Shinichi Miyagawa

Antidepressants are environmental contaminants widely prevalent in the environment that can affect aquatic organisms, but their underlying molecular mechanisms and species-specific sensitivities remain poorly understood. To address this knowledge gap, we performed a comprehensive molecular and pharmacological characterization of monoamine transporters (MATs), the primary targets of these pharmaceuticals, from two phylogenetically distinct teleost fish, medaka (Oryzias latipes) and ayu (Plecoglossus altivelis). We first cloned the cDNAs for two serotonin transporters (SERTa and SERTb), a dopamine transporter (DAT), and a norepinephrine transporter (NET) from both species. Pharmacological assays showed that fish SERTa is substantially more sensitive than SERTb to several antidepressants. While the overall inhibitory profiles of the antidepressants were generally conserved across species, fish SERTa orthologs were consistently and markedly more sensitive to human-targeted pharmaceuticals than the human SERT ortholog. Notably, inhibitory effects were also observed for several pharmaceuticals not traditionally recognized as targeting MATs in human. These molecular mechanisms findings help illustrate the vulnerability of aquatic wildlife to environmentally relevant concentrations of these pharmaceuticals.

Background: Chronic inflammation contributes to the development of lifestyle-related diseases, and dietary phytochemicals are recognized as important modulators of inflammatory responses. However, the synergistic anti-inflammatory effects of phytochemical combinations and their underlying mechanisms remain insufficiently understood. Methods: The anti-inflammatory activities of menthol (ME), 1,8-cineole (CI), β-eudesmol (EU), and capsaicin (CA) were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Pro-inflammatory gene expression was quantified by quantitative PCR, intracellular Ca²⁺ signaling was assessed by calcium imaging, and the involvement of transient receptor potential (TRP) channels was examined using selective inhibitors. Synergistic effects were analyzed based on changes in half-maximal effective concentrations (EC₅₀). Results: All compounds suppressed LPS-induced pro-inflammatory genes, including tumor necrosis factor-alpha (Tnf) and interleukin-6 (Il6), in a dose-dependent manner, with CA showing the lowest EC₅₀ for Tnf expression (0.087 µM). Notably, combinations of CA with ME or CI exhibited strong synergy, reducing their EC₅₀ values by 699-fold and 154-fold, respectively, without cytotoxicity. These effects likely resulted from the synergic interaction between ME/CI-induced TRP-mediated signaling and CA-activated, TRP-independent signaling. Conclusions: Specific combinations of plant-derived functional components can markedly enhance anti-inflammatory efficacy, supporting dietary strategies that harness multiple phytochemicals for inflammation control and disease prevention.

Constitutive stimulation of estrogen signaling in mice causes dilated uterine glands with activation of epithelial cell proliferation. In estrogen receptor β knockout (Esr2 KO) mice, cell proliferation in the uterine epithelium is permanently stimulated; therefore, we histologically analyzed the uterine structure in Esr2 KO mice with age. In the uterus of Esr2 KO mice, dilation of the uterine glands was accelerated and the collagen was accumulated in the stroma. The uterine glands were dilated with age even in wild-type (WT) mice; however, Esr2 KO accelerated the dilation of uterine glands quantitatively. The expression of FOXA2 transcription factor, which is essential for uterine glandular function, was diminished in dilated uterine glands of WT and Esr2 KO mice and decreased in the uterine glands of normal size in 12-month-old Esr2 KO mice. To investigate mechanisms of the collagen accumulation in the Esr2 KO uterus, we focused on collagen synthesis and degradation. In the uterine stroma of Esr2 KO mice, MMP8 expression in whole uteri and the number of MMP8-expressing macrophages were decreased. An analysis of the comprehensive gene expression suggested that increased expression of fibroblast growth factors and decreased expression of an aquaporin may be related to the dilation of uterine glands in Esr2 KO mice, and reduced infiltration or differentiation into the macrophages with MMP8 expression may be involved with the collagen accumulation in Esr2 KO mice with age. Taken together, the absence of ESR2 constitutively disrupts estrogen signaling and promotes aging in the uterus via stimulation of epithelial cell proliferation and a decrease of MMP8-expressing macrophages.

Among amniotes, reptiles are ectothermic and are clearly distinguished from mammals and birds. Reptiles show great diversity not only in species numbers, but also in ecological and physiological features. Although their physiological diversity is an interesting research topic, less effort has been made compared to that for mammals and birds, in part due to lack of established experimental models and techniques. However, progress, especially in the field of neuroendocrinology, has been steadily made. With this process, basic data on selected reptilian species have been collected. This review article presents the progress made in the last decade, which includes 1) behavioral regulation by sex steroid hormones, 2) regulation of seasonal reproduction by melatonin and GnRH, and 3) regulation of social interaction by arginine vasotocin. Through these research topics, we provide insights into the physiology of reptiles and the latest findings in the field of amniote neuroendocrinology.

Estrogens play important roles in uterine growth and homeostasis through estrogen receptors (ESR1 and ESR2). To address the role of ESR1-mediated tissue events in the murine uterus, we analyzed mice with a mesenchymal tissue-specific knockout of Esr1. Isl1-driven Cre expression generated Esr1 deletion in the uterine stroma and endometrium (Isl-Esr1KO). We showed that overall structure of the Isl1-Esr1KO mouse uterus developed normally, but estrogen responsiveness and subsequent growth were defective, suggesting that mesenchymal ESR1 is necessary for both epithelial and mesenchymal cell proliferation. Furthermore, RNA-seq analysis revealed that the majority of estrogen-induced genes were regulated by stromal ESR1. In control mice, E2 administration induced 9476 up-regulated differentially expressed genes (DEGs), whereas only 1801 up-regulated DEGs were induced by E2 in Isl1-Esr1KO mice. We further showed that stromal ESR1-regulated genes in the mouse uterus included several growth factors and cytokines, which are potential factors that regulate epithelial and stromal tissue interaction, and also genes involved in lipid homeostasis. Therefore, we infer that stromal ESR1 expression is indispensable for most estrogen actions in the mouse uterus and the current results provide new insights into estrogen-mediated homeostasis in female reproductive organs.