EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals.

The Endocrine Society's first Scientific Statement in 2009 provided a wake-up call to the scientific community about how environmental endocrine-disrupting chemicals (EDCs) affect health and disease. Five years later, a substantially larger body of literature has solidified our understanding of plausible mechanisms underlying EDC actions and how exposures in animals and humans-especially during development-may lay the foundations for disease later in life. At this point in history, we have much stronger knowledge about how EDCs alter gene-environment interactions via physiological, cellular, molecular, and epigenetic changes, thereby producing effects in exposed individuals as well as their descendants. Causal links between exposure and manifestation of disease are substantiated by experimental animal models and are consistent with correlative epidemiological data in humans. There are several caveats because differences in how experimental animal work is conducted can lead to difficulties in drawing broad conclusions, and we must continue to be cautious about inferring causality in humans. In this second Scientific Statement, we reviewed the literature on a subset of topics for which the translational evidence is strongest: 1) obesity and diabetes; 2) female reproduction; 3) male reproduction; 4) hormone-sensitive cancers in females; 5) prostate; 6) thyroid; and 7) neurodevelopment and neuroendocrine systems. Our inclusion criteria for studies were those conducted predominantly in the past 5 years deemed to be of high quality based on appropriate negative and positive control groups or populations, adequate sample size and experimental design, and mammalian animal studies with exposure levels in a range that was relevant to humans. We also focused on studies using the developmental origins of health and disease model. No report was excluded based on a positive or negative effect of the EDC exposure. The bulk of the results across the board strengthen the evidence for endocrine health-related actions of EDCs. Based on this much more complete understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability, these findings can be much better translated to human health. Armed with this information, researchers, physicians, and other healthcare providers can guide regulators and policymakers as they make responsible decisions.

Executive Summary to EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals.

This Executive Summary to the Endocrine Society's second Scientific Statement on environmental endocrine-disrupting chemicals (EDCs) provides a synthesis of the key points of the complete statement. The full Scientific Statement represents a comprehensive review of the literature on seven topics for which there is strong mechanistic, experimental, animal, and epidemiological evidence for endocrine disruption, namely: obesity and diabetes, female reproduction, male reproduction, hormone-sensitive cancers in females, prostate cancer, thyroid, and neurodevelopment and neuroendocrine systems. EDCs such as bisphenol A, phthalates, pesticides, persistent organic pollutants such as polychlorinated biphenyls, polybrominated diethyl ethers, and dioxins were emphasized because these chemicals had the greatest depth and breadth of available information. The Statement also included thorough coverage of studies of developmental exposures to EDCs, especially in the fetus and infant, because these are critical life stages during which perturbations of hormones can increase the probability of a disease or dysfunction later in life. A conclusion of the Statement is that publications over the past 5 years have led to a much fuller understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability. These findings will prove useful to researchers, physicians, and other healthcare providers in translating the science of endocrine disruption to improved public health.

Effects of chronic NMDA-NR2b inhibition in the median eminence of the reproductive senescent female rat.

Gonadotrophin-releasing hormone (GnRH) neurones of the hypothalamic-pituitary-gonadal (HPG) axis drive reproductive function and undergo age-related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signalling via the NMDA receptor (NMDAR) and may be a result of changing NMDAR stoichiometry to favour NR2b over NR2a subunit expression with ageing. We have previously shown that the systemic inhibition of NR2b-containing receptors with ifenprodil, an NR2b-specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle-aged females. In the present study, we chronically administered ifenprodil, an NR2b-specific antagonist, at the site of GnRH terminals in the ME or at GnRH perikarya in the preoptic area, in reproductively senescent middle-aged female rats, aiming to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b and phosphorylated NR2b (Tyr-1472) in the ME were measured. Chronic ifenprodil treatment in the ME (but not the preoptic area) altered oestrous cyclicity by increasing the percentage of days spent in pro-oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co-labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR-NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive ageing.

Designing Endocrine Disruption Out of the Next Generation of Chemicals.

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical's potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at "the drawing board." It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In this paper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a 'proof-of-principle' test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.

Endocrine-disrupting chemicals and public health protection: a statement of principles from The Endocrine Society.

An endocrine-disrupting chemical (EDC) is an exogenous chemical, or mixture of chemicals, that can interfere with any aspect of hormone action. The potential for deleterious effects of EDC must be considered relative to the regulation of hormone synthesis, secretion, and actions and the variability in regulation of these events across the life cycle. The developmental age at which EDC exposures occur is a critical consideration in understanding their effects. Because endocrine systems exhibit tissue-, cell-, and receptor-specific actions during the life cycle, EDC can produce complex, mosaic effects. This complexity causes difficulty when a static approach to toxicity through endocrine mechanisms driven by rigid guidelines is used to identify EDC and manage risk to human and wildlife populations. We propose that principles taken from fundamental endocrinology be employed to identify EDC and manage their risk to exposed populations. We emphasize the importance of developmental stage and, in particular, the realization that exposure to a presumptive "safe" dose of chemical may impact a life stage when there is normally no endogenous hormone exposure, thereby underscoring the potential for very low-dose EDC exposures to have potent and irreversible effects. Finally, with regard to the current program designed to detect putative EDC, namely, the Endocrine Disruptor Screening Program, we offer recommendations for strengthening this program through the incorporation of basic endocrine principles to promote further understanding of complex EDC effects, especially due to developmental exposures.

Prenatal exposure to vinclozolin disrupts selective aspects of the gonadotrophin-releasing hormone neuronal system of the rabbit.

Developmental exposure to the agricultural fungicide vinclozolin can impair reproductive function in male rabbits and was previously found to decrease the number of immunoreactive-gonadotrophin-releasing hormone (GnRH) neurones in the region of the organum vasculosum of the lamina terminalis and rostral preoptic area by postnatal week (PNW) 6. In the present study, in an aim to further examine the disruption of GnRH neurones by foetal vinclozolin exposure, pregnant rabbits were dosed orally with vinclozolin, flutamide or carrot paste vehicle for the last 2 weeks of gestation. Offspring were euthanised at birth (males and females), PNW 6 (females), PNW 26 (adult males) or PNW 30 (adult females) of age. At birth and in adults, brains were sectioned and processed for immunoreactive GnRH. The numbers of immunoreactive GnRH neuronal perikarya were significantly decreased in vinclozolin-treated rabbits at birth and in adult littermates. By contrast, there was an increase in GnRH immunoreactivity in the terminals in the region of the median eminence. Analysis of PNW 6 female brains by radioimmunoassay revealed a two-fold increase in GnRH peptide content in the mediobasal hypothalamus in vinclozolin-treated rabbits. This finding was complemented by immunofluorescence analyses, which revealed a 2.8-fold increase in GnRH immunoreactivity in the median eminence of vinclozolin compared to vehicle-treated females at PNW 30. However, there was no difference between treatment groups in the measures of reproduction that were evaluated: ejaculation latency, conception rates or litter size. These results indicate that sub-acute, prenatal vinclozolin treatment is sufficient to create perdurable alterations in the GnRH neuronal network that forms an important input into the reproductive axis. Finally, the effect of vinclozolin on the GnRH neuronal network was not comparable to that of flutamide, suggesting that vinclozolin was not acting through anti-androgenic mechanisms.

Age- and hormone-regulation of N-methyl-D-aspartate receptor subunit NR2b in the anteroventral periventricular nucleus of the female rat: implications for reproductive senescence.

Glutamate, acting through its N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin-releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b-expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and / or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3-5 months), middle-aged (9-12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b-immunoreactive cell numbers in the AVPV showed an age-associated decrease in the density of NR2b-immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi-quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1-colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co-expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in the AVPV undergo both age- and hormonal-regulation, which may be related to previous observations of changes in functional responses of reproductive neuroendocrine systems to NMDA receptor modulators with ageing.

The hypothalamic insulin-like growth factor-1 receptor and its relationship to gonadotropin-releasing hormones neurones during postnatal development.

Reproduction in vertebrates is controlled by hypophysiotropic gonadotropin-releasing hormone (GnRH) neurones. Pulsatile GnRH release increases during reproductive development, resulting in the onset and progression of puberty and, ultimately, the acquisition and maintenance of adult reproductive function. These changes in GnRH release are largely due to inputs to GnRH cells from other factors, including the neurotrophic factor, insulin-like growth factor-1 (IGF-1). Here, molecular studies were undertaken to quantify expression of IGF-1 receptor (IGF-1R) mRNA in the preoptic area-anterior hypothalamus (POA-AH) and mediobasal hypothalamus (MBH)-median eminence (ME), the sites of GnRH perikarya and neuroterminals, respectively. Immunocytochemical studies were also carried out to study the anatomical relationship between the IGF-1R and GnRH neurones. Experiments were performed in a developmental context using neonatal (P5), peripubertal ( approximately P30) and adult (P60) male and female mice. We found that IGF-1R mRNA levels in the POA-AH were significantly different among all age groups, with levels higher at P60 then P5 or approximately P30. Levels of IGF-1R mRNA in the MBH-ME were lower at P5 than approximately P30 or P60. Qualitative observations suggested that IGF-1R immunoreactivity in POA-AH increased from P5 through P60. Quantitative double-label immunocytochemistry studies showed that GnRH perikarya expressed IGF-1R. Taken together, the results demonstrate expression of, and developmental changes in, IGF-1R gene and protein in brain regions containing GnRH and other neuroendocrine cells. Moreover, the novel finding that the IGF-1R is expressed on GnRH perikarya in vivo suggests a potential direct anatomical locus where IGF-1 can regulate reproductive development and function.

A novel mechanism for endocrine-disrupting effects of polychlorinated biphenyls: direct effects on gonadotropin-releasing hormone neurones.

Polychlorinated biphenyls (PCBs) cause abnormal development and physiology of the reproductive system. We hypothesized that these effects may be mediated, at least in part, by neuroendocrine cells in the hypothalamus that integrate inputs to and outputs from the central nervous system and reproductive systems. The effects of two PCB mixtures, Aroclor 1221 and Aroclor 1254, were tested on the hypothalamic GT1-7 cells, which synthesize and secrete the key hypothalamic hormone, gonadotropin-releasing hormone (GnRH). GT1-7 cells were treated for 24 h in dose-response experiments and GnRH gene expression and release were quantified. Aroclor 1221 was stimulatory to GnRH gene expression, particularly at post-transcriptional levels (GnRH cytoplasmic mRNA), and increased GnRH peptide levels, suggesting a post-translational regulation of GnRH biosynthesis. It also caused a qualitative increase in GT1-7 neurite outgrowth and cell confluency. Aroclor 1254 had very different effects from Aroclor 1221. It inhibited GnRH nuclear mRNA levels at high dosages, and stimulated GnRH mRNA at low doses, suggesting a post-transcriptional mechanism of regulation. Aroclor 1254 did not alter GnRH peptide levels. Qualitatively, Aroclor 1254 caused a retraction of GT1-7 cell processes and neurotoxicity at high dosages. In order to gauge the involvement of the oestrogen receptor in these responses, the oestrogen receptor antagonist, ICI 182,780 (ICI) was coadministered in other studies with the PCBs. While effects of Aroclor 1221 on GnRH gene expression were not blocked by ICI, its effects on GnRH peptide levels were blocked by ICI, indicating that some but not all of the effects of Aroclor 1221 are mediated by the classical oestrogen receptor alpha and/or beta. The inhibitory effects of Aroclor 1254 on GnRH gene expression were not prevented by ICI, although ICI itself had stimulatory effects on GnRH gene expression that were blocked by cotreatment with Aroclor 1254. These results demonstrate a novel mechanism for effects of the two PCBs directly on GnRH gene expression, and indicate a hypothalamic level for endocrine disruption by these environmental toxicants.

Vasoactive intestinal polypeptide contacts on gonadotropin-releasing hormone neurones increase following puberty in female rats.

Successful reproduction requires precise temporal coordination among various endocrine and behavioural events. The circadian system regulates daily temporal organization in behaviour and physiology, including neuroendocrine rhythms. The main circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN sends direct efferents to the reproductive axis via monosynaptic projections to gonadotropin-releasing hormone (GnRH) neurones. This communication generates circadian endocrine rhythms as well as the preovulatory luteinizing hormone (LH) surge necessary for successful ovulation. One SCN peptide thought to be important for the regulation of oestrous cycles is vasoactive intestinal polypeptide (VIP). VIP neurones from the SCN contact GnRH cells, and these cells are preferentially activated during an LH surge in rats. Unlike adult rats, prepubertal females do not exhibit oestrous cycles, nor do they exhibit an LH surge in response to oestradiol positive-feedback. The present study was undertaken to determine the extent to which the development of a 'mature' reproductive axis in female rats is associated with modifications in VIP contacts on GnRH neurones. The brains of diestrus adult (approximately 60 days of age) and prepubertal (21 days of age) female rats were examined using double-label fluorescence immunohistochemistry for VIP and GnRH, with light and confocal microscopy. Although the total number of GnRH-immunoreactive neurones did not differ between adult and prepubertal females, adults had a significant increase in the percentage of GnRH cells receiving VIP contacts compared to juveniles. These data suggest that the development of reproductive hormone rhythms and oestrous cyclicity may be, in part, due to modifications of VIP input to the GnRH system.

Age-related changes in hypothalamic gonadotropin-releasing hormone and N-methyl-D-aspartate receptor gene expression, and their regulation by oestrogen, in the female rat.

During reproductive ageing, the oestrous cycles of female rats become irregular and eventually cease. The mechanisms for reproductive senescence in rodents are believed to involve changes in hypothalamic neurones, including gonadotropin-releasing hormone (GnRH) cells and their afferent inputs. In addition, effects of oestrogen on hypothalamic function may vary in animals of different ages. These issues were addressed using young (aged 4-5 months), middle-aged (12-14 months) and old (24-26 months) female Sprague-Dawley rats. Animals were ovariectomized and given oestrogen or vehicle replacement. They were killed and the preoptic area-anterior hypothalamus (POA-AH) and the medial basal hypothalamus-median eminence (MBH-ME) were dissected out, RNA extracted, and RNase protection assay used to quantify gene expression of several hypothalamic molecules. In the first experiment, GnRH RNA levels were measured in the POA-AH. No effects of ageing or oestrogen were observed on GnRH gene expression. This finding suggests that ageing and oestrogen may affect GnRH release from neuroterminals independently of de novo biosynthesis, and that this may involve other neurones that affect GnRH neurosecretory function. In the second experiment, we investigated changes in N-methyl-D-aspartate (NMDA) receptor subunit mRNA levels. These receptors play an important regulatory role in mediating effects of glutamate on GnRH function, and are themselves regulated by oestrogen and ageing. NMDA receptor subunit (NR) 1, 2a and 2b mRNA levels were quantified in the POA-AH and MBH-ME, the sites of GnRH perikarya and neuroterminals, respectively. In general, oestrogen had inhibitory effects on NR1 and NR2a, and differential effects on NR2b subunit mRNA levels. NMDA receptor subunit mRNA levels also changed during ageing: age-related decreases in NR1 mRNA occurred in the MBH-ME, and an age-related increase in NR2b mRNA occurred in the POA-AH. Taken together, these results demonstrate subunit- and region-specific changes in hypothalamic NMDA receptor subunit gene expression with oestrogen and ageing. These alterations could have implications for the physiological effects of glutamate on its NMDA receptor, and impact the regulation of reproductive and other neuroendocrine and autonomic functions by hypothalamic glutamatergic inputs.

Gonadotropin-releasing hormone neurons, NMDA receptors, and their regulation by steroid hormones across the reproductive life cycle.

The effects of ovarian steroid hormones on gonadotropin-releasing hormone (GnRH) neurons have been studied for many years. In addition to their regulation by sex steroids, GnRH neurons are affected by inputs from neurotransmitters such as glutamate, acting via the NMDA receptor (NMDAR). Moreover, the NMDAR itself is subject to estrogen regulation. Thus, effects of ovarian steroids on GnRH neurons and the NMDAR, and their interactions, are under intense investigation. Messenger RNA and protein levels of GnRH and NMDAR subunits were measured in neuroendocrine brain regions in response to estrogen treatment, or across the reproductive cycle. Stimulatory effects of ovarian steroids on GnRH gene expression occur during the preovulatory LH surge in young adult rats, and this is abolished in middle-aged rats that have an attenuated LH surge. Effects of estrogen on GnRH neurons have also been studied in the ovariectomized, estrogen-primed rat, and while results vary between laboratories, there appear to be age-related changes in the sensitivity of GnRH neurons to estrogen. Estrogen also has effects on NMDAR mRNA levels. In intact rats, mRNA levels of NMDAR decrease during reproductive aging in the preoptic area, the site of GnRH perikarya, while in the medial basal hypothalamus-median eminence, the site of GnRH neuroterminals, levels of NMDAR subunit mRNAs increase with aging. Thus, glutamatergic inputs to GnRH perikarya and neuroterminals and other neuroendocrine cells may change during reproductive aging in intact rats. In ovariectomized rats, NMDAR subunit mRNA levels also undergo age-related changes, and respond to estrogen replacement in a subunit- and age-specific manner. Notably, there are major differences in NMDAR gene expression during aging between intact and ovariectomized rats, suggesting that ovarian factors other than estrogen play a role in the regulation of this receptor.

Alterations in hypothalamic insulin-like growth factor-I and its associations with gonadotropin releasing hormone neurones during reproductive development and ageing.

Insulin-like growth factor-I (IGF-I) is thought to play a role in the onset of reproductive ability at puberty and the control of reproductive function throughout adult life. It is believed that these effects are mediated at least in part by the activation of gonadotropin releasing hormone (GnRH) neurones by IGF-I, but the interactions of IGF-I with GnRH neurones in vivo are largely unknown. We first examined the anatomical relationship between GnRH and IGF-I cells in neuroendocrine regions. Using double-label immunocytochemistry, we observed that in the preoptic area-anterior hypothalamus (POA-AH), the site of GnRH perikarya, the majority (78%) of GnRH cell bodies expressed IGF-I immunoreactivity. IGF-I immunoreactivity was also high in the median eminence, the site of GnRH release, and GnRH neuroterminals were seen to interweave among IGF-I-immunopositive cells. Due to this substantial overlap of GnRH and IGF-I immunoreactive elements, we then tested the hypothesis that changes in IGF-I may regulate the GnRH system. Animals were examined at the two important reproductive life transitions: puberty and reproductive senescence. IGF-I mRNA levels were measured in POA-AH and medial basal hypothalamus-median eminence (MBH-ME) and effects of IGF-I treatment on GnRH mRNA levels were quantified by RNase protection assay. Although IGF-I treatment did not alter GnRH gene expression, there were significant alterations in hypothalamic IGF-I gene expression at both puberty and reproductive senescence. During puberty, IGF-I mRNA levels in the MBH-ME of rats increased from the juvenile stage (P25) to the day of vaginal opening (P35), and from the day of vaginal opening to young adulthood (P45) in the POA-AH. During reproductive ageing, IGF-I mRNA levels were significantly lower in middle-aged than young rats, particularly in the MBH-ME. At all ages, IGF-I expression was greater in the MBH-ME than in the POA-AH. These experiments demonstrate that: (i) the majority of adult GnRH neurones are immunopositive for the IGF-I protein; (ii) hypothalamic IGF-I levels increase at the onset of reproductive function and decrease at reproductive senescence in a regionally specific manner; and (iii) despite the presence of IGF-I in GnRH perikarya, IGF-I does not affect GnRH gene expression, suggesting that IGF-I may act at the level of GnRH release rather than gene expression.

Length of postovariectomy interval and age, but not estrogen replacement, regulate N-methyl-D-aspartate receptor mRNA levels in the hippocampus of female rats.

Estrogens and N-methyl-D-aspartate (NMDA) receptors regulate multiple aspects of morphological and functional plasticity in young animals. For example, estrogens increase spine density in the hippocampus, and NMDA antagonists block these effects. Few studies have examined the effects of age, postovariectomy interval, and duration of estrogen replacement in the hippocampus and more specifically on NMDA receptor subunits. Therefore, the present study was designed to investigate the effects of short- and long-term estrogen replacement or deprivation on mRNA levels of three NMDA receptor subunits, NR1, NR2A, and NR2B, in the hippocampus of aging female Sprague-Dawley rats. Young (3- to 4-month-old) and middle-aged (12- to 13-month-old) rats were ovariectomized for 1 month and then treated with estrogen or vehicle for either 2 days or 2 weeks. Another set of middle-aged and aged (24-to 25-month-old) animals were ovariectomized for 6 months and treated with estrogen or vehicle for 2 days or 2 weeks. RNase protection assay was used to assess changes in the NMDA receptor subunit mRNA levels. Our results demonstrated significant effects of age and length of ovariectomy on NMDA receptor mRNA levels, with little effect of the estrogen status of the animals on these parameters. The largest effect was seen for the length of the postovariectomy interval, with the results demonstrating that rats with a short-term ovariectomy have substantially higher NMDA receptor subunit mRNA levels than animals with long-term ovariectomy. The most dramatic effects of aging were seen for NR1 and NR2B mRNAs in ventral hippocampus, with large age-related increases. These data suggest that age and duration of ovariectomy impact NMDA receptor mRNA levels in the hippocampus, potentially affecting the stoichiometry and/or function of these receptors. These findings have important implications for postmenopausal or hysterectomy/oophorectomy estrogen depletion and replacement in humans.