Additionally, our data suggest that estradiol acts in both age groups via blood flow-independent mechanisms, as basal and postinjury blood flow was equivalent between estradiol- and oil-treated young and aging rats. These data demonstrate that replacement with physiological levels of estradiol protects against stroke-related injury in young and aging female rats and strongly suggest that older animals remain responsive to the protective actions of estradiol.

Age-related changes in the diurnal rhythm of serotonin turnover in microdissected brain areas of estradiol-treated ovariectomized rats.

The purpose of this study was to determine whether 1) a diurnal rhythm in serotonin turnover is present in specific hypothalamic nuclei of middle-aged ovariectomized rats and 2) in middle-aged animals exposure to estrogen can induce the pattern of serotonin dynamics which appears necessary for the occurrence of an LH surge in young rats. Young (3-4 month old) and middle-aged (8-10 month old) rats which demonstrated estrous cyclicity were bilaterally ovariectomized. Seven days later half of the young and middle-aged animals received Silastic estradiol capsules. On day 9 post ovariectomy the groups were again divided; half of the rats in each group were killed at 0800, 1200, 1800, and 2400 h. Remaining animals were treated with pargyline (75 mg/kg BW, ip) at these times and were killed 10 min later, and the following brain areas were microdissected and analyzed for serotonin (5HT) and 5-hydroxyindole acetic acid: median eminence (ME), suprachiasmatic nucleus (SCN), medial preoptic nucleus (MPN), arcuate nucleus (AN), and globus pallidus. In young ovariectomized rats the SCN, MPN, and AN exhibited a diurnal rhythm in 5HT activity which was high during the light hours and low during the dark. The diurnal rhythm could not be detected in any hypothalamic nuclei of ovariectomized middle-aged animals. The loss in the circadian component of 5HT activity is not due to a loss in serotonergic function, since overall turnover rates were not reduced compared to young animals. Estrogen treatment modified the diurnal pattern of 5HT activity in the SCN, MPN, and AN in young rats but had no effect in the middle-aged rats. In young rats, estrogen induced a transitory rise in ME-5HT turnover at 1200 h, just before the expected onset of the LH surge. In middle-aged animals the increase in ME-5HT turnover did not occur until 1800 h and correlates with a delay in the initiation of the estradiol-induced LH surge. We conclude that: 1) there is a loss in the rhythm of 5HT activity in middle-aged rats and 2) the diurnal rhythmicity of 5HT turnover may be necessary for the maintenance of normal cyclic release of LH.

Aging progressively decreases the densities and alters the diurnal rhythms of alpha 1-adrenergic receptors in selected hypothalamic regions.

Associated with the decline in reproductive function that occurs with age in female rats are a diminished ability to secrete LH in response to ovariectomy as well as alterations in the preovulatory LH surge mechanisms. The noradrenergic system, an important regulator of LH release, exhibits age-related alterations in the pattern of neurotransmitter release. In addition, the density of alpha 1-receptors changes in intact old rats. To determine whether age-related changes in the pulsatile pattern of LH secretion are associated with declines in the densities and/or a loss of diurnal rhythm of alpha 1-receptors, we measured the density of alpha 1-receptors at various times of day in young, middle-aged, and old ovariectomized rats. By middle-age, mean concentrations of alpha 1-receptors decline in the median eminence and suprachiasmatic nucleus. By old age, mean concentrations of alpha 1-receptors decline in all regions of the hypothalamus examined but not in the dorsal lateral septum. In addition, the diurnal rhythms in alpha 1-receptors, which occur in the medial preoptic nucleus and the suprachiasmatic nucleus of young rats, are lost by middle-age. These data indicate that progressive alterations in the mean concentrations and diurnal rhythms of alpha 1-receptors occur with age in brain regions important for the regulation of reproductive functions and may contribute to the age-associated deficits in LH secretion.

Estrogen and progesterone regulate opiate receptor densities in multiple brain regions.

The negative and positive feedback, actions of estrogen and progesterone on the hypothalamus may be mediated in part by the endogenous opiate system. Estrogen and progesterone suppress physiological responses to the administration of opiate peptides. To determine whether this desensitization to opiates involves receptor down-regulation, we measured the density of naloxone-binding sites in hypothalamic regions regulating gonadotropin release and/or sexual behavior. Using autoradiographic techniques, we measured the density of naloxone-binding sites at various times of the day in intact proestrous, ovariectomized, or ovariectomized rats treated with estrogen or estrogen plus progesterone. Ovariectomy increased naloxone-binding sites compared to intact proestrous rats in all hypothalamic regions examined. Treatment with estrogen decreased and treatment with estrogen plus progesterone decreased even more the densities of naloxone-binding sites. This steroid-induced suppression of opiate receptors appears to have important physiological repercussions and provides a basis for the steroid-induced differential sensitivity to exogenous opiates that has been observed previously.

Effects of estradiol on the diurnal rhythm of serotonin activity in microdissected brain areas of ovariectomized rats.

The purpose of these studies was to determine whether diurnal rhythms in serotonin (5HT) activity are detectable in individual hypothalamic nuclei of ovariectomized rats and whether estradiol induces specific rhythms of 5HT which may be necessary to cyclic release of LH and/or PRL. Young (3- to 4-month old) rats were bilaterally ovariectomized and 7 days later half the animals received Silastic estradiol capsules. Two days later groups were again divided: half the animals in each group were killed at 0800, 1200, 1800, and 2400 h. The remaining animals received pargyline (75 mg/kg body weight, ip) at these times and were killed 10 min later. The median eminence (ME), suprachiasmatic nucleus (SCN), medial preoptic area (MPN), arcuate nucleus (AN), and globus pallidus (GP) were microdissected and assayed for 5HT by HPLC using electrochemical detection. A diurnal rhythm in 5HT turnover was found in the SCN, MPN, and AN of ovariectomized rats. 5HT turnover in these areas was significantly higher during the light hours (0800, 1200, and 1800 h) compared to the dark phase (2400 h). The ME and GP of ovariectomized rats did not exhibit a diurnal rhythm in 5HT activity. Exposure to estrogen altered the pattern of 5HT activity in all hypothalamic areas examined. In the ME, treatment with estradiol increased 5HT turnover at 1200 h, just before the predicted LH and PRL surge, and suppressed activity at all other times. In the SCN, estradiol reversed the 5HT rhythm: turnover was low during the light hours and high during the dark. In the AN and MPN, estradiol treatment increased 5HT activity and abolished the diurnal rhythm. 5HT activity in the GP was not altered by exposure to estrogen. We conclude from these data that specific brain nuclei exhibit diurnal rhythms in 5HT turnover and that the patterns of 5HT activity in specific hypothalamic nuclei exhibit individual and unique responses to the presence of estrogen. These data suggest that the estradiol-induced diurnal pattern of 5HT activity may be necessary for the induction of cyclic release of LH and/or PRL.

Effects of age and long term ovariectomy on prolactin secretion, as assessed by the reverse hemolytic plaque assay.

PRL secretion from pituitary lactotrophs was assessed using the reverse hemolytic plaque assay in young (2- to 3-month-old), middle-aged (10- to 12-month-old), and middle-aged long term ovariectomized (LT-OVX) rats to investigate whether 1) a change in the percentage of pituitary cells secreting PRL is detectable in middle-aged animals, 2) the amount of PRL secreted per cell changes with age, 3) aging involves a change in responsiveness to TRH and/or dopamine (DA), and 4) LT-OVX can prevent any of these changes. Young and middle-aged rats were OVX for 1 week. LT-OVX rats were OVX at 2-3 months of age and used when they were 10-12 months old. All animals were implanted with Silastic capsules containing estradiol (E2) in sesame oil and killed 3 or 8 days later. Anterior pituitaries were collected, and cells were dispersed and prepared for the reverse hemolytic plaque assay. Three days after E2 was implanted, the percentage of anterior pituitary cells that secrete PRL was higher in middle-aged compared to young rats. LT-OVX prevented this increase; the percentage of cells secreting PRL was significantly lower in LT-OVX than in both young and middle-aged rats. Basal secretion of PRL per cell was not different in young compared to middle-aged rats and was significantly lower in LT-OVX than in either young or middle-aged rats. TRH induced similar increases in plaque size in young and middle-aged rats, but had no effect in LT-OVX rats. DA (10(-7) M) inhibited plaque size only in LT-OVX rats; however, higher concentrations of DA were equally effective in the three experimental groups. Eight days after E2 was implanted, the percentage of cells that secrete PRL increased in LT-OVX rats, but was still significantly lower than that in middle-aged animals. After 8 days of E2 treatment, PRL release was similar in the three experimental groups under basal conditions. In LT-OVX rats TRH produced a small increase in PRL secretion (30-40%); DA suppressed PRL release in a similar manner in the three groups. These data demonstrate that middle-aged rats exhibit an increase in the percentage of cells secreting PRL without a concomitant detectable change in the amount of PRL released by single cells and/or a change in responsiveness to TRH or DA. Long term estrogen deprivation prevents this age-related change, suppresses responsiveness to TRH, and enhances sensitivity to DA.

Changes in concentrations of estradiol nuclear receptors in the preoptic area, medial basal hypothalamus, amygdala, and pituitary gland of middle-aged and old cycling rats.

This study determined whether the biochemical characteristics of the estradiol (E2) nuclear receptor located in three different brain areas and the pituitary gland (PIT) change in aging rats undergoing declining reproductive function. To measure maximal E2 nuclear receptor concentrations, groups of young (3-4 months old), middle-aged (8-11 months old), and old (16-18 months old) cycling rats were injected iv with E2 to translocate maximally cytosolic receptors into the nucleus. One hour later they were killed and nuclear extracts were prepared. Maximal E2 nuclear receptor concentrations and dissociation constants were assessed in the preoptic area (POA), medial basal hypothalamus (MBH), amygdala (AMYG), and PIT using an in vitro exchange assay and covariance analyses of double reciprocal plots (Lineweaver-Burk). Middle-aged cycling rats exhibited decreased E2 nuclear receptor concentrations in the POA, but no change in the MBH, AMYG, or PIT. Old rats exhibited decreased E2 nuclear receptor concentrations in the POA, MBH, and PIT and an unexpected increase in the AMYG. There was no change in the affinity of the receptor with age, although an apparent decrease in the pituitary E2 nuclear receptor of middle-aged rats was observed. This difference was not detected when saturation analyses were performed using varying dilutions of the pituitary nuclear extract. These results demonstrate that changes occur in maximal numbers of E2 nuclear receptors in the POA of middle-aged rats. As animals age further, the changes encompass a larger brain area and include the PIT. The data suggest that changes in E2 nuclear receptor concentrations may contribute to the age-related decline in reproductive function.

Nuclear estradiol and cytosol progestin receptor concentrations in the brain and the pituitary gland and sexual behavior in ovariectomized estradiol-treated middle-aged rats.

We wished to determine whether the altered nuclear estradiol (E2) receptor concentrations in middle-aged rats can explain the diminished responsiveness to E2 observed in aging rats. Therefore, we measured receptor concentrations in various brain areas and the pituitary gland of young and middle-aged ovariectomized rats 2 and 4 days after implantation of Silastic capsules containing E2. To determine whether any observable changes had physiological consequences, we correlated age-dependent changes in E2 nuclear receptor concentrations with two E2-dependent parameters: cytosol progestin receptor levels in equivalent brain areas and pituitary gland and progesterone-facilitated reproductive behaviors. Young (3-4 months old) and middle-aged (10-12 months old) Sprague-Dawley rats were ovariectomized and received Silastic capsules containing E2 dissolved in oil 1 week later (day 0). Groups of rats were killed at 1200 h on either day 2 or day 4. Nuclear E2 and cytosol progestin receptor concentrations were assessed in a nuclear and cytoplasmic extract from the medial basal hypothalamus, preoptic area, amygdala, and pituitary gland. To test steroid-induced mating behavior, ovariectomized young and middle-aged rats were treated with E2-containing capsules for 2 or 4 days. At 0900 h progesterone (0.2 mg/kg BW) was injected sc and receptive and proceptive behaviors were observed when experienced males were introduced 4-6 h later. Two days after implantation of E2 capsules, middle-aged rats exhibited lower nuclear E2 receptor concentrations in the medial basal hypothalamus and preoptic area than young rats. By day 4, there were no significant age-related differences in any brain area or in the pituitary gland. Parallel age-related differences were observed in cytosol progestin receptor concentrations on day 2 but they were not evident by day 4. Similarly, middle-aged rats exhibited deficits in proceptive behavior, lordosis quotient, and lordosis quality score on day 2, but there were no differences compared to young rats on day 4. These data demonstrate that E2-induced nuclear E2 receptor concentrations are lower in selected areas of the brain of middle-aged rats. Such changes appear to be physiologically important because they are correlated with changes in E2-induced cytosol progestin receptor concentrations and steroid-induced behaviors. Furthermore, they may partially account for age-related differences in E2-induced LH surges on day 2.(ABSTRACT TRUNCATED AT 400 WORDS)