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.     

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.     

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.     

The expression of α-internexin and peripherin in the developing mouse pineal gland

Summary The mammalian pineal gland contains pinealocytes, interstitial glial cells, perivascular macrophages, neurons and neuron-like cells. The neuronal identity of neurons and neuron-like cells was an enigma. α-Internexin and peripherin are specific neuronal intermediate filament proteins and are expressed differentially in the CNS and PNS. We investigated the development of immunoreactivity and expression patterns of mRNAs for α-internexin and peripherin in the mouse pineal gland to determine the neuronal identity of these cells. Both α-internexin- and peripherin-immunoreactive cells were readily visualized only after birth. Both proteins were at the highest level on the postnatal day 7 (P7), rapidly declined at P14, and obtained their adult level at P21. Both protein and mRNA of α-internexin are expressed in some cells and nerve processes, but not all, of adult mouse pineal gland. Less number of peripherin immunoreactive or RNA-expressing cells and nerve processes were identified. Accumulations of α-internexin and peripherin proteins were also found in the cells from the aged pineal gland (P360). We concluded that some cells in the developing mouse pineal gland may differentiated into neurons and neuron-like cells expressing both α-internexin and/or peripherin only postnatally, and these cells possess dual properties of CNS and PNS neurons in nature. We suggested that they may act as interneurons between the pinealocyte and the distal neurons innervating the pinealocytes, or form a local circuitry with pinealocytes to play a role of paracrine regulatory function on the pinealocytes.     

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.     

Ontogenetic expression of CART-peptides in the central nervous system and the periphery: a possible neurotrophic role?

Little attention has been devoted to the expression of CART during development. However, a few studies in the central nervous system and periphery provide a clear indication that these peptides may play significant roles during histogenesis, and may have trophic actions.     

Subcellular localization of β-catenin and cadherin expression in the cap-stage enamel organ of the mouse molar

We analyzed the subcellular distribution of -catenin in the cap-stage enamel organ and compared it with the expression of E- and P-cadherin by using confocal laser microscopy. The amounts of the molecules in the cytoplasm and the nucleus showed regional variations in the enamel organ, whereas cell surface-associated -catenin was ubiquitous. In both the enamel knot and the inner dental epithelium, -catenin was detected in the cytoplasm and in the nucleus. However, the amount of nuclear -catenin was apparently higher in the enamel knot than in the inner dental epithelium. P-cadherin also gave a stronger signal in the enamel knot than in other parts of the enamel organ. In the stellate reticulum, where E-cadherin was preferentially expressed, as well as in the cervical loop and outer dental epithelium, -catenin was localized in the cytoplasm but not in the nucleus. The nuclear localization of -catenin in the enamel knot suggests a specific activation of the canonical Wnt signaling pathway. A coincident upregulation of P-cadherin was observed in this area. Altogether, these observations suggest the possibility of a linkage between cell adhesion and Wnt signaling in the enamel knot.     

Developmental changes in functional expression and β-adrenergic regulation of If in the heart of mouse embryo

The hyperpolarization-activated current (If) plays an important role in determining the spontaneousrate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart,therefore we studied developmental changes in functional expression and β-adrenergic regulation of If inembryonic mouse heart. The expression of If is high in early developmental stage (EDS) (10.5 d after coitus)ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytesand even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating thatthese cells of the EDS embryonic heart have some properties of pacemaker cells. β-adrenergic agonistisoproterenol (ISO) stimulates If in LDS but not in EDS cardiomyocytes, indicating that theβ-adrenergicregulation of If is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase)and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of If in EDS cells,indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore,the results demonstrate that If is modulated by phosphorylation via cAMP dependent PKA both in EDSand LDS cells.