Contributions of estrogen receptor-α and estrogen receptor-β to the regulation of behavior

Studies of the mechanisms by which estrogens influence brain function and behavior have advanced from the explication of individual hormone receptors, neural circuitry and individual gene expression. Now, we can report patterns of estrogen receptor subtype contributions to patterns of behavior. Moreover, new work demonstrates important contributions of nuclear receptor coactivator expression in the central nervous system. In this paper, our current state of knowledge is reviewed.     

Immunohistochemical localization of estrogen receptor-α in sex ducts and gonads of newborn piglets

Estrogen receptor-alpha (ER-alpha) expression in piglet uteri has previously been reported from day 15 after birth. Nevertheless, uterine tissue has been reported to be estrogen sensitive from the day of birth. Since estrogen action in the uterine tissue is suggested to be mediated principally by ER-alpha, the present study aimed to evaluate the presence of ER-alpha in uteri of 1- to 2-day-old piglets by means of immunohistochemistry. In addition, sex ducts and gonads of both sexes were examined. The results clearly demonstrate the presence of ER-alpha immunopositive cells in uterine tissue, which explains its estrogen responsiveness. Immunostaining was most intense in the glandular epithelial cells and is suggested to indicate participation of ER-alpha in adenogenesis. In oviducts, almost all epithelial cells were immunostained moderately positive, while the stroma cells were stained comparably more positive. The functional significance of this intensity difference is uncertain but could indicate that part of the estrogen action on the epithelium is mediated through the stroma cells, as is known for the uterus. In ovaries, the surface epithelium and stroma cells were immunostained, whereas germ and granulosa cells were immunonegative. It is speculated that ER-alpha might be involved in yet unknown intraovarian mechanisms. In male sex ducts, immunostaining was virtually confined to the epithelium of efferent ducts. All cells in the epididymis as well as in vas deferens were immunonegative. The unique presence of ER-alpha in efferent ducts corresponds with localization in other species, where it has been shown to be involved in fluid reabsorption. The obtained data on localization of ER-alpha correspond with the present knowledge, obtained in ER-alpha knockout mice, of the biological function of ER-alpha within male and female gonads and sex ducts.     

Is regulation of aromatase expression in reptiles the key to understanding temperature-dependent sex determination?

A brief review of our current understanding (or lack of understanding) of the molecular basis of temperature-dependent sex determination (TSD) in reptiles is presented. Current theories are discussed: yolk steroids as sex determinants, the brain as the driver for TSD and the enzyme aromatase and estrogen production as the possible determinants of sex. There is little evidence to support the first two theories, but enough evidence to keep the third theory in play. As yet, however, we have no molecular understanding of how a two-degree difference in temperature during the temperature-sensitive phase of egg incubation can initiate the molecular cascade that determines whether the indifferent gonad develops as an ovary or a testis.     

Serotonin receptor expression along the dorsal–ventral axis of mouse hippocampus

Using in situ hybridization, we describe, for the first time, the profiles of expression of serotonin receptors (Htr/5-HTR) along the dorsal–ventral axis of mouse hippocampus. cRNA probes for most Htrs, excluding Htr6, were used. All hippocampal subregions and the entorhinal cortex cells providing input into the hippocampus were examined. The study shows that some, but not all, Htrs are expressed in the cells of the hippocampal circuitry. At both the subfield and the cell type levels, a somewhat overlapping pattern is observed. Four serotonin receptors, Htr1a, Htr2a, Htr2c and Htr7, display an expression pattern that changes along the dorsal–ventral axis of the hippocampus. Given the proposed functional differentiation of the hippocampus along its long axis, with the dorsal pole more involved in cognitive functions and the ventral pole more involved in mood and anxiety, our results suggest that serotonin receptors enriched in the ventral pole probably contribute to mood- and anxiety-related behaviours.     

Neuregulin 1-β regulates cell adhesion molecule L1 expression in the cortex and hippocampus of mice

Neuregulin 1 (Nrg1) functions in neuronal migration, survival and differentiation as well as synaptogenesis during ontogenetic development and maintenance of synaptic functions in the adult mammalian brain. The neural adhesion molecule L1 (L1CAM) functions in similar overlapping, but also non-overlapping roles in the nervous system. In the present study, we therefore investigated some aspects of the functional relationship between Nrg1 and L1 in mammalian neural cells. Nrg1 regulates the expression of L1 in cultures of both human neuroblastoma SK-N-SH cells and mouse cortical and hippocampal neurons. To analyze the role of Nrg1 on L1 expression in vivo, young adult male mice received intraperitoneal injections of Nrg1 or PBS (vehicle control). The correlation between Nrg1 and L1 expression was tested by qPCR, Western blot analysis, and immunocytology. Our data indicate that neuregulin 1-β (Nrg1β) increases L1 expression in neurons of the cerebral cortex, and decreases expression in neurons of the hippocampus in vitro and in vivo. In addition, Nrg1 induces phosphorylation of its receptors, ErbB2 and ErbB4, the predominant ErbB receptors in the nervous system. These results show that Nrg1β affects expression of L1 in the central nervous system and in parallel activates the ErbB receptors for Nrg1, suggesting a crosstalk between molecules that are of prime importance for nervous system functions.     

Sexually dimorphic estrogen receptor α mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards

Estradiol (E2) is important in activation of male reproductive behaviors, and masculinizes morphology of associated brain regions in a number of mammalian and avian species. In contrast, it is testosterone, rather than its metabolites, that is the most potent activator of male sexual behavior in green anole lizards. As in other vertebrate groups, however, E2 is critical for receptivity in females of this species. Aromatase, the enzyme which converts testosterone to E2, is more active in the male than female green anole brain, and appears to be actively regulated on a seasonal basis, suggesting some role for E2 in males. This study was designed to enhance our understanding of potential E2 actions by localizing and quantifying relative levels of estrogen receptor-alpha (ERα) mRNA in forebrain regions involved in masculine and feminine behaviors in anoles. These areas include the preoptic area (POA), ventromedial amygdala (AMY) and ventromedial hypothalamus (VMH). In situ hybridization was conducted in adult males and females collected during both breeding and non-breeding seasons. ERα mRNA was expressed in each brain region across sexes and seasons. However, expression was up to 3 times greater in the VMH compared to the POA and AMY. In the POA and VMH, expression was higher in females compared to males, independent of season. The increased receptor expression in females is consistent with E2 playing a larger role in female than male reproductive behaviors.     

Effects of lead exposure on the expression of amyloid β and phosphorylated tau proteins in the C57BL/6 mouse hippocampus at different life stages

The objective of this study was to investigate the effects of lead exposure on spatial learning and memory capacity and the expression of amyloid β and phosphorylated tau proteins in the mouse hippocampus. A total of 24 adult C57BL/6 mice (12 of each sex) were mated at a 1:1 ratio. After delivery, the litters were normalised to 6 pups per litter. During the lactation period, the pups were randomly separated into four groups: control, early exposure, late exposure, or long-term exposure. These groups were not exposed to lead, exposed to lead from birth to week 24, exposed to lead from week 24 to week 48, or exposed to lead from birth to 48 weeks of age, respectively. Lead exposure was induced by providing Pb-contaminated drinking water at a concentration of 0.1%. All of the pups were fed until 72 weeks of age, at which time their spatial learning and memory capacity was evaluated via the Morris water maze test. Then, the lead levels in their blood and hippocampus were measured via graphite furnace atomic absorption spectrometry. The protein expression of amyloid β and phosphorylated tau in the hippocampus was detected via Western blot. The results revealed that the hippocampal and blood lead levels were significantly higher in all of the groups exposed to lead than the control group (P < 0.05). The spatial learning and memory performances of the lead-exposed groups were much poorer than those of the control group (P < 0.05). The expression levels of amyloid β and phosphorylated tau proteins were increased in the lead-exposed groups compared to the control group (P < 0.05). The enhanced expressions of amyloid β and phosphorylated tau proteins might contribute to the impairment in spatial learning and memory in the lead-exposed mice.     

Molecular cloning and expression of TRα and TRβ in the protandrous cinnamon clownfish,Amphiprion melanopus during sex reversal

We divided the process of sex reversal from immature male to mature female in the protandrous cinnamon clownfish (Amphiprion melanopus) into six developmental stages as follows: I, immature male; II, mature male; III, male at 60 days after female removal; IV, male at 90 days after female removal; V, male at 120 days after female removal; and VI, mature female. Thyroid hormone receptors α (TRα) and β (TRβ) cDNAs were cloned from the ovary and mRNA expression levels were compared during the sex-reversal process. The nucleotide sequences of the TRα and TRβ cDNA were 1230 and 1188 bp in length with open reading frames encoding peptides of 409 and 395 amino acids, respectively. We observed that TRα mRNA and protein levels were high in all stages except the immature gonad, while TRβ mRNA levels were higher in the mature ovary than in any other gonadal stage. We injected gonadotropin-releasing hormone analogue to identify its effects on TRs mRNA in immature fish. The mRNA levels of TRs increased significantly. We therefore propose that TRs are related to testicular development as well as ovarian development in cinnamon clownfish. The present study also provides basic data on the role of TRs during sex reversal in fish.     

Altered responsiveness to TGF-β results in reduced Papss2 expression and alterations in the biomechanical properties of mouse articular cartilage

Introduction

Previous studies have indicated that transforming growth factor β (TGF-β) signaling has a critical role in cartilage homeostasis and repair, yet the mechanisms of TGF-β''s chondroprotective effects are not known. Our objective in this study was to identify downstream targets of TGF-β that could act to maintain biochemical and biomechanical properties of cartilage.

Methods

Tibial joints from 20-week-old mice that express a dominant-negative mutation of the TGF-β type II receptor (DNIIR) were graded histologically for osteoarthritic changes and tested by indentation to evaluate their mechanical properties. To identify gene targets of TGF-β, microarray analysis was performed using bovine articular chondrocytes grown in micromass culture that were either treated with TGF-β or left untreated. Phosphoadenosine phosphosynthetase 2 (PAPSS2) was identified as a TGF-β-responsive gene. Papss2 expression is crucial for proper sulfation of cartilage matrix, and its deficiency causes skeletal defects in mice and humans that overlap with those seen in mice with mutations in TGF-β-signaling genes. Regulation of Papss2 was verified by real time RT-PCR and Western blot analyses. Alterations in sulfation of glycosaminoglycans were analyzed by critical electrolyte concentration and Alcian blue staining and immunofluorescence for chondroitin-4-sulfate, unsulfated chondroitin and the aggrecan core protein.

Results

DNIIR mutants showed reduced mechanical properties and osteoarthritis-like changes when compared to wild-type control mice. Microarray analysis identified a group of genes encoding matrix-modifying enzymes that were regulated by TGF-β. Papss2 was upregulated in bovine articular chondrocytes after treatment with TGF-β and downregulated in cartilage from DNIIR mice. Articular cartilage in DNIIR mice demonstrated reduced Alcian blue staining at critical electrolyte concentrations and reduced chondroitin-4-sulfate staining. Staining for unsulfated chondroitin sulfate was increased, whereas staining for the aggrecan core protein was comparable in DNIIR and wild-type mice.

Conclusion

TGF-β maintains biomechanical properties and regulates expression of Papss2 and sulfation of glycosaminoglycans in mouse articular cartilage.