Estradiol-induced adult anovulatory syndrome in female C57BL/6J mice: age-like neuroendocrine, but not ovarian, impairments

Long-term effects of elevated plasma estradiol (E2) on ovarian and neuroendocrine functions were examined in 4-month-old cycling female C57BL/6J mice injected s.c. with 0.2 or 0.05 mg estradiol valerate (EV), or oil. Within 7 days, EV-injected mice became permanently acyclic, exhibiting the persistent vaginal cornification (PVC) characteristic of reproductive senescence in rodents. Four months after injection, ovaries from EV-injected mice exhibited no corpora lutea, but ovulated in response to an injection of human chorionic gonadotropin (hCG) (as do older, spontaneously PVC mice). When grafted into young mice, ovaries from EV-injected mice supported as many estrous cycles as ovaries from oil-injected controls. EV did not alter the suppression of luteinizing hormone (LH) by E2, LH response to injected LH releasing hormone (LHRH), or plasma prolactin (Prl). However, EV-injected mice exhibited impairments in LH regulation similar to those seen in old, acyclic mice. Plasma LH 30 days after ovariectomy was 40% lower, and E2-induced LH surges were 60% lower, in EV-injected mice versus controls. Furthermore, EV-injected mice were unable to support estrous cycles given young ovarian grafts, in contrast to controls. Effects of sustained but physiological levels (15-20 pg/ml) of plasma E2, were examined in intact cycling mice given sham or E2 implants. Six weeks after implantation, the implants were removed; only 50% of the E2-implanted mice subsequently exhibited estrous cycles, compared with 100% of sham-implanted controls. Furthermore, those E2-implanted mice which did cycle had fewer cycles than controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Supernumerary ovarian grafts in aging C57BL/6J mice reveal complexities in the neuroendocrine impairments of acyclic mice

Determinants of the age-related acyclic state, persistent vaginal cornification (PVC), were studied in reproductively senescent mice using a 2-stage ovarian transplantation procedure, whereby ovaries from young mice were grafted to older mice without removing their autogenous ovaries until 8 wk later. In contrast to the usual ("1-stage") procedure, in which the autogenous ovaries are removed at the time of grafting, the 2-stage approach is postulated to circumvent potential effects of the reduction in steroids during the ovariprival phase prior to vascularization of the grafted ovary, which may reverse age-related hypothalamic-pituitary impairments. The 2-stage transplantation procedure was validated in young C57BL/6J mice. Estrous cycles were not disrupted by removal of the autogenous ovaries 8 wk after the grafting, indicating that grafted ovaries began functioning before or within days after ovariectomy. No difference in estrous cycle frequency or distribution was detected between the young mice with 2-stage and those with 1-stage transplants for at least 3 mo after removal of the autogenous ovaries. Most older (15- to 18-mo-old) mice with PVC (70%) remained acyclic after receiving young ovaries by either the 1-stage or the 2-stage procedure, indicating that extra-ovarian, presumably neuroendocrine, impairments are sufficient to maintain acyclicity in most older mice once it is initiated. However, 30% of the older mice from each transplantation group began cycling after receiving young ovaries by either the 1-stage or 2-stage procedure, as observed before 1-stage transplants. Therefore, cycle reactivation was not a result of the transient ovariprival phase incurred during 1-stage transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Polyovular follicles in mouse ovaries exposed neonatally to diethylstilbestrol in vivo and in vitro

In 35-day-old C57BL/Tw female mice given daily injections of 1 microgram diethylstilbestrol (DES) for 5 days from the day of birth, a significantly higher incidence of polyovular follicles (PF) were found in the ovaries than in those of age-matched control mice. Ovaries of prepubertal mice treated neonatally with oil or DES (DES mice) showed an enhancement of ovulation and luteinization following a combined treatment with eCG and hCG. Tubal ova in DES mice treated with eCG plus hCG were surrounded by many granulosa cells. Incidence of PF in control mice was not changed by eCG plus hCG treatment. In contrast, PF incidence in DES mice was reduced by prepubertal injections of eCG plus hCG. A high incidence of PF was also found in newborn mouse ovaries transplanted for 30 days into ovariectomized adult hosts given DES injections, but not in ovaries transplanted into intact or ovariectomized DES-untreated hosts. When neonatal ovaries were cultivated in a serum-free medium containing DES for 5 days and then transplanted into ovariectomized hosts, PF were formed in the grafts, but not in DES-unexposed grafts. Oocytes from PF in DES mice were found to have a smaller capacity for fertilization when examined in vitro. The present study also demonstrated that neonatal ovaries exposed to estrogen in vivo or in vitro (which produces PF in prepubertal hosts) are capable of responding to gonadotropins given later, resulting in a reduction of PF incidence, and that exogenous estrogen acts directly on neonatal ovaries to induce PF.     

Age-related decline in deciduogenic ability of the rat uterus

To study age-related changes in uterine responsiveness to deciduogenic stimuli, virgin female rats of the T strain were ovariectomized at 4, 8, 10, or 12 mo of age and given daily s.c. injections of 3 mg progesterone for 7 days, commencing on the day after operation, and a single s.c. injection of 0.1 microgram estradiol-17 beta on the third day of the period. Endometrial stimulation was effected by either endometrial traumatization or intraluminal instillation of sesame oil or prostaglandin E2 (PGE2), applied 16 h after the injection of estradiol. Decidual response began to decrease at 8 mo of age and completely disappeared between 8 and 12 mo, regardless of the type of induction stimulus. At 8 mo of age, females formed deciduomata in response to instillation of oil or PGE2, only when they had been cycling regularly at the time of ovariectomy. In 10-mo-old rats, instillation of oil or PGE2 invariably failed to elicit a positive response, regardless of the pattern of estrous cycles at surgery. However, if an ovary was transplanted s.c. 5 or 7 mo after ovariectomy at 4 mo of age, the uteri responded positively to oil instillation at 10 and 12 mo of age, after the ovarian grafts had been removed and steroid treatments had been administered. Moreover, a 2-mo interval between ovariectomy at 8 mo of age and the commencement of the standard treatment schedule restored or maintained the uterus's ability to form deciduomata by 10 mo of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early treatment of young female rats with progesterone delays the aging-associated reproductive decline: a counteraction by estradiol

We have recently reported that successive treatments of young virgin rats with progesterone (P) implants produce elevated circulating P and consistently low estradiol (E2) concentrations, and subsequently delay the aging-associated reproductive decline. Inasmuch as E2 has been implicated in causing the loss of regular estrous cyclicity in aging rats, the present study examined if the concomitant presence of moderately increased circulating E2 levels could counteract the effects of P implants on reproductive aging. Starting at 3 1/2 mo and continuing to 8 mo of age, regularly cyclic, virgin rats received either s.c. Silastic implants of P (P-implanted), blank Silastic implants (virgin controls), or P + E2 implants (P + E2-implanted) for 3 wk, followed by implant removal for 1 wk. Each of these implant treatments was repeated in the same female rats 5 times. Blood samples were obtained on different days of the estrous cycle from the control group and on Day 11 of successive treatments with P or P + E2 implants for measurements of serum P and E2 values. At 8 1/2 and 10 mo of age, estrous cyclicity of these same virgin rats was again monitored, and 10-mo-old regularly cyclic females from each treatment group were mated with young fertile males to complete term pregnancies. While virgin controls showed cyclic increases in E2 and P secretion during the estrous cycle, P-implanted virgins exhibited consistently low serum E2 and moderately increased P levels during 5 successive treatments. The latter indicates a potent inhibition of ovarian E2 secretion by P implants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influences of age and ovarian follicular reserve on estrous cycle patterns, ovulation, and hormone secretion in the Long-Evans rat

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E(2)) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E(2) secretion, as preovulatory E(2) levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.     

Deceleration of age-associated changes in the preovulatory but not secondary follicle-stimulating hormone surge by progesterone

Recently, it has been reported that mating can delay the age-associated decline in reproductive function of female rats. Since circulating progesterone (P) levels are elevated for a 2- to 3-wk interval during pregnancy, the following study was conducted to determine whether intermittent elevation in P levels can alter the rate of reproductive aging in female rats. Beginning at 2 mo of age, 4-day-cycling, virgin rats were divided into two groups. In one group, 3 Silastic capsules containing crystalline P were inserted s.c. into each rat while rats in another group each received 1 empty capsule. After 2 wk, the capsules were removed for 2 wk. Thereafter, implantation and removal of capsules was repeated 5 additional times. Rats receiving P capsules became acyclic 3-4 days after exposure to P and resumed cyclicity 4-7 days after removal of P-capsules. One month after the last series of capsules was removed (rats approximately 8-mo-old), rats exhibiting consecutive 4-day cycles were inserted with indwelling atrial cannulae and bled at 4-h intervals from 1400 h on proestrus (Pr) to 1000 h on estrus (E). At 1600 h E, rats were killed and trunk blood was collected. For comparison, a group of 3-mo-old (young) rats was bled on Pr and E. In 8-mo-old rats that received empty capsules, 27% exhibited 4-day cycles compared to 66% of the young rats. However, in contrast to rats that received empty capsules, 63.1% of P-treated rats exhibited 4-day cycles. Surges of preovulatory luteinizing hormone (LH) and follicle-stimulating hormone (FSH) surges were attenuated in 8-mo-old rats given empty capsules compared to young rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oral administration of estradiol to young C57BL/6J mice induces age-like neuroendocrine dysfunctions in the regulation of estrous cycles

Exogenous estradiol (E2) can accelerate the onset of acyclicity and other age-related neuroendocrine changes in rodents. The present study demonstrates that chronic oral administration of E2 (850 micrograms/kg body wt/day) induces premature acyclicity in intact C57BL/6J mice. After 6 wk of E2, mice regained cyclicity but ceased cycling prematurely, whereas 12 wk of E2 caused permanent acyclicity in all mice. The acyclicity after 12 wk of E2 was not reversed by ovarian replacement from young donors, which implies extra-ovarian (neuroendocrine) lesions. The above studies with intact mice can not identify the contributions from exogenous E2 and endogenous ovarian sections and the possible impact of oral E2 on the ovary. Therefore, mice were ovariectomized (OVX) to remove ovarian contributions, treated for 12 wk with oral E2, and then were given young ovarian grafts and assayed for neuroendocrine functions. Approximately 50% of the E2-treated and grafted mice were permanently acyclic, whereas controls cycled well. Thus, oral E2 causes irreversible neuroendocrine damage. However, the presence of the ovary during E2 treatment increases the loss of cyclicity, implying a dose effect. We conclude that the induction of acyclicity is dependent on the dose and duration of E2 exposure.     

Levels of alpha-inhibin in aging female mice.

alpha-inhibin was immunocytochemically localized in granulosa cells of different stages of developing follicles, freshly formed corpora lutea, and scattered interstitial cells (pigmented or ceroid cells) in ovaries of 6-, 14-, and 23-25-mo-old C57BL/6NNia mice. Developing follicles exhibited the greatest amount of staining. Quantitation of the stain using an image analysis system indicated the staining intensity within ovarian follicles of 14-mo-old mice was greater than that in 23-25-mo-old mice. The levels of plasma alpha-inhibin and estradiol (E2) decreased with age. The number of follicles present in ovaries of middle-aged mice was comparable to those of 6-mo-old mice, yet plasma levels of FSH were significantly higher than those of 6-mo-old mice. This may be due to an age-related loss in the sensitivity of the hypothalamus and/or pituitary of middle-aged mice to ovarian hormones. In contrast, ovaries of 23-25-mo-old mice contained few antral follicles and consequently produced little alpha-inhibin. There appeared to be little negative feedback regulation of FSH secretion in 23-25-mo-old mice as a result of age-related ovarian impairments. This study supports an earlier hypothesis from our laboratory [Biol Reprod 1985; 32:989-997] that the primary defect(s) limiting age-related reproductive performance in mice appears to reside within the hypothalamo-hypophyseal axis, whereas secondary defects arise from the ovary.     

Transient shortening of estrous cycles in aging C57BL/6J mice: effects of spontaneous pseudopregnancy, progesterone, L-dihydroxyphenylalanine, and hydergine

Regular estrous cycles can be reinitiated in old acyclic female rats by pharmacologic, hormonal, and environmental manipulations. The most responsive acyclic states are persistent vaginal cornification (PVC) and spontaneous pseudopregnancy (SP). However, it is not known if the irregular cyclicity that precedes acyclicity during aging can also be alleviated. We found that transient shortening of estrous cycles follows smear sequences indicative of pseudopregnancy in C57BL/6J mice, aged 9-15 mo, suggesting a role for progesterone. This phenomenon was investigated through a limited model of pseudopregnancy in which intact aging mice with lengthened cycles were given progesterone implants (yielding 70 ng progesterone/ml plasma) that suppressed estrous cycles; upon removal of the implants, cycles were transiently shortened in aging mice. Therefore, we hypothesize that withdrawal from the progesterone elevations associated with SP is the mechanism in shortening subsequent estrous cycles. Effects of central-acting drugs, similar to those used to reinitiate cyclicity in acyclic old rats, were also examined. Hydergine, an ergot mixture with partial dopaminergic and serotonergic agonist activities, suppressed SP when fed to 10- to 12-mo-old, middle-aged mice. Hydergine did not otherwise affect estrous cycle length, prevent PVC, or reinitiate cycling in acyclic PVC mice. Feeding L-dihydroxyphenylalanine to middle-aged mice did not suppress SP, affect estrous cycle lengths, or reinitiate cycles from PVC.     

Age-related alterations in pulsatile luteinizing hormone release: effects of long-term ovariectomy, repeated pregnancies and naloxone

Aging of the female reproductive system may be regulated by changes at the hypothalamic, pituitary, and ovarian levels. Long-term ovariectomy (LT-OVX) and/or multiple pregnancies delay age-related deterioration of several parameters of reproductive potential in rodents. We tested whether long-term suppression of cyclic ovarian hormone release that is normally associated with the 4- to 5-day estrous cycle decelerates age-related decreases in the frequency of luteinizing hormone (LH) pulses to assess whether hormonal milieu influences the rate of aging of the pulse generator. We determined the percentage of rats exhibiting pulsatile LH secretion, mean LH levels, and amplitude and frequency of LH pulses in seven groups of ovariectomized (OVX) rats. Young (3-4 mo), middle-aged (8-10 mo), and old (18-22 mo) virgin rats, ovariectomized 4 wk (4WK-OVX) prior to experimentation, were used to determine the effect of age. The effect of long-term ovarian hormone deprivation was tested by ovariectomizing rats at 2-3 mo of age and using them when they were middle-aged (8-10 months) or old (18-22 mo). The effect of deprivation of cyclic increases in ovarian hormones associated with repeated estrous cycles was tested by using retired breeder (RB) rats that had been ovariectomized 4 wk prior to experimentation. Each rat was implanted with a right atrial cannula and bled the next day at 10-min intervals for 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effectiveness of estradiol and progesterone in inducing LH release in different stages in rat estrous cycles

The effectiveness of estradiol (E2) and progesterone (P4) in inducing the release of the luteinizing hormone (LH) in the acutely ovariectomized (OVx) rats was studied. Female rats were Ovx in different stages of the estrous cycles and received a series of injections of E2 and P4. LH dynamic changes in the blood were examined in the afternoon of the following day after Ovx. Intact rats treated with oil vehicle or E2 and P4 were used as controls. The surgical operation and oil treatment did not interfere with the normal reproductive rhythm and LH secretion, but treatment with E2 and P4 did facilitate the LH release in some intact rats. E2 and P4 were very effective in inducing LH release in Ovx rats as compared with controls. Results indicated that E2 and P4 are essential substances in eliciting the LH surge, but their efficacies are dependent on the stage in the estrous cycles.     

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)     

The relation of ovarian steroid levels in young female rats to subsequent estrous cyclicity and reproductive function during aging

In multiparous rats, the incidence of regular estrous cyclicity and fertility decreases markedly at middle age. However, recent studies have shown that repeated pregnancies or progesterone (P) implants can subsequently cause retired breeder females to maintain regular cyclicity for an extended period of time; these results suggest a P-mediated deceleration of reproductive aging. In the present study, we examined the relation of ovarian steroid levels in young virgin females to their subsequent estrous cyclicity and reproductive function during aging as compared to multiparous females. Beginning at 4 mo of age and continuing to 6 mo of age, regularly cyclic virgin rats received either consecutive P implants (n = 41) or no implants (controls, n = 45) for 3 wk, followed by implant removal for 1 wk. Additional females (n = 72) were mated and allowed to undergo repeated pregnancies at 4, 6 1/2, and 8 mo of age. Blood samples were obtained throughout the estrous cycle (virgin females), during pregnancy (multiparous rats), and on Day 11 of successive treatments with P implants (virgins with P implants) for P, estradiol (E2), and testosterone (T) measurements. Subsequently, regularly cyclic females from all three groups were mated with fertile males to undergo term pregnancies at 10 and 12 mo of age. While the virgin controls showed cyclic increases in P, T, and E2 secretion during their estrous cycles, the P-implanted females had persistently low E2 and high P and T levels during treatment, which indicates an inhibition of ovarian E2 synthesis by P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian reproductive function after exposure to diethylstilbestrol in neonatal life

Inbred and random-bred NMRI mice were treated with diethylstilbestrol (DES, 5 micrograms per day) or vehicle (olive oil) on Days 1-5 after birth. At the age of 8 wk, females were treated with saline or eCG and hCG to induce ovulation. Ova never occurred in the ampulla of the uterine tube of saline-treated, DES-treated females when these mice were not mated. After gonadotropin treatment, ova were found in the ampulla of all olive oil-treated females and in approximately 80% of DES-treated females. The number of ovulated ova was similar in both groups. Twenty percent of gonadotropin-treated, DES-treated females had ova in the ampulla and a vaginal plug after being caged with males but none became pregnant. Ovaries from inbred control or DES-treated females were grafted to the ovarian bursa of control or DES-treated ovariectomized hosts. DES-treated hosts, carrying control or DES-exposed ovaries, never became pregnant. Control females, with control ovaries or DES-exposed ovaries, became pregnant; pregnancy rate and litter size were similar for control mice regardless of whether they were supporting DES-exposed or control ovaries. Oocytes from ovaries exposed neonatally to DES can thus give rise to apparently normal offspring. The results also indicate DES-induced nonovarian disturbances, e.g. tubal and/or endometrial function, both of which are important for fertility. In the grafting experiments, a high mortality rate was found in inbred DES-exposed females caged with males. All deaths were associated with vaginal concrements (vaginal stones) and intestinal complications.     

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.     

Adverse effects on female development and reproduction in CD-1 mice following neonatal exposure to the phytoestrogen genistein at environmentally relevant doses

Outbred female CD-1 mice were treated with genistein (Gen), the primary phytoestrogen in soy, by s.c. injections on Neonatal Days 1-5 at doses of 0.5, 5, or 50 mg/kg per day (Gen-0.5, Gen-5, and Gen-50). The day of vaginal opening was observed in mice treated with Gen and compared with controls, and although there were some differences, they were not statistically significant. Gen-treated mice had prolonged estrous cycles with a dose- and age-related increase in severity of abnormal cycles. Females treated with Gen-0.5 or Gen-5 bred to control males at 2, 4, and 6 mo showed statistically significant decreases in the number of live pups over time with increasing dose; at 6 mo, 60% of the females in the Gen-0.5 group and 40% in the Gen-5 group delivered live pups compared with 100% of controls. Mice treated with Gen-50 did not deliver live pups. At 2 mo, >60% of the mice treated with Gen-50 were fertile as determined by uterine implantation sites, but pregnancy was not maintained; pregnancy loss was characterized by fewer, smaller implantation sites and increased reabsorptions. Mice treated with lower doses of Gen had increased numbers of corpora lutea compared with controls, while mice treated with the highest dose had decreased numbers; however, superovulation with eCG/hCG yielded similar numbers of oocytes as controls. Serum levels of progesterone, estradiol, and testosterone were similar between Gen-treated and control mice when measured before puberty and during pregnancy. In summary, neonatal treatment with Gen caused abnormal estrous cycles, altered ovarian function, early reproductive senescence, and subfertility/infertility at environmentally relevant doses.