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.

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.

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.

Impact of Machine Learning-Associated Research Strategies on the Identification of Peptide-Receptor Interactions in the Post-Omics Era.

BACKGROUNDS: Elucidation of peptide-receptor pairs is a prerequisite for many studies in the neuroendocrine, endocrine, and neuroscience fields. Recent omics analyses have provided vast amounts of peptide and G protein-coupled receptor (GPCR) sequence data. GPCRs for homologous peptides are easily characterized based on homology searching, and the relevant peptide-GPCR interactions are also detected by typical signaling assays. In contrast, conventional evaluation or prediction methods, including high-throughput reverse-pharmacological assays and tertiary structure-based computational analyses, are not useful for identifying interactions between novel and omics-derived peptides and GPCRs. SUMMARY: Recently, an approach combining machine learning-based prediction of novel peptide-GPCR pairs and experimental validation of the predicted pairs have been shown to breakthrough this bottleneck. A machine learning method, logistic regression for human class A GPCRs and the multiple subsequent signaling assays led to the deorphanization of human class A orphan GPCRs, namely, the identification of 18 peptide-GPCR pairs. Furthermore, using another machine learning algorithm, the support vector machine (SVM), the peptide descriptor-incorporated SVM was originally developed and employed to predict GPCRs for novel peptides characterized from the closest relative of vertebrates, Ciona intestinalis Type A (Ciona robusta). Experimental validation of the predicted pairs eventually led to the identification of 11 novel peptide-GPCR pairs. Of particular interest is that these newly identified GPCRs displayed neither significant sequence similarity nor molecular phylogenetic relatedness to known GPCRs for peptides. KEY MESSAGES: These recent studies highlight the usefulness and versatility of machine learning for enabling the efficient, reliable, and systematic identification of novel peptide-GPCR interactions.

Characterization and localization of relaxin-like gonad-stimulating peptide in the crown-of-thorns starfish, Acanthaster cf. solaris.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) is the gonadotropin responsible for final gamete maturation. RGP comprises two different peptides, A- and B-chains with two interchain and one intrachain disulfide bonds. The existence of two isomers of RGP in the crown-of-thorns starfish, Acanthaster planci, has been reported previously, but it was recently shown that A. planci represents a species complex with four different species. Here we elucidated the authentic sequence of the Pacific species, Acanthaster cf. solaris, RGP (Aso-RGP). The Aso-RGP precursor encoded by a 354 base pair open reading frame was composed of 117 amino acids (aa). The amino acid identity of Aso-RGP to Patiria pectinifera RGP (Ppe-RGP) and Asterias amurensis RGP (Aam-RGP) was 74% and 60%, respectively. Synthetic Aso-RGP induced spawning of ovarian fragments from A. cf. solaris. Ppe-RGP and Aam-RGP also induced spawning by A. cf. solaris ovaries. In contrast, Ppe-RGP and Aso-RGP induced spawning by P. pectinifera ovaries, but Aam-RGP was inactive. Notably, anti-Ppe-RGP antibodies recognized Aso-RGP as well as Ppe-RGP. Localization of Aso-RGP was observed immunohistochemically using anti-Ppe-RGP antibodies, showing that Aso-RGP was mainly present in the radial nerve cords of A. cf. solaris. Aso-RGP was distributed not only in the epithelium of the ectoneural region but also in the neuropile of the ectoneural region. These results suggest that Aso-RGP is synthesized in the epithelium of the ectoneural region, then transferred to fibers in the neuropile of the ectoneural region in radial nerve cords.

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.

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.

Functional characterization and related evolutionary implications of invertebrate gonadotropin-releasing hormone/corazonin in a well-established model species.

In vertebrates, gonadotropin-releasing hormone (GnRH) peptide is the central mediator of reproduction. Homologous peptides have previously also been identified in molluscan species. However, emerging evidence suggests that these molecules might serve diverse regulatory functions and proposes to consider them as corazonin (CRZ). We previously isolated the full-length cDNA of the invGnRH/CRZ peptide (termed ly-GnRH/CRZ) in the well-established invertebrate model species, the great pond snail Lymnaea stagnalis; however, its predicted functions remain to be verified. In this study, we first confirmed the presence of the deduced active peptide from the central nervous system of L. stagnalis. Further, we performed in vivo and in vitro studies to explore the functions of ly-GnRH/CRZ. Injection of sexually mature specimens with synthetic active peptide had an inhibitory effect on locomotion and an acceleratory effect on egg-laying, but had no effect on feeding. The previously predicted modulatory effect of ly-GnRH/CRZ was supported by its identified co-localization with serotonin on the surface of the heart atria. Lastly, we demonstrated not only the presence of ly-GnRH/CRZ in the penial complex but also that ly-GnRH/CRZ-containing neurons project to the efferent penis nerve, suggesting ly-GnRH/CRZ may directly modulate the motor output of this peripheral tissue. Overall, our findings strongly support that ly-GnRH/CRZ is a multifunctional neuropeptide. These results contribute to the understanding of the GnRH superfamily and, more broadly, disciplines such as comparative endocrinology and neurobiology.

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.

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.

A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

The ventral peptidergic system of the adult ascidian Ciona robusta (Ciona intestinalis Type A) insights from a transgenic animal model.

Ascidians are the sister group of vertebrates and occupy a critical position in explorations of the evolution of the endocrine and nervous systems of chordates. Here, we describe the complete ventral peptidergic system in adult transgenic Ciona robusta (Ciona intestinalis Type A) which expresses the Kaede reporter gene driven by the prohormone convertase 2 (PC2) gene promoter. Numerous PC2 promoter-driven fluorescent (Kaede-positive) non-neural cells were distributed in the blood sinus located at the anterior end of the pharynx, suggesting the acquisition of a peptidergic circulatory system in Ciona. Kaede-positive ciliated columnar cells, rounded cells, and tall ciliated cells were observed in the alimentary organs, including the endostyle, pharynx, esophagus, stomach, and intestine, suggesting that digestive functions are regulated by multiple peptidergic systems. In the heart, Kaede-positive neurons were located in the ring-shaped plexus at both ends of the myocardium. Nerve fiber-like tracts ran along the raphe and appeared to be connected with the plexuses. Such unique structures suggest a role for the peptidergic system in cardiac function. Collectively, the present anatomic analysis revealed the major framework of the ventral peptidergic system of adult Ciona, which could facilitate investigations of peptidergic regulation of the pharynx, endostyle, alimentary tissues, and heart.

Mechanism of gamete shedding in starfish: Involvement of acetylcholine in extracellular Ca2+-dependent contraction of gonadal walls.

Starfish are suitable animals for the study of hormonal regulatory mechanism of oocyte maturation and ovulation. Although contraction of the gonadal walls is essential for the shedding gametes, little was known about the mechanism. When ovaries of starfish Patiria pectinifera were incubated in Ca2+-free seawater in the presence of 1-methyladenine (1-MeAde), the germinal vesicles in oocytes broke down, but no ovulation occurred. Verapamil, a potent inhibitor of voltage-dependent Ca2+ channels, inhibited 1-MeAde-induced ovulation. These results suggest that extracellular Ca2+ and its influx are indispensable for gamete shedding. Furthermore, acetylcholine (ACh) was involved in extracellular Ca2+-dependent contractions of gonadal walls. Although 1-MeAde failed to induce contraction of the gonadal walls in normal seawater containing L-glutamic acid, application of ACh or carbachol, an agonist for ACh receptor, could bring about shedding of mature oocytes. Atropine, a competitive antagonist of the muscarinic ACh receptor, inhibited 1-MeAde-induced ovulation, but a nicotinic ACh receptor antagonist mecamylamine had no effect. Furthermore, ACh was detected in the ovaries and testes in P. pectinifera. These findings suggest that ACh acts on muscarinic ACh receptors in gonadal walls to induce peristaltic contractions caused by Ca2+ influx via Ca2+ channels in the gonadal wall muscle for gamete shedding. The present study also provides new insight into the regulatory mechanism that 1-MeAde acts on secretion of ACh in ovaries and testes.

Distribution and physiological effect of enterin neuropeptides in the olfactory centers of the terrestrial slug Limax.

In gastropods, the function of neuropeptides has been studied primarily in the peripheral motor systems. Their functional roles in the central nervous system have received less attention. The procerebrum, the secondary olfactory center of the terrestrial slug Limax, consists of several hundred thousand interneurons, and plays a pivotal role in olfactory learning and memory. In the present study, we found that enterin, known as a myoactive peptide functioning in the enteric and vascular system of Aplysia, is expressed in the procerebrum of Limax. These enterin-expressing neurons primarily make projections within the cell mass layer of the procerebrum. The oscillatory frequency of the local field potential in the procerebrum was reduced by an exogenous application of enterin. The local field potential oscillation in the tentacular ganglion, the primary olfactory center, was also modulated by enterin. Whole-cell patch-clamp recordings revealed that the modulatory effect in the procerebrum was due to the inhibitory effect of enterin on the bursting neurons, which function as the kernels determining the oscillatory activity of the procerebrum. Our results revealed the novel role of the myoactive neuropeptide enterin in the higher olfactory function in terrestrial gastropods.

Fractionation of Ovarian Follicles and in vitro Oocyte Maturation and Ovulation Assay of Ciona intestinalis Type A.

Ascidians are the closest living relatives of vertebrates ( Delsuc et al., 2006 ; Satoh et al., 2014 ) and are important for the evolutionary study of the ovarian follicle development including oocyte maturation and ovulation. However, neither the endogenous factors nor the molecular mechanisms underlying the oocyte maturation and ovulation had been elucidated mainly due to the lack of efficient procedure for isolating ovarian follicles. Here, we present the protocol for the effective fractionation and isolation of the ovarian follicle of Ciona intestinalis type A using stainless steel sieves with various particle size-meshes, and the simple incubation method of Ciona follicles for evaluating oocyte maturation and ovulation. Combined with the RNA-seq data from each fraction, the current methods lead us to investigate ovarian follicle development including oocyte maturation and ovulation in a stage-specific manner.

Neuropeptides, Peptide Hormones, and Their Receptors of a Tunicate, Ciona intestinalis.

The critical phylogenetic position of the ascidian, Ciona intestinalis, as the closest relative of vertebrates, suggested its potential applicability as a model organism in a wide variety of biological events including the nervous, neuroendocrine, and endocrine regulation. To date, approximately 40 neuropeptides and/or peptide hormones and several cognate receptors have been identified. These peptides are categorized into two types: (1) orthologs of vertebrate peptides, such as cholecystokinin, GnRH, tachykinin, vasopressin, and calcitonin, and (2) novel family peptides such as LF peptides and YFL/V peptides. Ciona GnRH receptors (Ci-GnRHR) were found to be multiplicated in the Ciona-specific lineages and to form unique heterodimers between Ci-GnRHR1 and R4 and between Ci-GnRHR2 and R4, leading to fine-tuning of the generation of second messengers. Furthermore, Ciona tachykinin was shown to regulate a novel protease-associated follicle growth pathway. These findings will pave the way for the exploration of both conserved and diversified endocrine, neuroendocrine, and nervous systems in the evolutionary lineage of invertebrate deuterostomes and/or chordates. In this chapter, we provide an overview of primary sequences, functions, and evolutionary aspects of neuropeptides, peptide hormones, and their receptors in C. intestinalis.

The regulation of oocyte maturation and ovulation in the closest sister group of vertebrates.

Ascidians are the closest living relatives of vertebrates, and their study is important for understanding the evolutionary processes of oocyte maturation and ovulation. In this study, we first examined the ovulation of Ciona intestinalis Type A by monitoring follicle rupture in vitro, identifying a novel mechanism of neuropeptidergic regulation of oocyte maturation and ovulation. Ciona vasopressin family peptide (CiVP) directly upregulated the phosphorylation of extracellular signal-regulated kinase (CiErk1/2) via its receptor. CiVP ultimately activated a maturation-promoting factor, leading to oocyte maturation via germinal vesicle breakdown. CiErk1/2 also induced expression of matrix metalloproteinase (CiMMP2/9/13) in the oocyte, resulting in collagen degradation in the outer follicular cell layer and liberation of fertile oocytes from the ovary. This is the first demonstration of essential pathways regulating oocyte maturation and ovulation in ascidians and will facilitate investigations of the evolutionary process of peptidergic regulation of oocyte maturation and ovulation throughout the phylum Chordata.

Identification of a New Theca/Interstitial Cell-Specific Gene and Its Biological Role in Growth of Mouse Ovarian Follicles at the Gonadotropin-Independent Stage.

Theca/interstitial cells are responsible for the growth and maturation of ovarian follicles. However, little is known about the theca/interstitial cell-specific genes and their functions. In this study, we explored transcriptomes of theca/interstitial cells by RNA-seq, and the novel biological roles of a theca cell marker, asporin (Aspn)/periodontal ligament-associated protein 1 (PLAP-1). RNA-seq detected 432 and 62 genes expressed specifically in theca/interstitial cells and granulosa cells isolated from 3-weeks old mouse ovaries. Gene ontology analysis demonstrated that these genes were largely categorized into four major groups: extracellular matrix organization-related terms, chemotaxis-related terms, the angiogenesis-related terms, and morphogenesis-related terms. In situ hybridization demonstrated that the newly detected representative gene, Aspn/PLAP-1, was detected specifically in the outer layer of theca cells in contrast with the expression of the basal lamina-specific gene, Nidgen-1. Intriguingly, an Aspn/PLAP-1 antibody completely arrested the growth of secondary follicles that is the gonadotropin-independent follicle developmental stage. Furthermore, transforming growth factor-ß (TGF-ß)-triggered signaling was induced by the Aspn/PLAP-1 antibody treatment, which is consistent with the inhibitory effect of Aspn/PLAP-1 on TGF-ß. Altogether, these results suggest that theca cells are classified into subpopulations on the basis of new marker genes and their biological functions, and provide evidence that Aspn/PLAP-1 is expressed exclusively in the outer layer of theca cells and plays a pivotal role in the growth of secondary follicles via downregulation of the canonical TGF-ß signaling cascade.

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.

Repertoires of G protein-coupled receptors for Ciona-specific neuropeptides.

Neuropeptides play pivotal roles in various biological events in the nervous, neuroendocrine, and endocrine systems, and are correlated with both physiological functions and unique behavioral traits of animals. Elucidation of functional interaction between neuropeptides and receptors is a crucial step for the verification of their biological roles and evolutionary processes. However, most receptors for novel peptides remain to be identified. Here, we show the identification of multiple G protein-coupled receptors (GPCRs) for species-specific neuropeptides of the vertebrate sister group, Ciona intestinalis Type A, by combining machine learning and experimental validation. We developed an original peptide descriptor-incorporated support vector machine and used it to predict 22 neuropeptide-GPCR pairs. Of note, signaling assays of the predicted pairs identified 1 homologous and 11 Ciona-specific neuropeptide-GPCR pairs for a 41% hit rate: the respective GPCRs for Ci-GALP, Ci-NTLP-2, Ci-LF-1, Ci-LF-2, Ci-LF-5, Ci-LF-6, Ci-LF-7, Ci-LF-8, Ci-YFV-1, and Ci-YFV-3. Interestingly, molecular phylogenetic tree analysis revealed that these receptors, excluding the Ci-GALP receptor, were evolutionarily unrelated to any other known peptide GPCRs, confirming that these GPCRs constitute unprecedented neuropeptide receptor clusters. Altogether, these results verified the neuropeptide-GPCR pairs in the protochordate and evolutionary lineages of neuropeptide GPCRs, and pave the way for investigating the endogenous roles of novel neuropeptides in the closest relatives of vertebrates and the evolutionary processes of neuropeptidergic systems throughout chordates. In addition, the present study also indicates the versatility of the machine-learning-assisted strategy for the identification of novel peptide-receptor pairs in various organisms.