Ovarian Follicle Development in Ascidians.

Ovarian follicle development is an essential process for continuation of sexually reproductive animals, and is controlled by a wide variety of regulatory factors such as neuropeptides and peptide hormones in the endocrine, neuroendocrine, and nervous systems. Moreover, while some molecular mechanisms underlying follicle development are conserved, others vary among species. Consequently, follicle development processes are closely related to the evolution and diversity of species. Ciona intestinalis type A (Ciona rubusta) is a cosmopolitan species of ascidians, which are the closest relative of vertebrates. However, unlike vertebrates, ascidians are not endowed with the hypothalamus-pituitary-gonadal axis involving pituitary gonadotropins and sexual steroids. Combined with the phylogenetic position of ascidians as the closest relative of vertebrates, such morphological and endocrine features suggest that ascidians possess both common and species-specific regulatory mechanisms in follicle development. To date, several neuropeptides have been shown to participate in the growth of vitellogenic follicles, oocyte maturation of postvitellogenic follicles, and ovulation of fully mature follicles in a developmental stage-specific fashion. Furthermore, recent studies have shed light on the evolutionary processes of follicle development throughout chordates. In this review, we provide an overview of the neuropeptidergic molecular mechanism in the premature follicle growth, oocyte maturation, and ovulation in Ciona, and comparative views of the follicle development processes of mammals and teleosts.

Prediction of acute lung injury assessed by chest computed tomography, oxygen saturation/fraction of inspired oxygen ratio, and serum lactate dehydrogenase in patients with COVID-19.

INTRODUCTION: In treating acute hypoxemic respiratory failure (AHRF) caused by coronavirus disease 2019 (COVID-19), clinicians choose respiratory therapies such as low-flow nasal cannula oxygenation, high-flow nasal cannula oxygenation, or mechanical ventilation after assessment of the patient's condition. Chest computed tomography (CT) imaging contributes significantly to diagnosing COVID-19 pneumonia. However, the costs and potential harm to patients from radiation exposure need to be considered. This study was performed to predict the quantitative extent of COVID-19 acute lung injury using clinical indicators such as an oxygenation index and blood test results. METHODS: We analyzed data from 192 patients with COVID-19 AHRF. Multiple logistic regression was used to determine correlations between the lung infiltration volume (LIV) and other pathophysiological or biochemical laboratory parameters. RESULTS: Among 13 clinical parameters, we identified the oxygen saturation/fraction of inspired oxygen ratio (SF ratio) and serum lactate dehydrogenase (LD) concentration as factors associated with the LIV. In the binary classification of an LIV of ≥20 % or not and with the borderline LD = 2.2 × [SF ratio]-182.4, the accuracy, precision, diagnostic odds ratio, and area under the summary receiver operating characteristic curve were 0.828, 0.818, 23.400, and 0.870, respectively. CONCLUSIONS: These data suggest that acute lung injury due to COVID-19 pneumonia can be estimated using the SF ratio and LD concentration without a CT scan. These findings may provide significant clinical benefit by allowing clinicians to predict acute lung injury levels using simple, minimally invasive assessment of oxygenation capacity and biochemical blood tests.

A Novel Hemocyte-Derived Peptide and Its Possible Roles in Immune Response of Ciona intestinalis Type A.

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1-4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates.

A dual enhancer-silencer element, DES-K16, in mouse spermatocyte-derived GC-2spd(ts) cells.

The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16 gene as the chromatin highly marked with epigenetic modifications of both enhancers (H3K4me1 and H3K27ac) and silencers (H3K27me3) in mouse spermatocytes. In vitro reporter gene assay demonstrated that DES-K16 exhibited significant enhancer activity in spermatocyte-derived GC-2spd(ts) and hepatic tumor-derived Hepa1-6 cells, and a deletion of this sequence in GC-2spd(ts) cells resulted in a decrease and increase of Yipf5 and Kctd16 expression, respectively. This was consistent with increased and decreased expression of Yipf5 and Kctd16, respectively, in primary spermatocytes during testis development. While known dual enhancer-silencers exert each activity in different tissues, our data suggest that DES-K16 functions as both enhancer and silencer in a single cell type, GC-2spd(ts) cells. This is the first report on a dual enhancer-silencer element which activates and suppresses gene expression in a single cell type.

A relaxin-like gonad-stimulating peptide identified from the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B-chain (21 aa), C-peptide (47 aa), and A-chain (24 aa). There are three putative processing sites (Lys-Arg) between the B-chain and C-peptide, between the C-peptide and A-chain, and within the C-peptide. This structural organization revealed that the mature AscRGP is composed of A- and B-chains with two interchain disulfide bonds and one intrachain disulfide bond. The C-terminal residues of the B-chain are Gln-Gly-Arg, which is a potential substrate for formation of an amidated C-terminal Gln residue. Non-amidated (AscRGP-GR) and amidated (AscRGP-NH2 ) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP-GR and AscRGP-NH2 induced oocyte maturation and ovulation in similar dose-dependent manners. This is the first report on a C-terminally amidated functional RGP. Collectively, these results suggest that AscRGP-GR and AscRGP-NH2 act as a natural gonadotropic hormone in A. scoparius.

Two cases of novel coronavirus infection (COVID-19) with transient viral elevation using semi-quantitative real-time reverse transcription PCR and symptom relapse after completion of 10 days of favipiravir treatment.

The coronavirus disease of 2019 (COVID-19), which began in Wuhan, China, at the end of 2019, is spreading around the world and causing many deaths, mainly from pneumonia. Currently, there are no specific drugs to treat COVID-19, and existing antiviral drugs are being used as an alternative. One of these is favipiravir, a new type of influenza drug. However, its efficacy, dosage, and duration of administration are still under study. In this case study, we administered favipiravir to patients with COVID-19 and determined the viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pathogen, using semi-quantitative real-time reverse transcription PCR in sputum samples. We report on two patients in whom the viral load increased again after completion of 10 days of favipiravir treatment and a transient relapse of symptoms was observed.

Transcriptional activation of the mouse Scd2 gene by interdependent enhancers and long noncoding RNAs in ovarian granulosa cells.

Specific gene expression in granulosa cells is key for the function of ovary, but the molecular mechanism of transcriptional activation is not well studied. Here we investigated the regulatory mechanism of the mouse stearoyl-CoA desaturase 2 (Scd2) gene encoding an enzyme for lipid metabolism. Northern blot and in situ hybridization indicated that the mouse Scd2 mRNA was highly expressed in ovarian granulosa cells. We found four conserved noncoding sequences (CNSs) and two long noncoding RNAs (lncRNAs) transcribed from regions upstream of the Scd2 gene as candidates of regulatory elements/factors. These lncRNAs were predominantly transcribed in the opposite direction to Scd2 and localized in nuclei and showed the correlation with Scd2 expression, raising the possibility of their transcriptional regulatory roles. Indeed, knockdown of both lncRNAs, lncRNA-sc1 and lncRNA-sc2, significantly decreased the Scd2 mRNA level in primary granulosa cells. Then, we investigated the histone modification pattern at this locus by a chromatin immunoprecipitation assay, and two CNSs, CNS1 and CNS2, were found to be marked with high levels of histone H3K9/K27 acetylation in primary granulosa cells. By a reporter gene assay, both CNS1 and CNS2 interdependently exhibited enhancer activity for the Scd2 promoter in primary granulosa cells. These data suggest that the mouse Scd2 gene is activated by two lncRNAs and interdependent enhancers in ovarian granulosa cells, which provides a new insight into transcriptional activation in granulosa cells.

Invertebrate Gonadotropin-Releasing Hormone Receptor Signaling and Its Relevant Biological Actions.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproduction via the hypothalamus-pituitary-gonad axis (HPG axis) in vertebrates. GnRHs and their receptors (GnRHRs) are also conserved in invertebrates lacking the HPG axis, indicating that invertebrate GnRHs do not serve as "gonadotropin-releasing factors" but, rather, function as neuropeptides that directly regulate target tissues. All vertebrate and urochordate GnRHs comprise 10 amino acids, whereas amphioxus, echinoderm, and protostome GnRH-like peptides are 11- or 12-residue peptides. Intracellular calcium mobilization is the major second messenger for GnRH signaling in cephalochordates, echinoderms, and protostomes, while urochordate GnRHRs also stimulate cAMP production pathways. Moreover, the ligand-specific modulation of signal transduction via heterodimerization between GnRHR paralogs indicates species-specific evolution in Ciona intestinalis. The characterization of authentic or putative invertebrate GnRHRs in various tissues and their in vitro and in vivo activities indicate that invertebrate GnRHs are responsible for the regulation of both reproductive and nonreproductive functions. In this review, we examine our current understanding of and perspectives on the primary sequences, tissue distribution of mRNA expression, signal transduction, and biological functions of invertebrate GnRHs and their receptors.

Peptide receptors and immune-related proteins expressed in the digestive system of a urochordate, Ciona intestinalis.

The digestive system is responsible for nutrient intake and defense against pathogenic microbes. Thus, identification of regulatory factors for digestive functions and immune systems is a key step to the verification of the life cycle, homeostasis, survival strategy and evolutionary aspects of an organism. Over the past decade, there have been increasing reports on neuropeptides, their receptors, variable region-containing chitin-binding proteins (VCBPs) and Toll-like receptors (TLRs) in the ascidian, Ciona intestinalis. Mass spectrometry-based peptidomes and genome database-searching detected not only Ciona orthologs or prototypes of vertebrate peptides and their receptors, including cholecystokinin, gonadotropin-releasing hormones, tachykinin, calcitonin and vasopressin but also Ciona-specific neuropeptides including Ci-LFs and Ci-YFVs. The species-specific regulation of GnRHergic signaling including unique signaling control via heterodimerization among multiple GnRH receptors has also been revealed. These findings shed light on the remarkable significance of ascidians in investigations of the evolution and diversification of the peptidergic systems in chordates. In the defensive systems of C. intestinalis, VCBPs and TLRs have been shown to play major roles in the recognition of exogenous microbes in the innate immune system. These findings indicate both common and species-specific functions of the innate immunity-related molecules between C. intestinalis and vertebrates. In this review article, we present recent advances in molecular and functional features and evolutionary aspects of major neuropeptides, their receptors, VCBPs and TLRs in C. intestinalis.

Functional characterization of an invertebrate gonadotropin-releasing hormone receptor in the Yesso scallop Mizuhopecten yessoensis.

The neuropeptide control of bivalve reproduction with particular reference to gonadotropin-releasing hormone (invGnRH) is a frontier yet to be investigated. Bivalves are unique because they have two forms of the invGnRH peptide; however, there has been no functional characterization of the peptide-receptor pair. Therefore, the identification of a cognate receptor is a preliminary step toward exploring the biological roles of invGnRHs in bivalves. In this study, we functionally characterize an invGnRH receptor (invGnRHR) of a bivalve, the Yesso scallop Mizuhopecten yessoensis. In the receptor assay, HEK293 cells were transfected to transiently express the M. yessoensis invGnRHR (my-invGnRHR), which was found to be localized on the plasma membrane, confirming that my-invGnRHR, similar to other G-protein-coupled receptors, functions as a membrane receptor. Using both forms of invGnRH as ligands in a function-receptor assay, my-invGnRH11aa-NH2 stimulated intracellular Ca2+ mobilization but not cyclic AMP production, whereas my-invGnRH12aa-OH did not induce increase in Ca2+ levels. Therefore, we concluded that my-invGnRHR is an endogenous receptor specific to my-invGnRH11aa-NH2 which is hypothesized to be the mature peptide. To the best of our knowledge, this is the first study reporting the functional characterization of a bivalve invGnRHR.

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish.

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10-7M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10-9 to 10-7M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10-7M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts.

The significance of Ciona intestinalis as a stem organism in integrative studies of functional evolution of the chordate endocrine, neuroendocrine, and nervous systems.

Ascidians are the closest phylogenetic neighbors to vertebrates and are believed to conserve the evolutionary origin in chordates of the endocrine, neuroendocrine, and nervous systems involving neuropeptides and peptide hormones. Ciona intestinalis harbors various homologs or prototypes of vertebrate neuropeptides and peptide hormones including gonadotropin-releasing hormones (GnRHs), tachykinins (TKs), and calcitonin, as well as Ciona-specific neuropeptides such as Ciona vasopressin, LF, and YFV/L peptides. Moreover, molecular and functional studies on Ciona tachykinin (Ci-TK) have revealed the novel molecular mechanism of inducing oocyte growth via up-regulation of vitellogenesis-associated protease activity, which is expected to be conserved in vertebrates. Furthermore, a series of studies on Ciona GnRH receptor paralogs have verified the species-specific regulation of GnRHergic signaling including unique signaling control via heterodimerization among multiple GnRH receptors. These findings confirm the remarkable significance of ascidians in investigations of the evolutionary processes of the peptidergic systems in chordates, leading to the promising advance in the research on Ciona peptides in the next stage based on the recent development of emerging technologies including genome-editing techniques, peptidomics-based multi-color staining, machine-learning prediction, and next-generation sequencing. These technologies and bioinformatic integration of the resultant "multi-omics" data will provide unprecedented insights into the comprehensive understanding of molecular and functional regulatory mechanisms of the Ciona peptides, and will eventually enable the exploration of both conserved and diversified endocrine, neuroendocrine, and nervous systems in the evolutionary lineage of chordates.

PTBP2 binds to a testis-specific long noncoding RNA, Tesra, and activates transcription of the Prss42/Tessp-2 gene.

Long noncoding RNAs (lncRNAs) have recently been proved to be functional in the testis. Tesra, a testis-specific lncRNA, was suggested to activate the transcription of Prss42/Tessp-2, a gene that is involved in meiotic progression, in mouse spermatocytes. To reveal the molecular mechanism underlying the activation, we searched for Tesra-binding proteins by a Ribotrap assay followed by LC-MS/MS analysis and identified polypyrimidine tract binding protein 2 (PTBP2) as a candidate. Analysis of public RNA-seq data and our qRT-PCR results indicated that Ptbp2 mRNA showed an expression pattern similar to the expression patterns of Tesra and Prss42/Tessp-2 during testis development. Moreover, PTBP2 was found to be associated with Tesra in testicular germ cells by RNA immunoprecipitation. To evaluate the effect of PTBP2 on the Prss42/Tessp-2 promoter, we established an in vitro reporter gene assay system in which Tesra expression could be induced by the Tet-on system and thereby Prss42/Tessp-2 promoter activity could be increased. In this system, the Prss42/Tessp-2 promoter activity was significantly decreased by the knockdown of PTBP2. These results suggest that PTBP2 contributes to Prss42/Tessp-2 transcriptional activation by Tesra in spermatocytes. The finding provides a precious example of a molecular mechanism of testis lncRNA functioning in spermatogenesis.

Characterization of a novel species-specific 51-amino acid peptide, PEP51, as a caspase-3/7 activator in ovarian follicles of the ascidian, Ciona intestinalis Type A.

Invertebrates lack hypothalamic-pituitary-gonadal axis, and have acquired species-specific regulatory systems for ovarian follicle development. Ascidians are marine invertebrates that are the phylogenetically closest living relatives to vertebrates, and we have thus far substantiated the molecular mechanisms underlying neuropeptidergic follicle development of the cosmopolitan species, Ciona intestinalis Type A. However, no ovarian factor has so far been identified in Ciona. In the present study, we identified a novel Ciona-specific peptide, termed PEP51, in the ovary. Immunohistochemical analysis demonstrated the specific expression of PEP51 in oocyte-associated accessory cells, test cells, of post-vitellogenic (stage III) follicles. Immunoelectron microscopy revealed that PEP51 was localized in the cytosol of test cells in early stage III follicles, which lack secretory granules. These results indicate that PEP51 acts as an intracellular factor within test cells rather than as a secretory peptide. Confocal laser microscopy verified that activation of caspase-3/7, the canonical apoptosis marker, was detected in most PEP51-positive test cells of early stage III. This colocalization of PEP51 and the apoptosis marker was consistent with immunoelectron microscopy observations demonstrating that a few normal (PEP51-negative) test cells reside in the aggregates of PEP51-positive apoptotic test cells of early stage III follicles. Furthermore, transfection of the PEP51 gene into COS-7 cells and HEK293MSR cells resulted in activation of caspase-3/7, providing evidence that PEP51 induces apoptotic signaling. Collectively, these results showed the existence of species-specific ovarian peptide-driven cell metabolism in Ciona follicle development. Consistent with the phylogenetic position of Ciona as the closest sister group of vertebrates, the present study sheds new light on the molecular and functional diversity of the regulatory systems of follicle development in the Chordata.

Omics Studies for the Identification of Ascidian Peptides, Cognate Receptors, and Their Relevant Roles in Ovarian Follicular Development.

Omics studies contribute to the elucidation of genomes and profiles of gene expression. In the ascidian Ciona intestinalis Type A (Ciona robusta), mass spectrometry (MS)-based peptidomic studies have detected numerous Ciona-specific (nonhomologous) neuropeptides as well as Ciona homologs of typical vertebrate neuropeptides and hypothalamic peptide hormones. Candidates for cognate G protein-coupled receptors (GPCRs) for these peptides have been found in the Ciona transcriptome by two ways. First, Ciona homologous GPCRs of vertebrate counterparts have been detected by sequence homology searches of cognate transcriptomes. Second, the transcriptome-derived GPCR candidates have been used for machine learning-based systematic prediction of interactions not only between Ciona homologous peptides and GPCRs but also between novel Ciona peptides and GPCRs. These data have ultimately led to experimental evidence for various Ciona peptide-GPCR interactions. Comparative transcriptomics between the wildtype and Ciona vasopressin (CiVP) gene-edited Ciona provide clues to the biological functions of CiVP in ovarian follicular development and whole body growth. Furthermore, the transcriptomes of follicles treated with peptides, such as Ciona tachykinin and cionin (a Ciona cholecystokinin homolog), have revealed key regulatory genes for Ciona follicle growth, maturation, and ovulation, eventually leading to the verification of essential and novel molecular mechanisms underlying these biological events. These findings indicate that omics studies, combined with artificial intelligence and single-cell technologies, pave the way for investigating in greater details the nervous, neuroendocrine, and endocrine systems of ascidians and the molecular and functional evolution and diversity of peptidergic regulatory networks throughout chordates.

Evidence for a functional role of Start, a long noncoding RNA, in mouse spermatocytes.

A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously showed that Start regulates steroidogenesis through controlling the expression of Star and Hsd3b1 genes in Leydig cells, but its function in germ cells was not known. Here we verified that a spermatocyte-specific protease gene, Prss43/Tessp-3, was downregulated in Start-knockout testes. To investigate the transcriptional regulatory activity of Start in spermatocytes, we first performed a series of reporter gene assays using a thymidine kinase promoter in spermatocyte-derived GC-2spd(ts) cells. A 5.4-kb genome sequence encompassing Start exhibited enhancer activity for this promoter, and the activity was decreased by knockdown of Start. Deletion of the Start promoter and replacement of the Start sequence abolished the enhancer activity and, consistently, the activity was detected in further experiments only when Start was actively transcribed. We then examined whether the Prss43/Tessp-3 gene could be a target of Start. A reporter gene assay demonstrated that the 5.4-kb sequence exhibited enhancer activity for a Prss43/Tessp-3 promoter in GC-2spd(ts) cells and that the activity was significantly decreased by knockdown of Start. These results suggest that Start functions in transcriptional activation of the Prss43/Tessp-3 gene in spermatocytes. Given that Start is presumed to regulate steroidogenic genes at the posttranscriptional level in Leydig cells, the function in spermatocytes is a novel role of Start. These findings provide an insight into multifunctionality of lncRNAs in the testis.

Cionin, a vertebrate cholecystokinin/gastrin homolog, induces ovulation in the ascidian Ciona intestinalis type A.

Cionin is a homolog of vertebrate cholecystokinin/gastrin that has been identified in the ascidian Ciona intestinalis type A. The phylogenetic position of ascidians as the closest living relatives of vertebrates suggests that cionin can provide clues to the evolution of endocrine/neuroendocrine systems throughout chordates. Here, we show the biological role of cionin in the regulation of ovulation. In situ hybridization demonstrated that the mRNA of the cionin receptor, Cior2, was expressed specifically in the inner follicular cells of pre-ovulatory follicles in the Ciona ovary. Cionin was found to significantly stimulate ovulation after 24-h incubation. Transcriptome and subsequent Real-time PCR analyses confirmed that the expression levels of receptor tyrosine kinase (RTK) signaling genes and a matrix metalloproteinase (MMP) gene were significantly elevated in the cionin-treated follicles. Of particular interest is that an RTK inhibitor and MMP inhibitor markedly suppressed the stimulatory effect of cionin on ovulation. Furthermore, inhibition of RTK signaling reduced the MMP gene expression in the cionin-treated follicles. These results provide evidence that cionin induces ovulation by stimulating MMP gene expression via the RTK signaling pathway. This is the first report on the endogenous roles of cionin and the induction of ovulation by cholecystokinin/gastrin family peptides in an organism.

Comparative analysis of transcriptomic profiles among ascidians, zebrafish, and mice: Insights from tissue-specific gene expression.

Tissue/organ-specific genes (TSGs) are important not only for understanding organ development and function, but also for investigating the evolutionary lineages of organs in animals. Here, we investigate the TSGs of 9 adult tissues of an ascidian, Ciona intestinalis Type A (Ciona robusta), which lies in the important position of being the sister group of vertebrates. RNA-seq and qRT-PCR identified the Ciona TSGs in each tissue, and BLAST searches identified their homologs in zebrafish and mice. Tissue distributions of the vertebrate homologs were analyzed and clustered using public RNA-seq data for 12 zebrafish and 30 mouse tissues. Among the vertebrate homologs of the Ciona TSGs in the neural complex, 48% and 63% showed high expression in the zebrafish and mouse brain, respectively, suggesting that the central nervous system is evolutionarily conserved in chordates. In contrast, vertebrate homologs of Ciona TSGs in the ovary, pharynx, and intestine were not consistently highly expressed in the corresponding tissues of vertebrates, suggesting that these organs have evolved in Ciona-specific lineages. Intriguingly, more TSG homologs of the Ciona stomach were highly expressed in the vertebrate liver (17-29%) and intestine (22-33%) than in the mouse stomach (5%). Expression profiles for these genes suggest that the biological roles of the Ciona stomach are distinct from those of their vertebrate counterparts. Collectively, Ciona tissues were categorized into 3 groups: i) high similarity to the corresponding vertebrate tissues (neural complex and heart), ii) low similarity to the corresponding vertebrate tissues (ovary, pharynx, and intestine), and iii) low similarity to the corresponding vertebrate tissues, but high similarity to other vertebrate tissues (stomach, endostyle, and siphons). The present study provides transcriptomic catalogs of adult ascidian tissues and significant insights into the evolutionary lineages of the brain, heart, and digestive tract of chordates.

Muscarinic receptor M3 contributes to intestinal stem cell maintenance via EphB/ephrin-B signaling.

Acetylcholine (ACh) signaling through activation of nicotinic and muscarinic ACh receptors regulates expression of specific genes that mediate and sustain proliferation, differentiation, and homeostasis in the intestinal crypts. This signaling plays a pivotal role in the regulation of intestinal stem cell function, but the details have not been clarified. Here, we performed experiments using type 3 muscarinic acetylcholine receptor (M3) knockout mice and their intestinal organoids and report that endogenous ACh affects the size of the intestinal stem niche via M3 signaling. RNA sequencing of crypts identified up-regulation of the EphB/ephrin-B signaling pathway. Furthermore, using an MEK inhibitor (U0126), we found that mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, which is downstream of EphB/ephrin-B signaling, is activated in M3-deficient crypts. Collectively, M3, EphB/ephrin-B, and the MAPK/ERK signaling cascade work together to maintain the homeostasis of intestinal epithelial cell growth and differentiation following modifications of the cholinergic intestinal niche.