RESUMO
The sea cucumber Holothuria scabra is both an economically important species in Asian countries and an emerging experimental model for research studies in regeneration and medicinal bioactives. Growth factors and their receptors are known to be key components that guide tissue repair and renewal, yet validation of their presence in H. scabra has not been established. We performed a targeted in silico search of H. scabra transcriptome data to elucidate conserved growth factor family and receptor genes. In total, 42 transcripts were identified, of which 9 were validated by gene cloning and sequencing. The H. scabra growth factor genes, such as bone morphogenetic protein 2A (BMP 2A), bone morphogenetic protein 5-like (BMP5-like), neurotrophin (NT) and fibroblast growth factor 18 (FGF18), were selected for further analyses, including phylogenetic comparison and spatial gene expression using RT-PCR and in situ hybridization. Expression of all genes investigated were widespread in multiple tissues. However, BMP 2A, BMP5-like and NT were found extensively in the radial nerve cord cells, while FGF18 was highly expressed in connective tissue layer of the body wall. Our identification and expression analysis of the H. scabra growth factor genes provided the molecular information of growth factors in this species which may ultimately complement the research in regenerative medicine.
RESUMO
The authors recently reported the presence and distribution of oxytocin/vasopressin-like peptide in Portunus pelagicus as well as demonstrated its function to inhibit ovarian steroid release (Saetan et al., 2018). Here, the full-length receptor of this peptide, namely oxytocin/vasopressin-like peptide receptor (PpelOT/VP-like peptide receptor) is reported. The coding region of the PpelOT/VP-like peptide receptor contained 1497 bp which translationally corresponded to 499 amino acids. Sequence analysis revealed its seven transmembrane characteristics, with -two N-linked glycosylation residues located before the first transmembrane domain (TM I). The phylogenetic tree revealed that the PpelOT/VP-like peptide receptor was placed in the group of invertebrate OT/VP-like receptors, and was clearly distinguishable from the V1R, V2R and OTR of vertebrates. Also, this receptor gene transcript was detected in several organs of the blue swimming crab with highest abundance found in brain tissue. In situ hybridization exhibited its distribution in all neuronal clusters of the eyestalk, brain, ventral nerve cord (VNC), as well as in the ovary. Comparative gene expressions between this receptor and its corresponding peptide in immature and mature female crabs revealed no significant difference of the PpelOT/VP-like peptide receptor gene expression in the central nervous system (CNS) and ovary. In contrast, the PpelOT/VP-like peptide gene was shown to significantly express higher in the VNC of immature crabs and in the ovary of mature crabs. Changes in expression of this peptide gene, but not its receptor, might result in ovarian steroid release inhibition. However, the detailed mechanism of this peptide in reproduction regulation will be included in our further studies.
Assuntos
Braquiúros/fisiologia , Ocitocina/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Vasopressinas/fisiologia , Vasopressinas/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Feminino , Perfilação da Expressão Gênica , Ovário/metabolismo , Peptídeos/química , Filogenia , RNA Mensageiro/metabolismo , Receptores de Peptídeos/genética , Receptores de Vasopressinas/metabolismoRESUMO
Serotonin (5-HT) plays pivotal roles in many physiological processes including reproduction of crustaceans, which are mediated 5-HT receptors. The distributions of 5-HT and its receptor have never been explored in Portunus pelagicus. To validate the targets which indirectly indicate the roles of 5-HT in this crab, we have investigated the distribution of 5-HT in the central nervous system (CNS) and ovary using immunohistochemistry and tissue expression of its receptor by RT-PCR. In the brain, 5-HT immunoreactivity (-ir) was detected in clusters 6, 7, 8, 11, 14, 15 and the fibers. In the ventral nerve cord (VNC), 5-HT-ir was detected in pairs of neurons and the fibers connected to the neurons. In the ovary, 5-HT-ir was intense in the oocyte step 1 (Oc1) and Oc2, and its intensity was slightly decreased in Oc3 and Oc4. The 5-HT receptor was molecularly characterized to be type 7, and it was strongly expressed in the eyestalk, brain, VNC, mature ovary and muscle. Due to the presence of 5-HT receptor we suggest that 5-HT acts primarily at the CNS and ovary, thus implicating its role in reproduction especially in the development of oocytes though its exact function in this crab needed to be explored further.
Assuntos
Proteínas de Artrópodes , Braquiúros , Sistema Nervoso Central/metabolismo , Ovário/metabolismo , Receptores de Serotonina , Serotonina , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Braquiúros/genética , Braquiúros/metabolismo , Clonagem Molecular , Feminino , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Serotonina/genética , Serotonina/metabolismoRESUMO
The vitellogenesis-inhibiting hormone (VIH), also known as gonad-inhibiting hormone, is a neuropeptide hormone in crustaceans that belongs to the crustacean hyperglycemic hormone (CHH)-family peptide. There is regulation vitellogenesis by VIH during gonad maturation in crustaceans. A full-length Scylla olivacea VIH (Scyol-VIH) was identified through reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The open reading frame consists of 378 nucleotides, which encodes a 126-amino acid precursor protein, including a 22-residue signal peptide and a 103-amino acid mature peptide in which 6 highly conserved cysteine residues are present. There was expression of the Scyol-VIH gene in immature female Scylla olivacea in the eyestalk, brain and ventral nerve cord. The Scyol-VIH gene expression was localized to the eyestalk X-organ, brain neuronal clusters 6 and 11, and in multiple neuronal clusters of the ventral nerve cord. The relative abundance of Scyol-VIH mRNA transcript in the eyestalk was relatively greater in immature stage females, then decreased as ovarian maturation progressed. Furthermore, eyestalk Scyol-VIH increased after dopamine (5⯵g/g BW) injection. The present research provides fundamental information about Scyol-VIH and its potential effect in controlling reproduction.
Assuntos
Braquiúros/fisiologia , Dopamina/farmacologia , Hormônios de Invertebrado/metabolismo , Ovário/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Braquiúros/genética , Clonagem Molecular , Dopamina/administração & dosagem , Dopaminérgicos/farmacologia , Antagonistas de Dopamina/administração & dosagem , Antagonistas de Dopamina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios de Invertebrado/genética , Ovário/metabolismo , Filogenia , RNA Mensageiro/genética , Serotonina/administração & dosagem , Serotonina/farmacologia , Serotoninérgicos/administração & dosagem , Serotoninérgicos/farmacologia , Maturidade Sexual , Espiperona/administração & dosagem , Espiperona/farmacologia , Fatores de TempoRESUMO
Prostaglandins have important physiological roles in marine invertebrates, including larval development and reproduction. The prostaglandin E concentration fluctuates during the ovarian development of crustaceans. The biosynthetic pathway of prostaglandin, however, has not been well studied in portunid crabs, including in the mud crab, Scylla olivacea. In this study, the aim was to investigate the presence of prostaglandin E synthase (PGES), enzyme that catalyzes the terminal conversion in the prostaglandin E2 (PGE2) biosynthesis, and its gene expression in the central nervous system (CNS) and ovary during ovarian maturation of S. olivacea. cDNA sequence encoding PGES was cloned from the S. olivacea ovary. The PGES transcript of S. olivacea (Scyol-PGES) consists of 1258 nucleotides, which encodes for 420 amino acid PGES protein precursor. Investigation of gene expression by RT-PCR indicated that Scyol-PGES was detected in all organs studied. Based on in situ hybridization, Scyol-PGES was detected in the I to III stages for oocyte development of Stage 3 of ovarian development, and in the CNS, including the various neuronal clusters of the brain. In the ventral nerve cord, the Scyol-PGES gene was expressed in the neurons within the subesophageal, thoracic and abdominal ganglia. The Scyol-PGES gene expression as indicated by relative abundance of mRNA in the Stage 4 of ovarian development was greater than that at Stages 1 to 3 of ovarian development. This is the first report on PGES in the mud crab, S. olivacea, and its gene expression suggested the involvement of PGES in the ovarian development of this species.