Transcriptome analysis of East Asian common octopus, Octopus sinensis, paralarvae.

BACKGROUND: The genes involved in cephalopod development and their association with hatching and survival during early life stages have been extensively studied. However, few studies have investigated the paralarvae transcriptome of the East Asian common octopus (Octopus sinen sis). OBJECTIVE: This study aimed to identify the genes related to embryonic development and hatching in O. sinensis using RNA sequencing (RNA-seq) and verify the genes most relevant to different embryonic stages. METHODS: RNA samples from hatched and 25 days post-hatching (dph) O. sinensis paralarvae were used to construct cDNA libraries. Clean reads from individual samples were aligned to the reference O. sinensis database to identify the differentially expressed genes (DEGs) between the 0- and 25-dph paralarvae libraries. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to supplement the RNA-seq data for embryogenic developmental stages. RESULTS: A total of 12,597 transcripts were annotated and 5,468 DEGs were identified between the 0- and 25-dph O. sinensis paralarvae, including 2,715 upregulated and 2,753 downregulated transcripts in the 25-dph paralarvae. Several key DEGs were related to transmembrane transport, lipid biosynthesis, monooxygenase activity, lipid transport, neuropeptide signaling, transcription regulation, and protein-cysteine S-palmitoyltransferase activity during the post-hatching development of O. sinensis paralarvae. RT-qPCR analysis further revealed that SLC5A3A, ABCC12, and NPC1 transcripts in 20 and/or 30 days post-fertilization (dpf) embryos were significantly higher (p < 0.05) than those in 10-dpf embryos. CONCLUSION: Transcriptome profiles provide molecular targets to understand the embryonic development, hatching, and survival of O. sinensis paralarvae, and enhance octopus production.

Characterization of myoinhibitory peptide signaling system and its implication in larval metamorphosis and spawning behavior in Pacific abalone.

Myoinhibitory peptides (MIPs) affect various physiological functions, including juvenile hormone signaling, muscle contraction, larval development, and reproduction in invertebrates. Although MIPs are ligands for MIP and/or sex peptide receptors (MIP/SPRs) in diverse arthropods and model organisms belonging to Lophotrochozoa, the MIP signaling system has not yet been fully investigated in mollusks. In this study, we identified the MIP signaling system in the Pacific abalone Haliotis discus hannai (Hdh). Similar to the invertebrate MIPs, a total of eight paracopies of MIPs (named Hdh-MIP1 to Hdh-MIP8), harboring a WX5-7Wamide motif, except for Hdh-MIP2, were found in the Hdh-MIP precursor. Furthermore, we characterized a functional Hdh-MIPR, which responded to the Hdh-MIPs, except for Hdh-MIP2, possibly linked with the PKC/Ca2+ and PKA/cAMP signaling pathways. Hdh-MIPs delayed larval metamorphosis but increased the spawning behavior. These results suggest that the Hdh-MIP signaling system provides insights into the unique function of MIP in invertebrates.

Functional Characterization of Gonadotropin-Releasing Hormone and Corazonin Signaling Systems in Pacific Abalone: Toward Reclassification of Invertebrate Neuropeptides.

INTRODUCTION: The proposed evolutionary origins and corresponding nomenclature of bilaterian gonadotropin-releasing hormone (GnRH)-related neuropeptides have changed tremendously with the aid of receptor deorphanization. However, the reclassification of the GnRH and corazonin (CRZ) signaling systems in Lophotrochozoa remains unclear. METHODS: We characterized GnRH and CRZ receptors in the mollusk Pacific abalone, Haliotis discus hannai (Hdh), by phylogenetic and gene expression analyses, bioluminescence-based reporter, Western blotting, substitution of peptide amino acids, in vivo neuropeptide injection, and RNA interference assays. RESULTS: Two Hdh CRZ-like receptors (Hdh-CRZR-A and Hdh-CRZR-B) and three Hdh GnRH-like receptors (Hdh-GnRHR1-A, Hdh-GnRHR1-B, and Hdh-GnRHR2) were identified. In phylogenetic analysis, Hdh-CRZR-A and -B grouped within the CRZ-type receptors, whereas Hdh-GnRHR1-A/-B and Hdh-GnRHR2 clustered within the GnRH/adipokinetic hormone (AKH)/CRZ-related peptide-type receptors. Hdh-CRZR-A/-B and Hdh-GnRHR1-A were activated by Hdh-CRZ (pQNYHFSNGWHA-NH2) and Hdh-GnRH (pQISFSPNWGT-NH2), respectively. Hdh-CRZR-A/-B dually coupled with the Gαq and Gαs signaling pathways, whereas Hdh-GnRHR1-A was linked only with Gαq signaling. Analysis of substituted peptides, [I2S3]Hdh-CRZ and [N2Y3H4]Hdh-GnRH, and in silico docking models revealed that the N-terminal amino acids of the peptides are critical for the selectivity of Hdh-CRZR and Hdh-GnRHR. Two precursor transcripts for Hdh-CRZ and Hdh-GnRH peptides and their receptors were mainly expressed in the neural ganglia, and their levels increased in starved abalones. Injection of Hdh-CRZ peptide into abalones decreased food consumption, whereas Hdh-CRZR knockdown increased food consumption. Moreover, Hdh-CRZ induced germinal vesicle breakdown in mature oocytes. CONCLUSION: Characterization of Hdh-CRZRs and Hdh-GnRHRs and their cognate peptides provides new insight into the evolutionary route of GnRH-related signaling systems in bilaterians.

Functional analysis of LFRFamide signaling in Pacific abalone, Haliotis discus hannai.

The invertebrate LFRFamide (LFRFa) and short neuropeptide F (sNPF), consisting of 6 to 10 amino acids, are orthologs for bilaterian NPF/Y, which consist of 36 to 40 amino acids. Recently, a molluscan G protein-coupled receptor (GPCR) for NPF was characterized in Pacific abalone (Haliotis discus hannai). To address the functional evolutionary route of the invertebrate LFRFa and NPF signaling system, in this study, we identified cDNAs encoding LFRFa precursors and the sNPF receptor (Hdh-sNPFR) in Pacific abalone. Four LFRFa mature peptides with 6 or 7 amino acids were predicted: GSLFRFa, GGLFRFa, GTLFRFa, and GSTLFRFa. Hdh-sNPFR was identified as a classical rhodopsin-like GPCR and classified into a molluscan sNPFR group. In HEK293 cells, Hdh-sNPFR was mainly localized in the cell membranes and internalized in the cytoplasm following treatment with LFRFa peptides. Reporter assays demonstrated that LFRFa peptides inhibit forskolin-stimulated cAMP accumulation in Hdh-sNPFR-expressing HEK293 cells. LFRFa precursor and Hdh-sNPFR transcripts were more strongly expressed in the cerebral and pleural-pedal ganglia of Pacific abalone than in the peripheral tissues such as the ovary, gills, intestine, and hepatopancreas. The levels of LFRFa transcripts in the ovary, intestine, and hepatopancreas were significantly higher in mature female abalone than in immature females. Injection of LFRFa induced the egg release and spawning behavior of mature abalone, but suppressed food intake. These results suggest that LFRFa peptides are endogenous ligands for Hdh-sNPFR involved in food intake and reproduction through a Gαi-protein dependent signaling pathway.

Mechanistic target of rapamycin (mTOR) implicated in plasticity of the reproductive axis during social status transitions.

The highly conserved brain-pituitary-gonadal (BPG) axis controls reproduction in all vertebrates, so analyzing the regulation of this signaling cascade is important for understanding reproductive competence. The protein kinase mechanistic target of rapamycin (mTOR) functions as a conserved regulator of cellular growth and metabolism in all eukaryotes, and also regulates the reproductive axis in mammals. However, whether mTOR might also regulate the BPG axis in non-mammalian vertebrates remains unexplored. We used complementary experimental approaches in an African cichlid fish, Astatotilapia burtoni, to demonstrate that mTOR is involved in regulation of the brain, pituitary, and testes when males rise in rank to social dominance. mTOR or downstream components of its signaling pathway (p-p70S6K) were detected in gonadotropin-releasing hormone (GnRH1) neurons, the pituitary, and testes. Transcript levels of mtor in the pituitary and testes also varied when reproductively-suppressed subordinate males rose in social rank to become dominant reproductively-active males, a transition similar to puberty in mammals. Intracerebroventricular injection of the mTORC1 inhibitor, rapamycin, revealed a role for mTOR in the socially-induced hypertrophy of GnRH1 neurons. Rapamycin treatment also had effects at the pituitary and testes, suggesting involvement of the mTORC1 complex at multiple levels of the reproductive axis. Thus, we show that mTOR regulation of BPG function is conserved to fishes, likely playing important roles in regulating reproduction and fertility across all male vertebrates.

Molecular characterization, expression analysis, and functional properties of multiple 5-hydroxytryptamine receptors in Pacific abalone (Haliotis discus hannai).

Neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT) in the central nervous system regulate diverse physiological functions, including reproduction, feeding, learning, and memory, in diverse animal phyla. 5-HT and the 5-HT1 subtype receptor play important roles in sexual maturation and in the initiation of gamete release in mollusks. However, little is known about the involvement of other 5-HT receptor subfamilies in the reproduction process. In the present study, we identified the cDNAs encoding eight subtypes of 5-HT receptors from the ganglia tissues of the Pacific abalone Haliotis discus hannai (Mollusca; Gastropoda; Haliotidae), and examined the gonadal expression of the transcripts of 5-HT receptors. A phylogenetic analysis indicated that the molluskan 5-HT receptors are largely classified into four major clades: 5-HT1/5/7, 5-HT2, 5-HT4, and 5-HT6. Among the H. discus hannai (Hdh) 5-HT1-7 transcripts, Hdh5-HT1B, 4A, 4B, and 6 were the major subtypes detected in the mature ovary. Estradiol-17ß injection into the pedal sinus induced the downregulation of 5-HT4B and upregulation of 5-HT6 transcripts in the ovary of mature abalone within 72 h. In HEK293 cells overexpressing Hdh5-HT1B, forskolin-stimulated cAMP response element luciferase (CRE-Luc) reporter activity was inhibited by 5-HT in a dose-dependent manner, whereas serum response element luciferase (SRE-Luc) activity was not affected. In Hdh5-HT4A-expressing HEK293 cells, forskolin-stimulated CRE-Luc and SRE-Luc reporter activities were both marginally increased by treatment with a high dose of 5-HT. Our results provide new insights into the roles of 5-HT through diverse G protein-coupled 5-HT receptors in the reproductive process of mollusks.

Transcriptional Activity of an Estrogen Receptor ß Subtype in the Medaka Oryzias dancena.

In vertebrate reproductive system, estrogen receptor (ER) plays a pivotal role in mediation of estrogenic signaling pathways. In the present study, we report the cDNA cloning, expression analysis, and transcriptional activity of ERß1 subtype from medaka Oryzias dancena. The deduced O. dancena ERß1 (odERß1; 519 amino acids) contained six characteristic A/B to E/F domains with very short activation function 2 region (called AF2). A phylogenetic analysis indicated that odERß1 was highly conserved among teleost ERß1 subgroup. A conventional RT-PCR revealed that the odERß1 transcripts were widely distributed in the multiple tissues, the ovary, brain, gill, intestine, kidney, and muscle. Further, the relatively higher odERß1 expressions in the ovary and brain were clearly reproduced in RT-qPCR assay. When HA-fused odERß1 expression vector was transfected into HEK293 cells, an immunoreactivity for odERß1 was mainly detected in the nucleus part. Finally, an estrogen responsive element driven luciferase reporter assays demonstrated that the transcriptional activity of odERß1 significantly increased by estradiol-17ß (E2) in a dose dependent manner (p<0.05). However, fold-activation of odERß1 in the presence of E2 was markedly weak, when it compared with those of O. latipes ERß1. Taken together, these data suggest that odERß1 represents a functional variant of teleost ERß subtype and provides a basic tool allowing future studies examining the function of F domain of ERß1 subtype and expanding our knowledge of ERß evolution.

Characterization and spatiotemporal expression of gonadotropin-releasing hormone in the Pacific abalone, Haliotis discus hannai.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in humans and other vertebrates. Recently, GnRH-like cDNAs and peptides were reported in marine mollusks, implying that GnRH-mediated reproduction is an ancient neuroendocrine system that arose prior to the divergence of protostomes and deuterostomes. Here, we evaluated the reproductive control system mediated by GnRH in the Pacific abalone Haliotis discus hannai. We cloned a prepro-GnRH cDNA (Hdh-GnRH) from the pleural-pedal ganglion (PPG) in H. discus hannai, and analyzed its spatiotemporal gene expression pattern. The open reading frame of Hdh-GnRH encodes a protein of 101 amino acids, consisting of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. This structure and sequence are highly similar to GnRH-like peptides reported for mollusks and other invertebrates. Quantitative polymerase chain reaction demonstrated that Hdh-GnRH mRNA was more strongly expressed in the ganglions (PPG and cerebral ganglion [CG]) than in other tissues (gonads, gills, intestine, hemocytes, muscle, and mantle) in both sexes. In females, the expression levels of Hdh-GnRH mRNA in the PPG and branchial ganglion (BG) were significantly higher at the ripe and partial spent stages than at the early and late active stages. In males, Hdh-GnRH mRNA levels in the BG showed a significant increase in the partial spent stage. Unexpectedly, Hdh-GnRH levels in the CG were not significantly different among the examined stages in both sexes. These results suggest that Hdh-GnRH mRNA expression profiles in the BG and possibly the PPG are tightly correlated with abalone reproductive activities.

Identification of Ran-binding protein M as a stanniocalcin 2 interacting protein and implications for androgen receptor activity.

Stanniocalcin (STC), a glycoprotein hormone originally discovered in fish, has been implicated in calcium and phosphate homeostasis. While fishes and mammals possess two STC homologs (STC1 and STC2), the physiological roles of STC2 are largely unknown compared with those of STC1. In this study, we identified Ran-binding protein M (RanBPM) as a novel binding partner of STC2 using yeast two-hybrid screening. The interaction between STC2 and RanBPM was confirmed in mammalian cells by immunoprecipitation. STC2 enhanced the RanBPM-mediated transactivation of liganded androgen receptor (AR), but not thyroid receptor ß, glucocorticoid receptor, or estrogen receptor ß. We also found that AR interacted with RanBPM in both the absence and presence of testosterone (T). Furthermore, we discovered that STC2 recruits RanBPM/AR complex in T-dependent manner. Taken together, our findings suggest that STC2 is a novel RanBPM-interacting protein that promotes AR transactivation.

Production and characterization of recombinant Manchurian trout thyrotropin.

Thyrotropin (thyroid-stimulating hormone, TSH), a heterodimeric glycoprotein hormone produced in the pituitary, stimulates the thyroid gland and release of thyroid hormones. In contrast to a well-known efficacy of recombinant mammalian TSHs, there is no report about the production of teleost recombinant TSH and its biological activity. In this study, we report the production of a single-chain recombinant TSH (mtTSH) of Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The mtTSH was produced in silkworm larvae and characterized as a form of N-linked glycosylation. The cAMP signaling system in transiently transfected COS-7 cells revealed that the mtTSH was recognized by their cognate receptors, salmon TSHα and TSHß receptors, but not LH receptor. The thyrotropic potency of the mtTSH was examined by rainbow trout basibranchial tissues containing thyroid follicles. The height of follicle epithelial cells was significantly increased by treatments of mtTSH in vivo and in vitro. In conclusion, the present study suggests that the mtTSH produced by baculovirus-silkworm larvae is a biologically active recombinant TSH.

9-Cis-retinoic acid induces growth inhibition in retinoid-sensitive breast cancer and sea urchin embryonic cells via retinoid X receptor α and replication factor C3.

There is widespread interest in defining factors and mechanisms that suppress the proliferation of cancer cells. Retinoic acid (RA) is a potent suppressor of mammary cancer and developmental embryonic cell proliferation. However, the molecular mechanisms by which 9-cis-RA signaling induces growth inhibition in RA-sensitive breast cancer and embryonic cells are not apparent. Here, we provide evidence that the inhibitory effect of 9-cis-RA on cell proliferation depends on 9-cis-RA-dependent interaction of retinoid X receptor α (RXRα) with replication factor C3 (RFC3), which is a subunit of the RFC heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp on DNA. An RFC3 ortholog in a sea urchin cDNA library was isolated by using the ligand-binding domain of RXRα as bait in a yeast two-hybrid screening. The interaction of RFC3 with RXRα depends on 9-cis-RA and bexarotene, but not on all-trans-RA or an RA receptor (RAR)-selective ligand. Truncation and mutagenesis experiments demonstrated that the C-terminal LXXLL motifs in both human and sea urchin RFC3 are critical for the interaction with RXRα. The transient interaction between 9-cis-RA-activated RXRα and RFC3 resulted in reconfiguration of the PCNA-RFC complex. Furthermore, we found that knockdown of RXRα or overexpression of RFC3 impairs the ability of 9-cis-RA to inhibit proliferation of MCF-7 breast cancer cells and sea urchin embryogenesis. Our results indicate that 9-cis-RA-activated RXRα suppresses the growth of RA-sensitive breast cancer and embryonic cells through RFC3.

Molecular characterization of tripartite motif protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii.

Tripartite motif-containing 25 (TRIM25), also known as estrogen-responsive finger protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii, designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4-28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo. Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart.

Steroidogenic response of carp ovaries to piscine FSH and LH depends on the reproductive phase.

The gonadotropins (GTHs) follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the key regulators of reproduction. We determined the competence of heterologous recombinant GTHs at eliciting steroid secretion from carp ovaries at different reproductive stages. We collected carp ovaries at: early, mid and end vitellogenesis, when most of the oocytes still contained a germinal vesicle (GV) at a central stage, and mature ovaries with a migrating GV. Plasma estradiol (E2) levels at early vitellogenesis were high and decreased thereafter. Basal secretion levels of E2 increased with oocyte diameter and GSI value, whereas 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) was detected only in females with mature follicles. Carp ovary fragments were exposed to recombinant fish GTHs belonging to different teleost orders: Japanese eel (Anguilla japonica, Anguilliformes), Manchurian trout (Brachymystax lenok, Salmoniformes), and Nile tilapia (Oreochromis niloticus); to mammalian GTHs (pFSH and hCG), or to carp and tilapia pituitary extract (CPE and TPE, respectively). All of the recombinant GTHs tested stimulated steroid secretion. However, the steroid secretion differed according to the type of GTH and the developmental state of the ovary. CPE increased the secretion of both E2 and DHP at almost all stages of ovarian maturity. In mature ovarian fragments, DHP secretion was higher in response to recombinant LHs (eel and tilapia) than to recombinant FSH. Early- and mid-vitellogenic ovaries showed no secretion of DHP and high secretion of E2 in response to all recombinant GTHs tested. This is in line with the hypothesis that LH regulates the final stages of maturation, when the involvement of FSH is marginal. These results may contribute to understanding the mechanisms that determine differential activation of steroid secretion and specificity in fish.

Molecular and expression analysis of the farnesoid X receptor in the urochordate Halocynthia roretzi.

The farnesoid X receptors (FXRs) are the major transcriptional regulators of bile salt synthesis in vertebrates. However, the structural conservation of invertebrate FXRs has only been studied for the major model organisms and studies on additional invertebrate FXRs are clearly required to obtain better resolution of FXR phylogeny and comparative developmental insights in chordates. In the present study, the cDNA encoding the farnesoid X receptor, HrFXR, was cloned from a marine invertebrate Halocynthia roretzi. The open reading frame of HrFXR encoded 688 amino acids including a longer N-terminal region and showed overall sequence identities of 28-41% to vertebrate and Ciona intestinalis FXRs. The N-terminal activation function 1 (AF-1) and hinge domains of HrFXR displayed relatively low identities (<20%), whereas the DNA-binding and ligand-binding domains showed relatively high (>73%) and intermediate (21-50%) identities, respectively. Based on a phylogenetic analysis, HrFXR belonged to a urochordate group, which was placed differently from vertebrate FXRα and FXRß subgroups. Real-time quantitative PCR analysis revealed that the HrFXR mRNA originated maternally and was highly expressed in adult gonads. Additionally, HrFXR mRNA levels in the gills and hepatopancreas showed significantly higher values in animals with soft tunic syndrome compared to those of normal individuals. Furthermore, direct injection of cholic acid significantly increased HrFXR transcript levels in vivo, although an expression vector containing HrFXR cDNA did not show a significant transactivation function in response to a well-known ligand for vertebrate FXR, GW4064, in HepG2 cells. These results suggest that the tunicate FXR has different structural and expressional characteristics compared to those of vertebrate FXRs.

Characterization of steroid receptor coactivator in sea urchin, Strongylocentrotus nudus, and its involvement in embryonic development.

Ligand-bound nuclear receptors (NRs) recruit coactivators such as members of the p160 steroid receptor coactivator (SRC) family and cyclic AMP responsive element binding protein (CREB)-binding protein (CBP) to specific enhancer elements and activate target gene transcription. In the present study, we isolated a novel SRC from the sea urchin Strongylocentrotus nudus (SnSRC) by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. The SnSRC and vertebrate SRCs are different in size but share the overall characteristic domains, such as NR interacting domain (NID), CBP-binding and glutamine-rich regions. SnSRC mRNA showed highest expression levels at the 32-cell, 64-cell and pluteus larval stages. Full-length SnSRC (1992 amino acids) interacted with several NRs, including sea urchin estrogen receptor-related receptor (ERR), human and masu salmon estrogen receptors (ERα), mouse ERRγ, rat glucocorticoid receptor α, and rat thyroid receptor ß. The SnSRC possesses two functional NIDs, both of which are dependent on their core LxxLL motifs. Furthermore, preferential interacting domains for ERα in the SnSRC are located in the central LxxLL motifs, revealed by the truncation and mutagenesis studies. Strikingly, the SnSRC has a single transcription activation domain, which interacts with CBP, a transcriptional integrator. In addition, transient knockdown of the SnSRC gene in the sea urchin embryo using morpholino antisense RNA induced abnormal phenotypes at gastrulation stage such as the lack of primary invagitation and exogastrulation. These results suggest that the SnSRC is a new member of the SRC family and plays an important role during early embryonic development.

Bi-functional induction of the quinone reductase and cytochrome P450 1A1 by youngiasides via Nrf2-ARE and AhR-XRE pathways.

Many phytochemicals are known to exert cancer chemopreventive activity by eliminating chemical carcinogens or toxic xenobiotics through the action of detoxification enzymes. In this study, we investigated the cancer chemopreventive effects of youngiasides isolated from Crepidiastrum denticulatum. These youngiasides significantly induced quinone reductase (QR) activity in mouse hepatoma Hepa-1c1c7 cells, and showed a relatively high chemoprevention index (CI; divided IC(50) value with CD value). The youngiasides also significantly induced transcriptional activation of QR in Hepa-QR-secreted alkaline phosphatase (SEAP) cells, which is a stable cell line containing the intact promoter region of QR. In order to determine if upregulation of QR by the youngiasides was mediated through a mono-functional or bi-functional mechanism, we examined the nuclear factor-E2 p45-related factor 2(Nrf2)-antioxidant response element (ARE) and aryl hydrocarbon receptor (AhR)-xenobiotic response element (XRE) pathways, which are two major pathways, involved in regulation of Phase I and/or Phase II detoxification enzymes. The youngiasides increased the cytochrome P450 1A1 (CYP1A1) mRNA and protein levels in human colorectal cancer Caco-2 cells and also increased the QR mRNA and protein levels in Caco-2 cells through ARE and XRE activation which resulted from translocation of Nrf2 and AhR into the nucleus. These results suggest that regulation of QR by the youngiasides was due to bi-functional induction through the Nrf2-ARE and AhR-XRE pathways. Thus, these youngiasides as bi-functional inducers of QR have potential as cancer chemopreventive agents.

Effects of shortened photoperiod on gonadotropin-releasing hormone, gonadotropin, and vitellogenin gene expression associated with ovarian maturation in rainbow trout.

Reproductive activities of salmonids are synchronized by changes in photoperiod, which control the endocrine system via the brain-pituitary-gonadal axis. Gonadotropin-releasing hormone (GnRH) in the brain regulates synthesis and release of the pituitary gonadotropins (GTHs; FSH and LH). FSH and LH in turn stimulate the production of sex steroids for oocyte growth and maturation-Inducing steroid hormones for oocyte maturation and ovulation, respectively, in female salmonids. To clarify effects of long-term photoperiod manipulations on the reproductive activity of salmonids from early recrudescence to ovulation, we Investigated the gene expression profiles of GnRH, GTHs, and vitellogenin (VTG), and plasma sex steroids in female rainbow trout (Oncorhynchus mykiss). In addition, the percentages of eyed embryos and hatched alevins were examined together with the number of ovulated eggs to evaluate the effects of photoperiod regimes on egg quality. During late summer, the mRNA levels of GnRHs, GTHalpha, and LHbeta, and the plasma level of a maturational steroid (17alpha,20beta-dihydroxy-4-pregnen-3-one; 17,20beta-P) were significantly elevated by a gradually shortened photoperiod under constant temperature, in accordance with accelerated sexual maturation. The percentages of eyed embryos and hatched alevins from fish ovulated in August were comparable to those of control fish observed in December. These results clearly indicate that syntheses of GnRHs, LH, VTG, and 17,20beta-P are effectively accelerated by a programmed long-short photoperiod regime in early recrudescent female rainbow trout, without a marked deterioration in egg quality.

Tectoridin, a poor ligand of estrogen receptor alpha, exerts its estrogenic effects via an ERK-dependent pathway.

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17beta-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER alpha as compared to 17beta-estradiol and genistein. Despite poor binding to ER alpha, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER alpha at Ser(118). It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.

Conserved properties of a urochordate estrogen receptor-related receptor (ERR) with mammalian ERRalpha.

Estrogen receptor-related receptors (ERRs) were the first orphan nuclear receptors identified on the basis of their sequence similarity to the estrogen receptors. Although unique ERRs were found in some marine invertebrates, the molecular functions of these receptors are not well understood. In the present study, we identified three transcript variants of the tunicate Halocynthia roretzi ERR (Hr-ERR), varying in their 3' untranslated regions, and putatively encoding a unique receptor deriving from an ancestor protein common to vertebrate ERRalpha/beta/gamma. Maternal mRNA of Hr-ERR was detected throughout the entire egg cytoplasm and early embryos. Zygotic Hr-ERR was predominantly expressed in the heart, and at lower levels in muscle, stomach, gonad and digestive glands. Electrophoretic mobility shift assay demonstrated that Hr-ERR directly binds to the estrogen-response element (ERE) and ERR-response element (ERRE). Gene reporter assays also showed that Hr-ERR activates transcription through ERE and ERRE. Hr-ERR-mediated transactivation was modulated by various cofactors for mammalian ERRs, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha and small heterodimer partner. In addition, the ERR antagonists 4-hydroxytamoxifen and diethylstilbestrol inhibited the Hr-ERR-mediated transactivation, whereas Hr-ERR activity on ERE was further induced by genistein, an ERRalpha agonist. Taken together, our results show that Hr-ERR is an unduplicated ERR that however, possesses functional properties common to ERRalpha and not to ERRbeta/gamma.

Biological activities of recombinant Manchurian trout FSH and LH: their receptor specificity, steroidogenic and vitellogenic potencies.

Gonadotropins (GTHs), FSH and LH, play central roles in vertebrate reproduction. Here, we report the production of biologically-active recombinant FSH (r-mtFSH) and LH (r-mtLH) of an endangered salmon species, Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The biological activities of the recombinant hormones were analyzed using COS-7 cell line transiently expressing either amago salmon FSH or LH receptor. The steroidogenic potency of the r-mtFSH and r-mtLH was examined by a culture system using rainbow trout follicles in vitro. In vivo, bioactivity was assessed by measuring ovarian weight, oocyte diameter, and plasma steroid hormone levels in female rainbow trout. Moreover, inducing potency of milt production were examined in vivo using goldfish. Our results demonstrated that the r-mtFSH and r-mtLH were successfully produced in the baculovirus-silkworm system and recognized by their cognate receptors specifically in vitro. The production of estradiol-17beta (E2) and testosterone (T) was stimulated by the r-mtFSH and r-mtLH respectively, from the full-grown follicles of rainbow trout, whereas both E2 and T were increased by relatively higher doses of the recombinant hormones from the follicles of the maturing stage. In in vivo assay, injection of the r-mtFSH but not r-mtLH increased ovarian weight, oocyte diameter, and plasma E2 levels in immature rainbow trout. Injection of both r-mtFSH and r-mtLH induced milt production in male goldfish. In conclusion, the present study strongly suggests that the r-mtFSH and r-mtLH have distinct biological properties, such as a specific responsiveness for the cognate receptor, steroidogenic, and vitellogenic activities for ovarian follicles in salmonids. These recombinant FSH and LH may be applied for future studies on the gonadal development and maturation in fishes as well as the endangered salmon species.