Gynecological surgery in adulthood imparts cognitive and brain changes in rats: A focus on hysterectomy at short-, moderate-, and long-term intervals after surgery.

Premenopausal hysterectomy is associated with a greater relative risk of dementia. We previously demonstrated cognitive impairments in adult rats six weeks after hysterectomy with ovarian conservation compared with intact sham-controls and other gynecological surgery variations. Here, we investigated whether hysterectomy-induced cognitive impairments are transient or persistent. Adult rats received sham-control, ovariectomy (Ovx), hysterectomy, or Ovx-hysterectomy surgery. Spatial working memory, reference memory, and anxiety-like behavior were tested either six-weeks post-surgery, in adulthood; seven-months post-surgery, in early middle-age; or twelve-months post-surgery, in late middle-age. Hysterectomy in adulthood yielded spatial working memory deficits at short-, moderate-, and long-term post-surgery intervals. Serum hormone levels did not differ between ovary-intact, but differed from Ovx, groups. Hysterectomy had no significant impact on healthy ovarian follicle or corpora lutea counts for any post-surgery timepoint compared with intact sham-controls. Frontal cortex, dorsal hippocampus, and entorhinal cortex were assessed for activity-dependent markers. In entorhinal cortex, there were alterations in FOSB and ΔFOSB expression during the early middle-age timepoint, and phosphorylated ERK1/2 levels at the adult timepoint. Collectively, results suggest a primary role for the uterus in regulating cognition, and that memory-related neural pathways may be modified following gynecological surgery. This is the first preclinical report of long-term effects of hysterectomy with and without ovarian conservation on cognition, endocrine, ovarian, and brain assessments, initiating a comprehensive framework of gynecological surgery effects. Translationally, findings underscore critical needs to decipher how gynecological surgeries, especially those involving the uterus, impact the brain and its functions, the ovaries, and overall aging from a systems perspective.

Clinically Used Hormone Formulations Differentially Impact Memory, Anxiety-Like, and Depressive-Like Behaviors in a Rat Model of Transitional Menopause.

A variety of U.S. Food and Drug Administration-approved hormone therapy options are currently used to successfully alleviate unwanted symptoms associated with the changing endogenous hormonal milieu that occurs in midlife with menopause. Depending on the primary indication for treatment, different hormone therapy formulations are utilized, including estrogen-only, progestogen-only, or combined estrogen plus progestogen options. There is little known about how these formulations, or their unique pharmacodynamics, impact neurobiological processes. Seemingly disparate pre-clinical and clinical findings regarding the cognitive effects of hormone therapies, such as the negative effects associated with conjugated equine estrogens and medroxyprogesterone acetate vs. naturally circulating 17ß-estradiol (E2) and progesterone, signal a critical need to further investigate the neuro-cognitive impact of hormone therapy formulations. Here, utilizing a rat model of transitional menopause, we administered either E2, progesterone, levonorgestrel, or combinations of E2 with progesterone or with levonorgestrel daily to follicle-depleted, middle-aged rats. A battery of assessments, including spatial memory, anxiety-like behaviors, and depressive-like behaviors, as well as endocrine status and ovarian follicle complement, were evaluated. Results indicate divergent outcomes for memory, anxiety, and depression, as well as unique physiological profiles, that were dependent upon the hormone regimen administered. Overall, the combination hormone treatments had the most consistently favorable profile for the domains evaluated in rats that had undergone experimentally induced transitional menopause and remained ovary-intact. The collective results underscore the importance of investigating variations in hormone therapy formulation as well as the menopause background upon which these formulations are delivered.

Characterizing the effects of tonic 17ß-estradiol administration on spatial learning and memory in the follicle-deplete middle-aged female rat.

17ß-estradiol (E2)-containing hormone therapy is a safe, effective way to alleviate unwanted menopause symptoms. Preclinical research has focused upon the role of E2 in learning and memory using a surgically menopausal rodent model whereby the ovaries are removed. Given that most women retain their reproductive tract and undergo a natural menopause transition, it is necessary to understand how exogenous E2 impacts a structurally intact, but follicle-deplete, system. In the current study, 8 month old female rats were administered the ovatoxin 4-vinylcyclohexene diepoxide (VCD), which accelerates ovarian follicular depletion, to model the human menopause transition. After follicular depletion, at 11 months old, rats were administered Vehicle or tonic E2 treatment for 12 days prior to behavioral evaluation on spatial working and reference memory tasks. Results demonstrated that E2 had both enhancing and impairing effects on taxed working memory depending upon the learning or retention phases of the water radial-arm maze, with no impact on reference memory. Relationships between memory scores and circulating estrogen levels were specific to follicle-depleted rats without E2 treatment. Collectively, findings demonstrate the complexity of E2 administration in a follicle-depleted background, with cognitive effects specific to working memory; furthermore, E2 administration altered circulating hormonal milieu and relationships between hormone profiles and memory. In sum, menopausal etiology impacts the parameters of E2 effects on cognition, complementing prior work with other estrogen compounds. Deciphering estrogenic actions in a system wherein the reproductive tract remains intact with follicle-depleted ovaries, thus modeling the majority or menopausal women, is critical for translational perspectives.

Hysterectomy Uniquely Impacts Spatial Memory in a Rat Model: A Role for the Nonpregnant Uterus in Cognitive Processes.

Approximately one-third of women experience hysterectomy, or the surgical removal of the uterus, by 60 years of age, with most surgeries occurring prior to the onset of natural menopause. The ovaries are retained in about half of these surgeries, whereas for the other half hysterectomy occurs concurrently with oophorectomy. The dogma is that the nonpregnant uterus is dormant. There have been no preclinical assessments of surgical variations in menopause, including hysterectomy, with and without ovarian conservation, on potential endocrine and cognitive changes. We present a novel rat model of hysterectomy alongside sham, ovariectomy (Ovx), and Ovx-hysterectomy groups to assess effects of surgical menopause variations. Rats without ovaries learned the working memory domain of a complex cognitive task faster than did those with ovaries. Moreover, uterus removal alone had a unique detrimental impact on the ability to handle a high-demand working memory load. The addition of Ovx, that is, Ovx-hysterectomy, prevented this hysterectomy-induced memory deficit. Performance did not differ amongst groups in reference memory-only tasks, suggesting that the working memory domain is particularly sensitive to variations in surgical menopause. Following uterus removal, ovarian histology and estrous cycle monitoring demonstrated that ovaries continued to function, and serum assays indicated altered ovarian hormone and gonadotropin profiles by 2 months after surgery. These results underscore the critical need to further study the contribution of the uterus to the female phenotype, including effects of hysterectomy with and without ovarian conservation, on the trajectory of brain and endocrine aging to decipher the impact of common variations in gynecological surgery in women. Moreover, findings demonstrate that the nonpregnant uterus is not dormant, and indicate that there is an ovarian-uterus-brain system that becomes interrupted when the reproductive tract has been disrupted, leading to alterations in brain functioning.

The opportunity for sexual selection and the evolution of non-responsiveness to pesticides, sterility inducers and contraceptives.

We illustrate a method for delaying and possibly eliminating the evolution of non-responsiveness to the treatments now used to control pest populations. Using simulations and estimates of the variance in relative fitness, i.e., the opportunity for selection, in a rat-like mammal, we show that the selection responsible for the evolution of non-responsiveness to pesticides and sterility-inducers, is similar in its action to sexual selection, and for this reason can be orders of magnitude stronger than that which exists for untreated populations. In contrast, we show that when contraceptives are used to reduce the fertility of a pest species, with non-responders embedded within such populations, the opportunity for selection favoring non-responsiveness is reduced to that which is expected by chance alone. In pest species with separate sexes, we show that efforts to control pest populations or to mitigate selection favoring non-responsiveness, are likely to be ineffective when members of one sex are sterilized or killed. We also show that while mating preferences can impede the rate at which resistance evolves, they are more likely to accelerate this process, arguing against the use of sterile male approaches for controlling pests. Our results suggest that contraceptives are more effective at controlling pest populations and slowing the evolution of non-responsiveness than treatments that cause sterilization or death in target species. Furthermore, our results indicate that contraceptives that work differentially on each sex will be most effective in mitigating selection favoring non-responders. Our results have significant implications for the development and application of treatments to manage pests, now and into the future.

COMPROMISED FERTILITY IN FREE FEEDING OF WILD-CAUGHT NORWAY RATS (RATTUS NORVEGICUS) WITH A LIQUID BAIT CONTAINING 4-VINYLCYCLOHEXENE DIEPOXIDE AND TRIPTOLIDE.

Wild rat pests in the environment cause crop and property damage and carry disease. Traditional methods of reducing populations of these pests involve poisons that can cause accidental exposures in other animals and humans. Fertility management with nonlethal chemicals would be an improved method of rat pest population control. Two chemicals known to target ovarian function in female rats are 4-vinylcyclohexene diepoxide (VCD) and triptolide. Additionally, triptolide impairs spermatogenesis in males. A liquid bait containing no active ingredients (control), or containing triptolide (0.001%) and VCD (0.109%; active) was prepared to investigate the potential use of these agents for wild rat pest population control. Liquid bait was made available to male (n = 8 control; n = 8 active) and female (n = 8 control; n = 8 active) Sprague Dawley rats ( Rattus norvegicus ) for oral consumption prior to breeding. Whereas, control bait-treated females produced normal-sized litters (10.0 ± 1.7 pups/litter), treated females delivered no pups. Wild Norway male (n = 20) and female (n = 20) rats ( Rattus norvegicus ) were trapped, individually housed, and one group given free access to control bait, one group to active bait. Following three cycles of treatment-matched mating pairs, females consuming control bait (control) produced normal litter sizes (9.73 ± 0.73 pups/litter). Females who had consumed active bait (treated) produced no litters on breeding cycles one and two; however, 2 of 10 females produced small litters on the third mating cycle. In a fourth breeding cycle, control females were crossmated with treated males, and treated females were crossmated with control males. In both groups, some dams produced litters, while others did not. The differences in response reflect a heterogeneity in return to cyclicity between females. These results suggest a potential approach to integrated pest management by compromising fertility, and could provide a novel alternative to traditional poisons for reducing populations of wild rat pests.

Cognitive changes across the menopause transition: A longitudinal evaluation of the impact of age and ovarian status on spatial memory.

Cognitive changes that occur during mid-life and beyond are linked to both aging and the menopause transition. Studies in women suggest that the age at menopause onset can impact cognitive status later in life; yet, little is known about memory changes that occur during the transitional period to the postmenopausal state. The 4-vinylcyclohexene diepoxide (VCD) model simulates transitional menopause in rodents by depleting the immature ovarian follicle reserve and allowing animals to retain their follicle-deplete ovarian tissue, resulting in a profile similar to the majority of perimenopausal women. Here, Vehicle or VCD treatment was administered to ovary-intact adult and middle-aged Fischer-344 rats to assess the trajectory of cognitive change across time with normal aging and aging with transitional menopause via VCD-induced follicular depletion, as well as to evaluate whether age at the onset of follicular depletion plays a role in cognitive outcomes. Animals experiencing the onset of menopause at a younger age exhibited impaired spatial memory early in the transition to a follicle-deplete state. Additionally, at the mid- and post- follicular depletion time points, VCD-induced follicular depletion amplified an age effect on memory. Overall, these findings suggest that age at the onset of menopause is a critical parameter to consider when evaluating learning and memory across the transition to reproductive senescence. From a translational perspective, this study illustrates how age at menopause onset might impact cognition in menopausal women, and provides insight into time points to explore for the window of opportunity for hormone therapy during the menopause transition period. Hormone therapy during this critical juncture might be especially efficacious at attenuating age- and menopause- related cognitive decline, producing healthy brain aging profiles in women who retain their ovaries throughout their lifespan.

Accelerated follicle depletion in vitro and in vivo in Sprague-Dawley rats using the combination of 4-vinylcyclohexene diepoxide and triptolide.

Rodent pests cause major damage to the world's agricultural crops and food stores. Rodenticides used since World War II did not lead to sustained reduction of rodent populations, and so fertility control is becoming attractive because rats reproduce with great efficiency. Chemical acceleration of ovarian failure via oral dosing also would improve management of rat pest populations. The chemical 4-vinylcyclohexene diepoxide (VCD) is orally efficacious, causing depletion of nonregenerating primordial ovarian follicles of Sprague-Dawley rats. However, to cause rapid reduction in pups in the first breeding cycle after dosing, all stages of ovarian follicle development must be targeted. To achieve this goal, the Chinese herb triptolide was tested because it can precipitate apoptosis and deplete growing follicles. The impact of triptolide was tested in cultured postnatal day 4 Sprague-Dawley rat pup ovaries. Triptolide at 5 nM caused 100% primordial, primary, and secondary follicle depletion after 8 days of culture, compared to 38% follicle depletion caused by VCD at 30 microM. Next, a palatable rat bait was developed, containing 1% VCD with increasing concentrations of triptolide at 25, 50, and 100 microg/kg body weight. Rats ate an average 3-6% of their body weight/day over 15 feeding days. Two days after the end of baiting, rats were euthanized to conduct necropsies and collect ovaries to count all follicular stages and corpora lutea. At 50 microg triptolide/kg body weight, there was significant reduction of all follicular stages; primordial follicles were 50% lower, secondary follicles were 64% lower, antral follicles were 80% lower, and there were no corpora lutea. These results suggest that combining VCD and triptolide in an oral bait leads to significantly compromised rat ovarian function and reduced ovulations, and is likely to reduce pup production.

A longitudinal study of the effect of genistein on bone in two different murine models of diminished estrogen-producing capacity.

This experiment was designed to assess the capacity of dietary genistein (GEN), to attenuate bone loss in ovariectomized (OVX) and ovary-intact VCD-treated mice. Pretreatment of mice with 4-vinylcyclohexene diepoxide (VCD) gradually and selectively destroys ovarian follicles whilst leaving ovarian androgen-producing cells largely intact. VCD induces a perimenopause-like condition prior to the onset of reproductive acyclicity. Sixteen-week-old C57BL/6J mice were randomized to five treatment groups: sham(SHM), OVX, SHM + VCD, OVX + GEN, and SHM + VCD + GEN. In vivo, blood samples were drawn for hormone and isoflavone analyses, estrous cycles were monitored, and X-ray imaging was performed to assess changes in bone parameters. Following sacrifice, ovaries were assessed histologically, bone microarchitecture was evaluated via microcomputed tomography, and bone mechanical properties were measured. Some effects of GEN were observed in OVX mice, but GEN effects were not able to be evaluated in VCD-treated mice due to the subtle diminution of bone during the 4 months of this experiment.

The cognitive effects of conjugated equine estrogens depend on whether menopause etiology is transitional or surgical.

The question of whether to take hormone therapy (HT) will impact every woman as she enters reproductive senescence. In women, studies suggest that ovarian hormone loss associated with menopause has deleterious cognitive effects. Results from clinical studies evaluating whether estrogen-containing HT mitigates these effects, and benefits cognition, are discrepant. Type of menopause, surgical vs. transitional, impacts cognitive outcome in women. However, whether type of menopause impacts cognitive effects of HT has not been methodically tested in women or an animal model. We used the 4-vinylcyclohexene diepoxide rodent model of ovarian follicle depletion, which mimics transitional menopause, and the traditional rat model of menopause, ovariectomy, to cognitively test the most commonly prescribed estrogen therapy in the United States, conjugated equine estrogens (Premarin). Here we show conjugated equine estrogens benefited cognition in surgically menopausal rats, but, in contrast, impaired cognition in transitionally menopausal rats. Androstenedione, released from the residual transitional menopausal ovary, was positively associated with impaired performance, replicating our previous findings in 4-vinylcyclohexene diepoxide animals. The current findings are especially salient given that no clinical study testing cognition has methodically separated these two populations of menopausal women for analysis. That we now show surgical vs. transitional modes of menopause result in disparate cognitive effects of HT has implications for future research and treatments optimizing HT for menopausal women.

Transitional versus surgical menopause in a rodent model: etiology of ovarian hormone loss impacts memory and the acetylcholine system.

Clinical research suggests that type of ovarian hormone loss at menopause influences cognition. Until recently ovariectomy (OVX) has been the primary rodent model to examine effects of ovarian hormone loss on cognition. This model limits evaluations to abrupt and complete ovarian hormone loss, modeling less than 13% of women who receive surgical menopause. The majority of women do not have their ovaries surgically removed and undergo transitional hormone loss via ovarian follicular depletion. 4-Vinylcyclohexene-diepoxide (VCD) produces gradual ovarian follicular depletion in the rodent, with hormone profiles more similar to naturally menopausal women vs. OVX. We directly compared VCD and OVX models to examine whether type of hormone loss (transitional vs. surgical) impacted cognition as assessed on a maze battery as well as the cholinergic system tested via scopolamine mnemonic challenge and brain acetylcholinesterase activity. Middle-aged rats received either sham surgery, OVX surgery, VCD, or VCD then OVX to assess effects of removal of residual ovarian output after transitional menopause and follicular depletion. VCD-induced transitional menopause impaired learning of a spatial recent memory task; surgical removal of residual ovarian hormones by OVX abolished this negative effect of transitional menopause. Furthermore, transitional menopause before OVX was better for memory than an abrupt loss of hormones via OVX only. Surgical ovarian hormone loss, regardless of menopause history, increased hippocampal acetylcholinesterase activity. Circulating gonadotropin and androstenedione levels were related to cognitive competence. Collectively, findings suggest that in the rat, initiation of transitional menopause before surgical ovary removal can benefit mnemonic function and could obviate some negative cognitive consequences of surgical menopause alone.

Premarin improves memory, prevents scopolamine-induced amnesia and increases number of basal forebrain choline acetyltransferase positive cells in middle-aged surgically menopausal rats.

Conjugated equine estrogen (CEE) is the most commonly prescribed estrogen therapy, and is the estrogen used in the Women's Health Initiative study. While in-vitro studies suggest that CEE is neuroprotective, no study has evaluated CEE's effects on a cognitive battery and brain immunohistochemistry in an animal model. The current experiment tested whether CEE impacted: I) spatial learning, reference memory, working memory and long-term retention, as well as ability to handle mnemonic delay and interference challenges; and, II) the cholinergic system, via pharmacological challenge during memory testing and ChAT-immunoreactive cell counts in the basal forebrain. Middle-aged ovariectomized (Ovx) rats received chronic cyclic injections of either Oil (vehicle), CEE-Low (10 microg), CEE-Medium (20 microg) or CEE-High (30 microg) treatment. Relative to the Oil group, all three CEE groups showed less overnight forgetting on the spatial reference memory task, and the CEE-High group had enhanced platform localization during the probe trial. All CEE groups exhibited enhanced learning on the spatial working memory task, and CEE dose-dependently protected against scopolamine-induced amnesia with every rat receiving the highest CEE dose maintaining zero errors after scopolamine challenge. CEE also increased number of ChAT-immunoreactive neurons in the vertical diagonal band of the basal forebrain. Neither the ability to remember after a delay nor interference, nor long-term retention, was influenced by the CEE regimen used in this study. These findings are similar to those reported previously for 17 beta-estradiol, and suggest that CEE can provide cognitive benefits on spatial learning, reference and working memory, possibly through cholinergic mechanisms.

Correlation of serum anti-Müllerian hormone with accelerated follicle loss following 4-vinylcyclohexene diepoxide-induced follicle loss in mice.

A chemically induced model of ovarian failure has been developed in rodents, and was used to test whether or not anti-Müllerian hormone (AMH) can be used as a non-invasive measure of primordial follicle numbers. Repeated exposures of mice to 4-vinylcyclohexene diepoxide (VCD) induce loss of primordial and earliest growing ovarian follicles. An accelerated exposure regimen was used to eliminate small ovarian follicles in C57BL6/J mice (240mg VCD/kg/day, 5 days, i.p.). Follicle populations were determined and correlated with circulating AMH levels. Exposures decreased only primordial and small primary follicles by 96% on day 16 after initiating exposures, followed by almost complete follicle elimination on days 37-100. AMH levels in VCD-exposed mice were similar to vehicle-treated mice on day 16, but became significantly lower or undetectable at later time points. Thus, AMH correlated well with growing follicle numbers. AMH only correlated with primordial follicles at time points after ovarian insult at which their loss led to decreased growing follicle numbers.

Behavioral consequences of ovarian atrophy and estrogen replacement in the APPswe mouse.

Cognitive performance was evaluated in a longitudinal study of APPswe2576 transgenic mice (APP) and a wildtype (WT) comparison group. Subgroups of the APP mice were treated with the ovarian toxicant 4-vinylcyclo-hexene diepoxide (VCD) at 60-75 days of age to induce ovarian atrophy and/or given estrogen (estradiol, 4 microg/day) continuously by pellet from 76 days of age. APP mice had a generally poorer radial maze performance than WT at 4.5, 7.5, 10.5 and 15 months of age. In separate tests, APP mice had a slight motor impairment, higher incidence of homecage stereotypy, hyperactivity in an open field and reduced object exploration relative to the WT group. Ovarian atrophy led to better maze performance at 7.5 months. The effect of estrogen on maze performance with aging could not be effectively evaluated due to poor survival (30%) of these mice. No effects of ovarian atrophy or estrogen treatment were identified for amyloid-beta accumulation or plaque formation at 15 months. Long-term longitudinal studies in animal models are needed to explore the consequences of menopause and hormone replacement on Alzheimer's disease, but they are complicated by considerations of survival, pre-aging deficits, testing experience and selection of appropriate estrogen treatment levels.

Drinking water with uranium below the U.S. EPA water standard causes estrogen receptor-dependent responses in female mice.

BACKGROUND: The deleterious impact of uranium on human health has been linked to its radioactive and heavy metal-chemical properties. Decades of research has defined the causal relationship between uranium mining/milling and onset of kidney and respiratory diseases 25 years later. OBJECTIVE: We investigated the hypothesis that uranium, similar to other heavy metals such as cadmium, acts like estrogen. METHODS: In several experiments, we exposed intact, ovariectomized, or pregnant mice to depleted uranium in drinking water [ranging from 0.5 microg/L (0.001 microM) to 28 mg/L (120 microM). RESULTS: Mice that drank uranium-containing water exhibited estrogenic responses including selective reduction of primary follicles, increased uterine weight, greater uterine luminal epithelial cell height, accelerated vaginal opening, and persistent presence of cornified vaginal cells. Coincident treatment with the antiestrogen ICI 182,780 blocked these responses to uranium or the synthetic estrogen diethylstilbestrol. In addition, mouse dams that drank uranium-containing water delivered grossly normal pups, but they had significantly fewer primordial follicles than pups whose dams drank control tap water. CONCLUSIONS: Because of the decades of uranium mining/milling in the Colorado plateau in the Four Corners region of the American Southwest, the uranium concentration and the route of exposure used in these studies are environmentally relevant. Our data support the conclusion that uranium is an endocrine-disrupting chemical and populations exposed to environmental uranium should be followed for increased risk of fertility problems and reproductive cancers.

Atherosclerotic lesion development in a novel ovary-intact mouse model of perimenopause.

OBJECTIVE: Since the unexpected results from the Women's Health Initiative, the possible protective role of estrogen in preventing heart disease in perimenopausal and postmenopausal women is uncertain. This study examined atherosclerotic lesion development in ovariectomized versus follicle-depleted ovary-intact cholesterol-fed female low-density lipoprotein (LDL) receptor-deficient mice. METHODS AND RESULTS: We studied lesion development in LDL receptor-deficient mice that were ovariectomized or follicle depleted with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure, then treated +/- exogenous 17beta-estradiol via pellet implant. At 120 days after start of cholesterol feeding, the extent of lesion in aorta and innominate artery was determined. Lesion area in both locations was similar in vehicle control, VCD-treated, and ovariectomized mice. Replacement with 17beta-estradiol caused lesion reduction (P<0.05) in both arterial locations, but it was most efficacious in suppressing innominate lesion area in VCD-treated mice (12.9+/-5.2%) compared with ovariectomized mice (40.0+/-6.04%). CONCLUSIONS: Endocrine status associated with the follicle-depleted ovary influences exogenous estradiol effects during the development of atherosclerotic lesions and, in particular, inhibits lesion progression in the innominate artery.

The follicle-deplete mouse ovary produces androgen.

The follicle-depleted postmenopausal ovary is enriched in interstitial cells that produce androgens. This study was designed to cause follicle depletion in mice using the industrial chemical, 4-vinylcyclohexene diepoxide (VCD), and characterize the steroidogenic capacity of cells in the residual ovarian tissue. From a dose-finding study, the optimal daily concentration of VCD was determined to be 160 mg/kg. Female B6C3F(1) immature mice were treated daily with vehicle control or VCD (160 mg kg(-1) day(-1), 15 days, i.p.). Ovaries were removed and processed for histological evaluation. On Day 15 following onset of treatment, primordial follicles were depleted and primary follicles were reduced to about 10% of controls. On Day 46, primary follicles were depleted and secondary and antral follicles were reduced to 0.7% and 2.6% of control, respectively. Seventy-five percent of treated mice displayed disruptions in estrous cyclicity. All treated mice were in persistent diestrus (acyclic) by Day 58. Plasma FSH levels were increased (P < 0.05) relative to controls on Day 37 and had plateaued by Day 100. Relative to age-matched cyclic controls, by Day 127, the significant differences in VCD-treated mice included reduced ovarian and uterine weights, elevated plasma LH and FSH, and reduced plasma progesterone and androstenedione. Furthermore, plasma 17beta-estradiol levels were nondetectable. Unlike controls, immunostaining for LH receptor, and the high density lipoprotein receptor (SR-BI), was diffuse in ovarian sections from VCD-treated animals. Ovaries from Day 120 control and VCD-treated animals were dissociated and dispersed cells were placed in culture. Cultured cells from ovaries of VCD-treated animals produced less LH-stimulated progesterone than control cells. Androstenedione production was nondetectable in cells from cyclic control animals. Conversely, cells from VCD-treated animals produced androstenedione that was doubled in the presence of insulin and LH (1 and 3 ng/ml). Collectively, these data demonstrate that VCD-mediated follicle depletion results in residual ovarian tissue that may be analogous to the follicle-deplete postmenopausal ovary. This may serve as a useful animal model to examine the dynamics of follicle loss in women as ovarian senescence ensues.

Exposure to 4-tert-octylphenol accelerates sexual differentiation and disrupts expression of steroidogenic factor 1 in developing bullfrogs.

Sex-specific gonadal steroidogenesis during development is critical to differentiation of the sexually dimorphic phenotype and reproductive function of adult organisms. Environmental contaminants may affect the process of sexual differentiation through disruption of steroid production and/or action. Control of the steroidogenic metabolic pathway is regulated partly by P450 cytochrome hydroxylases, and the expression of many of these enzymes is controlled by the orphan nuclear receptor, steroidogenic factor-1 (SF-1). In mammals, SF-1 expression is critical for development of the reproductive axis and adult reproductive function. In the bullfrog Rana catesbeiana, during sequential stages of development encompassing sexual differentiation, SF-1 protein expression becomes elevated in ovaries of sexually differentiating females, whereas expression in testes decreases. We exposed tadpoles to the industrial pollutant octylphenol (OP) for 24 hr before and during the critical stages of sexual differentiation to determine whether this known endocrine disruptor affects sex differentiation and SF-1 expression. We found that both females and males treated with an environmentally relevant low dose (10(-9)M) of OP underwent early gonadal differentiation. Furthermore, OP exposure disrupted the sexually dimorphic expression of SF-1 that occurs during sexual differentiation. Our results suggest that OP exposure may affect developmental processes that could ultimately influence adult reproductive function and that these disruptive effects may be mediated in partly through disturbances in gene regulation by SF-1.

Long-term effects of ovarian follicular depletion in rats by 4-vinylcyclohexene diepoxide.

4-Vinylcyclohexene diepoxide (VCD) destroys preantral ovarian follicles in rats. Female 28-day Fisher 344 (F344) rats were dosed (30 days) with VCD (80 mg/kg per day, i.p.) or vehicle, and animals were evaluated for reproductive function at subsequent time points for up to 360 days. At each time point animals were killed, and ovaries and plasma collected. VCD reduced (P<0.05) the number of preantral follicles by day 30 relative to control. There were no ultrastructural differences in morphology between VCD-treated and control ovaries. Circulating FSH levels in VCD-treated animals were greater (days 120, 240, and 360, P<0.05) than in controls. Cyclicity was disrupted in the VCD-treated group by day 360. These results show that VCD-induced follicular destruction in rats is associated with a sequence of events (loss of preantral follicles, increased plasma FSH, and cyclic disruption) preceding premature ovarian senescence that is similar to events that occur during the onset of menopause in women.

Sexually dimorphic expression of steroidogenic factor 1 (SF-1) in developing gonads of the American bullfrog, Rana catesbeiana.

Genetic sex determination leads to gonadal differentiation and ultimately the differences between the sexes in steroid hormone secretion. Gonadal steroidogenesis is critical for the development of a sexually dimorphic phenotype and adult reproductive function. Control of gonadal development and steroidogenesis is under the regulation, at least in part, of steroidogenic factor-1 (SF-1). We have begun to characterize SF-1 expression in an amphibian to determine the role of this protein in development and reproduction. We have detected a putative SF-1 protein from several tissues in the American bullfrog, Rana catesbeiana, that co-migrates with mouse SF-1 on a Western blot. Our results show that bullfrog SF-1 protein is expressed in steroidogenic and other reproductive tissues in a manner similar to that reported for other species, with high expression in the brain, pituitary, gonad, liver, and interrenal, but little or no expression in non-reproductive tissues such as skin and intestine. Using a quantitative Western blot analysis system, we documented changes in SF-1 protein in the gonads of developing tadpoles. Our results indicate that there is sexually dimorphic expression of SF-1 protein that becomes evident at the time of sexual differentiation of the gonads. In males, the expression of SF-1 decreases following testicular formation and in females the expression increases with the formation of ovaries. This is the first study to investigate changes in SF-1 during development at the protein level. The expression is similar to that reported for changes in SF-1 mRNA expression in chickens and alligators, however, opposite to that seen in mammals and turtles. These results indicate that SF-1 may play a pivotal role in development of the reproductive system in amphibians as it does in other vertebrate groups.