Constant Estrous Research Articles

Effects of age on GABA turnover rates in specific hypothalamic areas in female rats.

During aging in female rats the estrous cycle ceases and the animals develop phases of constant estrous (CE) or constant diestrous (CD) prior to the irreversible transition into anestrous. In young rats, gamma-aminobutyric acid (GABA) is of pivotal importance for the release of GnRH. In the medial-preoptic area (MPO) where the majority of the GnRH perikarya are located in the rat, GABA release decreases at the time of the preovulatory LH surge. The suprachiasmatic nucleus (SCN) contains numerous GABA neurons. Neurochemical signals from this hypothalamic nucleus provide temporal information to GnRH neurons and thereby influence the preovulatory LH surge and the length of estrous cycles. To investigate aging-related changes of the activity of hypothalamic GABAergic neurons, we determined GABA turnover rates in various hypothalamic nuclei of CE and CD rats and compared them to those determined in young estrous (E) or diestrous rats (D1). In old female rats, GABA activity in the MPO was significantly decreased compared to E and D1 rats. A selective increase of GABA turnover rates was observed in the SCN of CE animals. No age-related changes were observed in the other examined brain areas. These data provide the first evidence for alterations in GABAergic activity in specific hypothalamic areas that depend on age and reproductive status. These may cause changes in ability to induce preovulatory LH surges and to maintain regular estrous cyclicity.

Effects of age, reproductive status and ambient temperature on skin temperature regulation in the female rat

In the present study, we evaluated a large population of aged Long-Evans female rats for the occurrence of spontaneous tail skin temperature (TST) surges. Young (5-7 months) normally cycling (NC) rats and aged (19-29 months), repeated pseudopregnant (PP) and constant estrous (CE) rats were placed in an environmentally controlled room with ambient temperatures of 24-25 degrees C or 29-30 degrees C and TST was monitored at 5 min intervals for 2 h. In young NC and aged PP rats, the incidence of TST surges (flushes) was 8-17% at low and 28-33% at high ambient temperature. Old CE rats exhibited a flushing incidence of 21% and 74% at ambient temperatures of 24-25 degrees C and 29-30 degrees C, respectively. The observed flushing episodes did not differ in their duration or amplitude among the three groups of rats. However, at both low and high ambient temperature, the frequency of flushes were significantly higher in old CE rats. Collectively, these data indicate that reproductive status, rather than age, is the important determinant of TST stability in the female rat. Further, elevated estrogen associated with the CE state may compromise, while elevated progesterone associated with the PP state has a protective effect on the stability of TST in the female rat.

Synergistic effects of estrogen and serotonin-receptor agonists on the development of pituitary tumors in aging rats

—The purpose of this study was to determine if pituitary 5-HT levels change as a function of age or endocrine state, and further if such changes are associated with pituitary pathology. Middle-aged constant estrous (CE) rats had larger (p<0.05) pituitary glands containing more (p<0.05) serotonin (5-HT) than those from young females or comparably aged, irregularly cycling rats. Ovariectomy lowered pituitary 5-HT content in middle aged CE rats. In contrast, pituitary weight and 5-HT content were increased in young rats of both sexes bearing subcutaneous, steroid-containing capsules that produced elevated levels of serum estradiol 17β. However, exogenous estrogen failed to raise pituitary 5-HT concentration since pituitary weight increased more than 5-HT levels, even though the total amount of amine was significantly increased (p<0.05) compared with controls. These findings suggest that pituitary 5-HT increases during aging regardless of ovarian status and in addition, that total 5-HT content of the gland is increased further in hyperestrogenic states such as CE. Since pituitary adenomas occur more frequently in aged CE rats than in diestrous females or males, it was of interest to determine if 5-HT contributes to the tumorogenic effect of estrogen. Thus, the 5-HT receptor agonists zimelidine or quipazine were administered to ovariectomized rats bearing estrogen containing capsules. Rats treated with drugs had larger pituitaries containing more tumors than those receiving the steroid alone. However, these effects were dependent upon estrogen since pituitary pathology did not increase when ovariectomized rats were given 5-HT neuroleptics without the steroid. It is concluded that 5-HT levels in the pituitary increase during aging and in the presence of estrogen may contribute to the frequent occurrence of pituitary tumors in old female rats.

Progesterone and luteinizing hormone secretion following stress-induced interruption of constant estrus in aged rats.

Concentrations of progesterone and luteinizing hormone (LH) were measured during the first diestrus and subsequent proestrus after constant estrous was interrupted by restraint stress. Stress stimulated an acute increase in serum progesterone. Although stress resulted in changes in vaginal cytology characteristic of normal estrous cycles, stress-treated constant estrous (CE) rats had no midcycle (diestrous day 1) elevation of progesterone and markedly lower LH and progesterone during the afternoon of proestrous than age-matched normally cycling controls. The majority of the treated rats resumed CE without completing a cycle. In a separate experiment, progesterone injection during proestrous resulted in increased LH secretion in aging rats with normal estrous cycles. These data indicate that decreased proestrous progesterone secretion may contribute to the decrease in neuro-endocrine stimulation of proestrous gonadotropin secretion in the aged rat and may be associated with the initiation of CE.

Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariectomized long evans rats

The ability of several stimuli which augment central catecholamine (CA) neuronal activity to reinitiate estrous cycles in old constant estrous (CE) rats suggests CA neuronal function is impaired with advanced age. We examined the effects of age on dopamine (DA) and norepinephrine (NE) levels and turnover rates within microdissected brain regions of previously normally cycling young (3–4 months old) and middle-aged (10 months old) and CE old (20–22 months old) Long Evans 2 weeks after ovariectomy. Steady-state DA concentrations were significantly decreased in old compared to young rats in the nucleus accumbens (34%), anterior hypothalamic nucleus (54%, NHA), neurointermediate pituitary lobe (51%, NIL) and median eminence (74%, ME). The rate constant of DA loss, an estimate of neuronal activity, decreased in old versus young rats only in the preoptic area suprachiasmatica (60%, POAs) and NHA (60%) and was unchanged or augmented in the 7 other regions. In contrast, a decline in DA turnover rate of 29–67% was observed in 6 of 9 regions in middle-aged rats and 45–81% in 5 of 9 regions in old rats. Steady-state NE concentrations similarly were significantly decreased in old versus young rats in the POAs (54%), medial forebrain bundle (44%), nucleus suprachiasmatica (49%) and ME (59%). The rate constant of NE loss progressively decreased with increasing age only in the POAs and was unchanged or augmented in other regions. Turnover rate of NE was decreased from 21 to 98% in 4 of 8 regions from old animals. A strong positive correlation was noted between the rate constant of NE (but not DA) loss measured in young rats and the magnitude of the age-related depletion in NE concentrations within specific brain regions. Collectively these data indicate that with increasing age: (i) CA neuronal function is differentially altered in nuclei located along the preoptico-tuberal pathway; (ii) substantial declines in both DA and NE concentrations are the primary contributor to the reduced amine turnover noted in several of these regions; and (iii) the observed age-related alterations in CA turnover may contribute to impaired LH response and the persistent hyperprolactinemia in old CE rats.

Immunoreactive beta-endorphin in the plasma, pituitary and hypothalamus of young female rats on the day of estrus and intact and chronically castrated old constant estrous female rats

Immunoreactive beta-endorphin (IR-beta-ENDO) was compared in the plasma, pituitary and hypothalamus of young female rats on the day of estrus and old constant estrous (CE) female rats, and in intact and chronically castrated old CE female rats. The concentration of IR-beta-ENDO in the plasma and the content and concentration of IR-beta-ENDO in the neurointermediate lobe of the pituitary were significantly greater in the old CE female rats than in the young female rats on the day of estrus. The content and concentration of IR-beta-ENDO in the anterior pituitary and hypothalamus were similar in the two age groups. To determine if estrogen contributed to the increase in plasma and pituitary levels of IR-beta-ENDO observed in the old animals, a group of old CE female rats were castrated and compared to sham operated control CE rats. Thirty days after castration, levels of plasma, pituitary and hypothalamic IR-beta-ENDO were comparable in the intact and the chronically castrated old female rats. These data indicate that in old CE female rats, plasma and pituitary IR-beta-ENDO are significantly increased in comparison to young female rats on the day of estrus, and that these increased levels of IR-beta-ENDO observed in old female rats do not appear to be influenced by gonadal estrogen.

Resumption of pulsatile luteinizing hormone release after alpha-adrenergic stimulation in aging constant estrous rat.

The present studies attempt to define the nature of the impairment of the LH secretory response to ovariectomy in the old constant estrous (CE) rat. LH secretory patterns were characterized 2 weeks after ovariectomy in cannulated, previously normally cycling young (4–5 months old) and previously CE middle-aged (11–12 months old) and aged (21–23 months old) Long-Evans rats from which blood was sampled at 10-min intervals for 3 h. Young rats had the expected pulsatile LH secretion pattern, which was characterized by a mean LH level of 137 ± 8 ng/ml plasma, an LH pulse amplitude of 154 ± 12 ng/ ml plasma, and an LH pulse frequency of 1.16 ± 0.13 pulses/h. In marked contrast, only four of seven middle-aged and two of six old rats exhibited significant pulsatile LH secretion. Of these six animals with pulsatile LH profiles, only two had more than one LH pulse during the 3-h experimental period. The significant, large decreases in mean LH levels for middle-aged (45 ± 4 ng/ml plasma) and old (40 ± 4 ng/ml plasma...

Reinstatement of LH surges by serotonin neuroleptics in aging, constant estrous rats

Aged rats in constant estrous (CE) were treated with drugs which effect monoamine metabolism in an attempt to restore the positive-feedback response to estrogen. Prior to treatment, the rats were ovariectomized so as to eliminate indirect effects of drug: ovary interaction which might alter the steroid environment. They then received silastic capsules containing estradiol 17β, SC, immediately (IME, immediate implant group) or 4 weeks after surgery (DEL, delayed implant group), to provide a constant, stable source of estrogen, mimicking the CE condition. Blood samples were taken 3 days after estrogen capsules were implanted to determine if the exogenous steroid induced LH surges in IME or DEL rats. LH surges occurred in 80% of DEL rats, but no IME rats suggesting that a 30-day hiatus from ovarian steroids restored the positive-feedback response in previously acyclic rats. Progesterone (0.5 mg) administered at 1100 hr increased the size of the surge in DEL rats but did not change serum LH levels in IME rats. After 3 days of estrogen exposure, hypothalamic content of serotonin was significantly lower ( p <0.05) in DEL rats vs. IME rats, while norepinephrine was not different in either group. Differential effects of estrogen on norepinephrine were not apparent in IME or DEL rats. The changes in serotonin metabolism following estrogen treatment in DEL rats occurred with the onset of LH surges, suggesting a functional correlation. On the other hand, serotonin levels did not change in response to estrogen in IME rats, and LH surges were absent as well. Monoamine neuroleptics were administered to IME rats in an attempt to reestablish positive feedback. I-Dihydroxyphenylalanine (l-DOPA; 200 mg/kg at 1100 hr) stimulated LH surges in 31% of the rats; however, its effectiveness was diminished (8% responding with LH surges) by pretreatment with p-chlorophenylalanine (pCPA; 250 mg/kg at 1600 hr, 19 hours before l-DOPA). LH surges were reinstated in the majority (77%) of IME rats following combined treatment with pCPA + 5-hydroxytryptophan (5-HTP; 50 mg/kg at 1100 hr, 43 hours after pCPA). Since this combination of pCPA + 5-HTP enhances sensitivity to serotonin signals, the drugs may have restored the facilitatory effect of serotonin on LH secretion which is lost in aging constant estrous rats. In conclusion, loss of the positive-feedback response in aging female rats may follow changes in monoamine metabolism resulting from life-long exposure to estrogen. Although metabolic changes occur in all hypothalamic monoamines, this study suggests that a major deficit with regard to this feedback loss residues with serotonin.

Differential patterns of gonadotropin responses to ovarian steroids and to LH-releasing hormone between constant-estrous and pseudopregnant states in aging rats.

The stimulatory feedback actions of estradiol and progesterone on gonadotropin secretion were tested in acutely ovariectomized (OVX) old constant estrous (CE) and pseudopregnant (PSP) rats of similar age (20-26 months). Studies were also performed to determine the pituitary LH responses to synthetic LU-releasing hormone (LHRI-I) in both intact and OVX, estradiol-implanted CE and PSP females. Serial blood samples were collected through chronic intra-atrial cannulae for measuring plasma LU and FSH, while single blood samples were obtained for estrogen and progestin assays. Serum concentrations of estradiol were twice as high in CE (26 ± 3 pg/mI, mean ± SEM) as in PSP rats (11 ± 2 pg/mI), whereas progesterone and 20a-hydroxyprogesterone levels were many-fold greater in PSP than in CE rats. In acutely OVX-PSP rats, estradiol and progesterone administration elicited a significant increase in LH release. PSP females that had evidence of pituitary tumors and/or lesions showed an LH response to ovarian steroidssimilartothatof animals with normal pituitaries. In contrast, steroid administration failed to increase LH release in acutely OVX-CE rats, and this lack of LH response was found in animals regardless of whether pituitary pathology was evident. Despite these differences between the CE and PSP states, the pituitary LU responses to single iv. pulses of LHRH were the same in intact CE and PSP rats. Placement of estradiol implants into OVX-CE and PSP rats significantly increased serum estradiol concentrations in both groups, but enhanced the LU response to LHRH only in the PSP rats and not in the CE animals. The present study demonstrates that the stimulatory feedback actions of estradiol and progesterone on LH secretion, which are present in acutely OVX old PS? rats, are absent in old CE females immediately following ovariectomy. Together with other data the results suggest changes in central nervous function of CE rats, which are probably related to their ovarian steroid background rather than to advanced age per Se.

Effect of ovariectomy on plasma LH, FSH, estradiol, and progesterone and medial basal hypothalamic LHRH concentrations old and young rats.

Resting plasma concentrations of LH, FSH, estradiol, and progesterone and medial basal hypothalamic concentrations of LHRH (MBH-LHRH) were measured by RIA in 8- to 12-month-old female rats which had begun to exhibit constant estrous (CE) or prolonged diestrous (PD) vaginal smear patterns and compared to young cycling rats on proestrus, estrus, or diestrus. In addition, we examined the effect of ovariectomy on these hormonal profiles. Old CE rats have normal plasma LH, FSH and progesterone concentrations, but exhibit elevated estradiol levels and decreased MBH-LHRH concentrations compared to young cycling rats on the day of estrus. Ovariectomy results in an attenuated rise in plasma LH and FSH and a much lesser decrease in MBH-LHRH when compared to young rats, despite comparable steroid changes. Old PD rats have normal LH and FSH levels,but have elevated estradiol and progesterone concentrations and decreased MBH-LHRH levels when compared to young rats on the day of diestrus. Ovariectomy causes a normal decrease in MBH-LHRH; however, the increased gonadotropin levels are significantly less than seen in young controls.

Chronological changes in sex steroid, gonadotropin and prolactin secretions in aging female rats displaying different reproductive states.

Longitudinal studies were performed in a colony of aging female rats,from 4-33 months of age,to determine the chronologicalchange inreproductive patterns and the changes in sex steroid, prolactin and gonadotropin secretionassociatedwith different reproductive states. The present study demonstrates that the incidence (65%) of irregular estrous cycles in aging rats increased abruptly from 10-12 months of age. Subsequently, female rats became chronically anovulatory with persistentvaginalcornificationsand theirovariescontained developed follicles but no corpora lutea. The highest incidence (65%) of constant estrous (CE) rats occurred at the age of about 19 months. During the anovulatory state, CE rats displayed low to medium levels of serum estradiol, estrone, testosterone and androstenedione, low levels of progesterone and minimal levels of 20nhydroxyprogesterone. Preovulatory increasesin gonadotropin and prolactin release,similar to those seen in young cycling ratson proestrus,were not observed in CE rats.Whereas serum basal LH levels remained unaltered, morning FSH levelswere increased in CE rats.The lattermay account for the persistent follicular development in aging rats during chronic anovulatory state. Serum basal prolactin levels were normal in CE rats during the early phase (11-16 months of age) of the anovulatory state, but were subsequently increased 3 to 4-fold beyond 24 months of age. Moreover, ovariectomy at a young age prevented the increased pituitary prolactin release in old female rats. These results suggest that continuous exposure to medium levels of estrogens, particularly in the presence of sustained low progesterone secretion, may alter pituitary secretion of prolactin in aging rats. With further advance of age and following many months of anovulatory function, aging female rats exhibited ovulatory activity at irregular intervals. After each ovulation, formed corpora lutea were maintained for a prolonged period, presumably due in part to the existing high prolactin levels in the circulation of older female rats. These corpora lutea in old “pseudopregnant (PSP)” rats were functional as indicated by active secretion of progestins, with 20a-hydroxyprogesterone levels greater than those of progesterone in the circulation. These results indicate that the ovaries of aging rats retain their functional capacity to develop follicles and corpora lutea and to secrete steroid hormones. Although the cause(s) responsible for cessation of normal ovulatory cycles in aging female rats is unknown, the present study demonstrates that the chronic anovulatory state in aging female rats is characterized by significantly reduced ovarian secretion and the lack of cyclic increases in pituitary gonadotropin and prolactmn release. The causal relationships between the decreased ovarian steroid production and the absence of preovulatory surges of gonadotropin release in aging CE rats remain to be determined.

Relation of Aging to Hypothalamic LHRH Content and Serum Gonadal Steroids in Female Rats

SummaryThe relationships of age and reproductive state to hypothalamic LHRH content and gonadal steroids was studied in young and old female rats. LHRH content in the medial basal hypothalamus (MBH) of young female rats was maximal on the morning of proestrus (PE), but fell to a nadir on PE afternoon. MBH-LHRH rose again on the morning of estrus (E) and then dropped by 1000 hr on diestrous Day 2 (DE-2). Old constant estrous (CE) rats had MBH-LHRH levels that were significantly lower than in young E rats, while old pseudopregnant-like (PP) rats had intermediate MBH-LHRH levels, not significantly different from those in young E or DE-2 rats. LHRH content in the MBH of old anestrous (AN) rats was lower than in any other group. Anterior hypothalamic (AH)-LHRH in old CE, PP, and AN rats were similar and did not differ from those in young E or DE-2 rats.Serum estradiol and progesterone peaked at PE in young cycling rats, but showed no cyclic variations in old rats. Old CE and PP rats had moderate estradiol leve...

Patterns of sex steroid and gonadotropin secretion in aging female rats.

Serum estradiol, progesterone, LH, and FSH were determined by RIA in 20- to 30-month-old constant estrous (CE), irregular pseudopregnant (PP), and anestrous (AS) female rats and from 4- to 5-month-old cycling female rats. Disruption of the estrous cycle in aging rats was associated with major changes in secretion of pituitary gonadotropins and ovarian steroids. None of the old rats, in contrast to the young rats, showed cyclic changes in any of the hormones studied. Serum progesterone was much higher in the PP than in the other two old groups, serum estradiol averaged somewhat higher in the CE than in the other two aged groups, and all four hormones were lower in the AS rats than in any other group. Basal serum FSH values were higher in the old CE rats than in either of the other old age groups and were slightly higher than in young rats on the afternoon of proestrus or morning of estrus. Serum FSH values were lower in the old PP and AS rats than in young rats on the afternoon of proestrus or morning of estrus. Serum FSH values were lower in the old PP and AS rats than in young rats on the afternoon of proestrus or morning of estrus. Serum values in the old CE rats were about the same as in young rats on the morning of proestrus or estrus, about the same in old PP rats as in young rats during diestrus, and were undectable in old AS rats. Since the ovaries of old rats are capable of near normal function under appropriate gonadotropic stimulation, it is concluded that the major cause for cessation of regular estrous cycles in old rats lies in altered hypothalamo-pituitary function.

Influence of the thyroid gland on ovarian function in the aging rat.

The effects of underfeeding and manipulation of the thyroid axis on ovarian function were determined in young and old rats. The depressant effect of reduced food intake on ovarian cycling in young females was potentiated by chemical thyroidectomy, while young anestrous, underfed rats cycled when their diet was supplemented with thyroid extract. These observations iindicate that cycling aberrations in underfed rats may occur secondarily to an altered thyroid state. To determine if thyroid state influences ovarian function in old animals, constant estrous (CE) rats were underfed or chemically thyroidectomized. All underfed rats eventually cycled, while the response to chemical thyroidectomy alone, though still effective, was less dramatic. Realimented CE animals eventually returned to a pattern of constant vaginal cornification. Underfeeding had no effect on ovarian function in old recurrently pseudopregnant females however, these rats responded to thyroid treatment with renewed cycling. 4-6 (YC) 10-12 (PEP) month old females entered a persistent vaginal estrous condition when fed low doses of thyroid extract with their ad libitum diet. When the thyroid supplemented diet was discontinued, YC females resumed regular cycling, whereas the vaginal smear in 40% of the PEP rats remained cornified. Cycling could be restored in YC-thyroid induced CE rats by electrochemical stimulation of the medial preoptic area. These date suggest that senile deviations from normal cycling in the aging reproductive system may be affected by alterations in the thyroid state.