Julia A. Taylor

Roles of Estrogen Receptor-α Gene Expression in Reproduction-Related Behaviors in Female Mice1

The role of gene expression of the estrogen receptor-␣ form (ER␣) in the regulation of female reproductive behavior was investigated in estrogen receptor knockout (ERKO) mice, deficient specifically for the ER␣, but not the ER␤, gene. Estrogen-or estrogen-plus progesterone-treated gonadectomized ERKO mice did not show any lordosis response. Detailed behavioral analysis revealed that ERKO females were also deficient in sexual behavioral interactions preceding the lordosis response. They were extremely rejective toward attempted mounts by stud male mice, which could not show any intromissions. During resident-intruder aggression tests, gonadally intact ERKO females were more aggressive toward female intruder mice than wild-type (WT) mice. Gonadectomy did not influence the levels of aggressive behavior, and their genotype differences when mice were tested both before and after gonadectomy. However, when mice were tested after gonadectomy for the first time, very few ERKO mice showed aggression. In contrast to aggression, male-type sexual behavior shown by resident mice toward female intruder mice during aggression tests was not different between ERKO and WT mice and was completely abolished after gonadectomy of the resident mice. Finally, it was also found that ERKO females showed greatly reduced levels of parental behavior toward newborn pups placed in their home cage. These changes in parental behavior were not influenced by gonad-ectomy. ERKO females retrieved significantly fewer numbers of pups with longer latencies compared with wild-type (WT) or heterozygous (HZ) littermates when they were tested as gonadally intact or 20 – 65 days after gonadectomy. In addition, during parental behavior tests, a significantly higher percentage of ERKO mice exhibited infanticide compared with WT and HZ mice, which rarely showed infanticide. Taken together, these findings suggest that ER␣ gene expression plays a key role in female mice, not only for sexual behavior but also for other interrelated behaviors, such as parental and aggressive behaviors. In addition, persistence of genotype differences in parental and aggressive behavior after gonadectomy indicates that ER␣ activation during neural developmental processes may also be involved in the regulation of these behaviors. I T IS WELL established that the ovarian steroid, estrogen, regulates female reproduction and lordosis behavior by binding to intracellular estrogen receptors (ER) in the brain. This genomic action of estrogen is assumed to be mediated not only through the classical form of ER (now termed ER␣), but also possibly through the second form of ER, ER␤, which was recently cloned (1, 2). Recent studies have shown that …

Components of plastic: experimental studies in animals and relevance for human health

Components used in plastics, such as phthalates, bisphenol A (BPA), polybrominated diphenyl ethers (PBDE) and tetrabromobisphenol A (TBBPA), are detected in humans. In addition to their utility in plastics, an inadvertent characteristic of these chemicals is the ability to alter the endocrine system. Phthalates function as anti-androgens while the main action attributed to BPA is oestrogen-like activity. PBDE and TBBPA have been shown to disrupt thyroid hormone homeostasis while PBDEs also exhibit anti-androgen action. Experimental investigations in animals indicate a wide variety of effects associated with exposure to these compounds, causing concern regarding potential risk to human health. For example, the spectrum of effects following perinatal exposure of male rats to phthalates has remarkable similarities to the testicular dysgenesis syndrome in humans. Concentrations of BPA in the foetal mouse within the range of unconjugated BPA levels observed in human foetal blood have produced effects in animal experiments. Finally, thyroid hormones are essential for normal neurological development and reproductive function. Human body burdens of these chemicals are detected with high prevalence, and concentrations in young children, a group particularly sensitive to exogenous insults, are typically higher, indicating the need to decrease exposure to these compounds.

THE ESTROGENIC ENDOCRINE DISRUPTING CHEMICAL BISPHENOL A (BPA) AND OBESITY

There is increasing experimental and epidemiological evidence that fetal programming of genetic systems is a contributing factor in the recent increase in adult obesity and other components of metabolic syndrome. In particular, there is evidence that epigenetic changes associated with the use of manmade chemicals may interact with other factors that influence fetal and postnatal growth in contributing to the current obesity epidemic. The focus of this review is on the developmental effects of estrogenic endocrine disrupting chemicals (EDCs), and more specifically on effects of exposure to the estrogenic EDC bisphenol A (BPA), on adipocytes and their function, and the ultimate impact on adult obesity; BPA exposure also results in impaired reproductive capacity. We discuss the interaction of EDCs with other factors that impact growth during fetal and neonatal life, such as placental blood flow and nutrient transport to fetuses, and how these influence fetal growth and abnormalities in homeostatic control systems required to maintain normal body weight throughout life.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Elucidation of a Role for Stromal Steroid Hormone Receptors in Mammary Gland Growth and Development Using Tissue Recombinants

The use of tissue recombinants in conjunction with steroid receptor deficient mice is described as a tool to dissect the complex paracrine pathways of sex-hormone-regulated epithelial growth and ductal morphogenesis in the mammary gland and other hormone target organs. The basic methodology involves the construction of the four possible tissue recombinants composed of epithelium (E)6 and stroma (S) from wild-type (wt) and knock-out (KO) mice: wt-S + wt-S, wt-S + KO-E, KO-S + KO-E, and KO-S + wt-E. All tissue recombinants are grown as subrenal capsule grafts in nude mice. Following appropriate hormonal challenge epithelial growth can be studied in the four types of tissue recombinants. Such studies using estrogen receptor, androgen receptor and progesterone receptor knockout mice demonstrate that epithelial steroid receptors are neither necessary nor sufficient for hormonal regulation of epithelial proliferation. Instead, hormonal regulation of epithelial proliferation is a paracrine event mediated by hormone-receptor-positive stromal cells.

Similarity of bisphenol A pharmacokinetics in rhesus monkeys and mice: Relevance for human exposure

Objective Daily adult human exposure to bisphenol A (BPA) has been estimated at < 1 μg/kg, with virtually complete first-pass conjugation in the liver in primates but not in mice. We measured unconjugated and conjugated BPA levels in serum from adult female rhesus monkeys and adult female mice after oral administration of BPA and compared findings in mice and monkeys with prior published data in women. Methods Eleven adult female rhesus macaques were fed 400 μg/kg deuterated BPA (dBPA) daily for 7 days. Levels of serum dBPA were analyzed by isotope-dilution liquid chromatography–mass spectrometry (0.2 ng/mL limit of quantitation) over 24 hr on day 1 and on day 7. The same dose of BPA was fed to adult female CD-1 mice; other female mice were administered 3H-BPA at doses ranging from 2 to 100,000 μg/kg. Results In monkeys, the maximum unconjugated serum dBPA concentration of 4 ng/mL was reached 1 hr after feeding and declined to low levels by 24 hr, with no significant bioaccumulation after seven daily doses. Mice and monkeys cleared unconjugated serum BPA at virtually identical rates. We observed a linear (proportional) relationship between administered dose and serum BPA in mice. Conclusions BPA pharmacokinetics in women, female monkeys, and mice is very similar. By comparison with approximately 2 ng/mL unconjugated serum BPA reported in multiple human studies, the average 24-hr unconjugated serum BPA concentration of 0.5 ng/mL in both monkeys and mice after a 400 μg/kg oral dose suggests that total daily human exposure is via multiple routes and is much higher than previously assumed.

Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression.

Bisphenol A (BPA) is a plasticizer and an endocrine-disrupting chemical. It is present in a variety of products used daily including food containers, paper, and dental sealants and is now widely detected in human urine and blood. Exposure to BPA during development may affect brain organization and behavior, perhaps as a consequence of its actions as a steroid hormone agonist/antagonist and/or an epigenetic modifier. Here we show that BPA produces transgenerational alterations in genes and behavior. Female mice received phytoestrogen-free chow with or without BPA before mating and throughout gestation. Plasma levels of BPA in supplemented dams were in a range similar to those measured in humans. Juveniles in the first generation exposed to BPA in utero displayed fewer social interactions as compared with control mice, whereas in later generations (F(2) and F(4)), the effect of BPA was to increase these social interactions. Brains from embryos (embryonic d 18.5) exposed to BPA had lower gene transcript levels for several estrogen receptors, oxytocin, and vasopressin as compared with controls; decreased vasopressin mRNA persisted into the F(4) generation, at which time oxytocin was also reduced but only in males. Thus, exposure to a low dose of BPA, only during gestation, has immediate and long-lasting, transgenerational effects on mRNA in brain and social behaviors. Heritable effects of an endocrine-disrupting chemical have implications for complex neurological diseases and highlight the importance of considering gene-environment interactions in the etiology of complex disease.

Estrogen Receptor Function as Revealed by Knockout Studies: Neuroendocrine and Behavioral Aspects☆

Estrogens are an important class of steroid hormones, involved in the development of brain, skeletal, and soft tissues. These hormones influence adult behaviors, endocrine state, and a host of other physiological functions. Given the recent cloning of a second estrogen receptor (ER) cDNA (the ER beta), work on alternate spliced forms of ER alpha, and the potential for membrane estrogen receptors, an animal with a null background for ER alpha function is invaluable for distinguishing biological responses of estrogens working via the ER alpha protein and those working via another ER protein. Data generated to date, and reviewed here, indicate that there are profound ramifications of the ER alpha disruption on behavior and neuroendocrine function. First, data on plasma levels of estradiol (E2), testosterone (T), and luteinizing hormone (LH) in wild-type (WT) versus ER alpha- mice confirm that ER alpha is essential in females for normal regulation of the hypothalamic-pituitary gonadal axis. Second, ovariectomized female ER alpha- mice do not display sexual receptivity when treated with a hormonal regime of estrogen and progesterone that induces receptivity in WT littermates. Finally, male sexual behaviors are disrupted in ER alpha- animals. Given decades of data on these topics our findings may seem self-evident. However, these data represent the most direct test currently possible of the specific role of the ER alpha protein on behavior and neuroendocrinology. The ER alpha- mouse can be used to ascertain the specific functions of ER alpha, to suggest functions for the other estrogen receptors, and to study indirect effects of ER alpha on behavior via actions on other receptors, neurotransmitters, and neuropeptides.

Paracrine Regulation of Epithelial Progesterone Receptor by Estradiol in the Mouse Female Reproductive Tract

Abstract Regulation of progesterone receptor (PR) by estradiol-17β (E2) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E2 induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor α (ERα) knockout (ERKO) mice showed that ERα is essential for E2-induced PR expression in both vaginal epithelium and stroma, and for E2-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E2 was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E2 only in wild-type epithelium and/or stroma. Hence, in vagina, E2 induces PR directly via ERα within the tissue. Conversely, E2 down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E2 requires stromal, but not epithelial, ERα. In vitro, isolated uterine epithelial cells retained a high PR level with or without E2, which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E2 down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERα.

Estrogen Receptor α Has a Functional Role in the Mouse Rete Testis and Efferent Ductules

Abstract Previous studies of the estrogen receptor-alpha knockout (αERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the αERKO mouse lacks a functional estrogen receptor alpha (ERα) throughout development, it was not known whether the morphological and physiological abnormalities observed in the αERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ERα in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in αERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent αERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in αERKO siblings of the same age. Based on this study, we conclude that ERα has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the αERKO mouse is likely the result of a concurrent response to the lack of functional ERα, and not solely due to the lack of ERα during early developmental times.