Tomomi Sato

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.

Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix–loop–helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.

In the mouse ovary, interactions between oocytes and somatic cells are essential for folliculogenesis and subsequent follicle development. The polyovular follicle (PF), which contains more than two oocytes in a follicle, can be induced in the neonatal mouse ovary when interactions between oocytes and somatic cells are disrupted by agents such as the potent synthetic estrogen diethylstilbestrol (DES) acting through estrogen receptor (ER) β. Hedgehog signaling is known to regulate granulosa cell proliferation, thecal cell differentiation, and follicle growth. To investigate the role of hedgehog signaling in the early folliculogenesis and in PF induction by DES, neonatal mouse ovaries were cultured with or without 10 μM cyclopamine (CPA), an inhibitor of hedgehog signaling, and grafted under the kidney capsule of adult ovariectomized host mice. The number and the incidence of PFs were significantly increased in organ-cultured ovaries post-grafting. Expression of procollagen type IV, alpha 1 (Col4a1) in organ-cultured ovaries was significantly reduced by CPA, but not by DES. The expression of two hedgehog ligands, Desert hedgehog (Dhh) and Indian hedgehog (Ihh), and a target gene, Hedgehog interacting protein (Hhip), was significantly increased by DES both in WT and ERβ KO mice. Therefore, we infer that DES can affect expression of those genes through ERα but not via suppression of hedgehog signaling. Thus, PFs are induced by DES or CPA, but the induction mechanism is different. Our results revealed an important role of hedgehog signaling in basement membrane remodeling during folliculogenesis even before thecal cell differentiation.

Background/Aim: Perinatal diethylstilbestrol (DES) treatment induces the polyovular follicle containing two or more oocytes in a follicle of mouse ovary through estrogen receptor (ER) β. The aim of the study was to investigate the direct effects of DES on the neonatal mouse ovary and the gene expression of activins. Materials and Methods: Ovaries from neonatal wild-type (WT) or ERβ- knockout (ERβKO) mice were organ-cultured in a serum-free medium with or without DES, and polyovular follicle induction and expression of activin signaling related genes were examined. Results: The polyovular follicle and cyst incidence in DES-treated organ-cultured ovaries from WT mice, but not from ERβKO mice, was significantly higher than that of control non-treated cultures. DES altered inhibin (Inh) a, Inhba and Inhbb expression in organ-cultured ovaries from C57BL/6J mice, while no change in Inha and an increase of Inhbb were observed by DES, in both WT and ERβKO mice. Conclusion: Alterations in activin signaling are involved in the polyovular follicle induction by DES.