Nancy A. Gee

Estrogen and Progesterone Metabolites and Follicle-Stimulating Hormone in the Aged Macaque Female

Abstract The study presented characterizes the ovarian and pituitary function of the aged female macaque through a complete annual reproductive cycle to compare hormone dynamics during the human and nonhuman primate menopausal transition. Data collected over an entire year from aged macaque females indicated that urinary FSHβ subunit baseline levels statistically significantly increased in females after age-related abnormal menstrual cycles occurred. These abnormal cycles were followed by anovulation and complete cessation of follicular activity. No statistically significant difference in urinary FSHβ subunit levels was seen between females that exhibited year-round normal ovarian cycles and those that exhibited seasonal ovarian cycles followed by an interval of anovulation during the nonbreeding season. Basal urinary estrogen metabolite levels were not observed to decrease until ovarian cycles became abnormal and FSHβ subunit levels began to rise. Early follicular phase circulating inhibin β levels were statistically significantly reduced only when ovariectomized females were compared to the year-round normally cycling females. A statistically nonsignificant trend toward decreased inhibin secretion, however, was apparent in aged females with normal cycles, aged females with abnormal cycles, anovulatory aged females, and finally, ovariectomized females. Whereas decreased circulating levels of dehydroepiandrosterone sulfate showed a general decline over the 1-yr study period in all groups, they were lowest in the year-round normally cycling group, progressively higher in the normal-to-anovulatory group and abnormal-to-anovulatory group, and highest in the anovulatory group. Finally, the nonbreeding season was associated with the highest number of abnormal cycles, suggesting that onset of complete ovarian senescence in these study macaques was more likely to occur during that time (i.e., females were less likely to return to normal ovarian cycles the following breeding season and more likely to exhibit permanent ovarian quiescence).

Costs and Consequences of Cellular Compartmentalization and Substrate Competition among Human Enzymes Involved in Androgen and Estrogen Synthesis

ABSTRACT The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs. Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.

Androstenediol Complements Estrogenic Bioactivity during the Menopausal Transition

ObjectiveThe perimenopausal increase in circulating dehydroepiandrosterone sulfate (DHEAS) levels during the menopausal transition (MT) is accompanied by other adrenal steroids that have the potential to alter estrogen/androgen balance and explain the wide interwoman range of estrogen-related symptoms experienced during the MT. MethodsAnnual serum samples from the Study of Women’s Health Across the Nation, which had previously been analyzed for immunoreactive estradiol (E2), testosterone, DHEAS, and sex hormone–binding globulin, were selected based on DHEAS concentration and analyzed for immunoreactive and bioactive estrogens and androgens, including immunoreactive androstenedione, dehydroepiandrosterone, and 5-androstene-3&bgr;,17&bgr;-diol (androstenediol [Adiol]). ResultsA two-fold increase in circulating androstenedione and testosterone was found to rise in parallel with the rise in circulating DHEAS, whereas dehydroepiandrosterone and Adiol concentrations rose seven- to eight-fold. Circulating Adiol, which has both androgenic and estrogenic biological activity, was significantly associated (P < 0.02) with circulating estrogen bioactivity only when E2 concentrations were low and Adiol levels were high. ConclusionsThe wide range of circulating levels of Adiol and its contribution to total circulating estrogenicity during the MT is consistent with the observed interwoman difference in symptoms at this time. Therefore, we conclude that Adiol contributes to circulating estrogenicity when E2 production falls at menopause and may contribute significantly to the endocrine changes experienced by midlife women.

Effects of Triclocarban on Intact Immature Male Rat: Augmentation of Androgen Action

Triclocarban (TCC; 3,4,4′-trichlorocarbanilide) is an antimicrobial agent used widely in various personal hygiene products including soaps. Recently, TCC has been shown to enhance testosterone-induced effects in vitro and to enlarge accessory sex organs in castrated male rats. This study was designed to evaluate the effects of TCC on intact age-matched male rats and on human prostate LNCaP and C4–2B cells. Seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with TCC (0.25% in diet) for 10 days. Triclocarban induced hyperplasia of accessory sex organs in the absence of significant qualitative histological changes. Serum luteinizing hormone (LH) and testosterone were not significantly altered by TCC treatment. In prostate cancer-derived LNCaP and C4–2B cells, TCC potentiated androgen actions via androgen receptor-dependent actions. In conclusion, TCC significantly affects intact male reproductive organs and potentiates androgen effects in prostate cancer cells.

Multiple clinically relevant hormone therapy regimens fail to improve cognitive function in aged ovariectomized rhesus monkeys

Preclinical studies in aged, surgically-menopausal rhesus monkeys have revealed powerful benefits of intermittent estrogen injections on prefrontal cortex-dependent working memory, together with corresponding effects on dendritic spine morphology in the prefrontal cortex. This contrasts with the inconsistent effects of hormone therapy (HT) reported in clinical studies in women. Factors contributing to this discrepancy could include differences in the formulation and sequence of HT regimens, resulting in different neurobiological outcomes. The current study evaluated, in aging surgically menopausal rhesus monkeys, the cognitive effects of 4 HT regimens modeled directly on human clinical practice, including continuous estrogen treatment opposed by progesterone. None of the regimens tested produced any cognitive effect, despite yielding physiologically relevant serum hormone levels, as intended. These findings have implications for the design of regimens that might optimize the benefits of hormone treatment for healthy aging, and suggest that common HT protocols used by women may fail to result in substantial cognitive benefit, at least via direct effects on the prefrontal cortex.

Clinically Relevant Hormone Treatments Fail to Induce Spinogenesis in Prefrontal Cortex of Aged Female Rhesus Monkeys

Preclinical animal models have provided strong evidence that estrogen (E) therapy (ET) enhances cognition and induces spinogenesis in neuronal circuits. However, clinical studies have been inconsistent, with some studies revealing adverse effects of ET, including an increased risk of dementia. In an effort to bridge this disconnect between the preclinical and clinical data, we have developed a nonhuman primate (NHP) model of ET combined with high-resolution dendritic spine analysis of dorsolateral prefrontal cortical (dlPFC) neurons. Previously, we reported cyclic ET in aged, ovariectomized NHPs increased spine density on dlPFC neurons. Here, we report that monkeys treated with cyclic E treatment paired with cyclic progesterone (P), continuous E combined with P (either cyclic or continuous), or unopposed continuous E failed to increase spines on dlPFC neurons. Given that the most prevalent form of ET prescribed to women is a combined and continuous E and P, these data bring into convergence the human neuropsychological findings and preclinical neurobiological evidence that standard hormone therapy in women is unlikely to yield the synaptic benefit presumed to underlie the cognitive enhancement reported in animal models.

Early Life Triclocarban Exposure During Lactation Affects Neonate Rat Survival

Triclocarban (3,4,4′-trichlorocarbanilide; TCC), an antimicrobial used in bar soaps, affects endocrine function in vitro and in vivo. This study investigates whether TCC exposure during early life affects the trajectory of fetal and/or neonatal development. Sprague Dawley rats were provided control, 0.2% weight/weight (w/w), or 0.5% w/w TCC-supplemented chow through a series of 3 experiments that limited exposure to critical growth periods: gestation, gestation and lactation, or lactation only (cross-fostering) to determine the susceptible windows of exposure for developmental consequences. Reduced offspring survival occurred when offspring were exposed to TCC at concentrations of 0.2% w/w and 0.5% w/w during lactation, in which only 13% of offspring raised by 0.2% w/w TCC dams survived beyond weaning and no offspring raised by 0.5% w/w TCC dams survived to this period. In utero exposure status had no effect on survival, as all pups nursed by control dams survived regardless of their in utero exposure status. Microscopic evaluation of dam mammary tissue revealed involution to be a secondary outcome of TCC exposure rather than a primary effect of compound administration. The average concentration of TCC in the milk was almost 4 times that of the corresponding maternal serum levels. The results demonstrate that gestational TCC exposure does not affect the ability of dams to carry offspring to term but TCC exposure during lactation has adverse consequences on the survival of offspring although the mechanism of reduced survival is currently unknown. This information highlights the importance of evaluating the safety of TCC application in personal care products and the impacts during early life exposure.

Daily Immunoactive and Bioactive Human Chorionic Gonadotropin Profiles in Periimplantation Urine Samples

Abstract A need exists for broadly applicable biomarkers of pregnancy outcome in population-based studies that assess environmental hazards to human reproduction. Previous studies have demonstrated that during the periimplantation period, measures of the circulating levels of immunoreactive hCG (IhCG) are not predictive of pregnancy outcome, whereas measurements of the circulating levels of bioactive hCG (BhCG) provide information relating to pregnancy outcome and might provide the basis for an early biomarker of pregnancy outcome. However, for this biomarker to have broad application in population-based studies, it must be adapted to urinary hCG metabolites. The principle objective of the present study was to characterize the periimplantation excretion patterns of urinary hCG metabolites of pregnancies that resulted in live birth (LB), early pregnancy loss (EPL), and recognized clinical abortion (CAB) with an immunoenzymometric assay specific to intact hCG and an LH/chorionic gonadotropin cellular bioassay as the basis for a preliminary comparison between successful (LB) and failing (EPL and CAB) outcome groups. Automated immunoassays for FSH and hCG were used to define each conceptive cycles implantation window. The timing of first hCG detection was significantly later for the EPL group. Pregnancies that resulted in LB had consistently rising average daily IhCG and BhCG levels, with no significant differences when average daily IhCG and BhCG measurements were compared (Student t-test, P > 0.05), whereas pregnancies that resulted in CAB and EFL had lower average daily IhCG and BhCG levels that increased inconsistently. These findings demonstrate that critical information related to pregnancy outcome may be present when multiple urinary hCG isoforms are measured. Further data suggest that the rate of change for the ratio of daily BhCG over IhCG levels might be useful as the basis of a broadly applicable early biomarker for pregnancy outcome.

Continuously delivered ovarian steroids do not alter dendritic spine density or morphology in macaque dorsolateral prefrontal cortical neurons

Aged ovariectomized (OVX) female monkeys, a model for menopause in humans, show a decline in spine density in the dorsolateral prefrontal cortex (dlPFC) and diminished performance in cognitive tasks requiring this brain region. Previous studies in our laboratory have shown that long-term cyclic treatment with 17β-estradiol (E) produces an increase in spine density and in the proportion of thinner spines in layer III pyramidal neurons in the dlPFC of both young and aged OVX rhesus monkeys. Here we used 3D reconstruction of Lucifer yellow-loaded neurons to investigate whether clinically relevant schedules of hormone therapy would produce similar changes in prefrontal cortical neuronal morphology as long-term cyclic E treatment in young female monkeys. We found that continuously delivered E, with or without a cyclic progesterone treatment, did not alter spine density or morphology in the dlPFC of young adult OVX rhesus monkeys. We also found that the increased density of thinner spines evident in the dlPFC 24h after E administration in the context of long-term cyclic E therapy is no longer detectable 20days after E treatment. When compared with the results of our previously published investigations, our results suggest that cyclic fluctuations in serum E levels may cause corresponding fluctuations in the density of thin spines in the dlPFC. By contrast, continuous administration of E does not support sustained increases in thin spine density. Physiological fluctuations in E concentration may be necessary to maintain the morphological sensitivity of the dlPFC to E.

Modulation of higher-primate adrenal androgen secretion with estrogen-alone or estrogen-plus-progesterone intervention.

ObjectiveCirculating adrenal steroids rise during the menopausal transition in most middle-aged women and may contribute to differences in between-women symptoms and ultimate health outcomes. However, the mechanisms for this shift in adrenal steroid production in middle-aged women are not known. This study aims to determine whether hormone therapy (HT) for 1 year can modulate adrenal androgen production. MethodsYounger (9.8 [0.4] years, n = 20) and older (22.7 [0.4] years, n = 37) female laboratory macaques were ovariectomized, and each group was treated with different regimens of HT for up to 1 year. Changes in adrenal histology and circulating adrenal androgens were monitored after estrogen-alone (E) or estrogen plus progesterone (E + P) treatment, and these changes were compared with the same measures in similarly aged animals given vehicle. ResultsZona reticularis area, serum dehydroepiandrosterone (DHEA), and serum dehydroepiandrosterone sulfate (DHEAS) were higher in younger vehicle-treated animals compared with older vehicle-treated animals (P < 0.02). Both E and E + P treatments decreased circulating DHEAS in the younger group (P < 0.05). Although E treatment also decreased DHEAS in the older group, this was not statistically significant. In contrast, E + P treatment in the older group resulted in a rise in DHEAS over vehicle, which was significantly higher than the results of E treatment (P < 0.01). Circulating concentrations of DHEA exhibited similar trends, but these changes did not reach statistical significance. ConclusionsThese data demonstrate that intervention with ovarian steroids can modulate adrenal androgen production in female higher primates and that both animal age and type of HT regimen determine adrenal response.