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Endocrinology Vol. 149, No. 10 5254-5261
Copyright © 2008 by The Endocrine Society

Unique Form and Osmoregulatory Function of a Neurohypophysial Hormone in a Urochordate

Kazuyoshi Ukena, Eiko Iwakoshi-Ukena and Akira Hikosaka

Section of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan

Address all correspondence and requests for reprints to: Kazuyoshi Ukena, Ph.D., Associate Professor, Section of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan. E-mail: ukena{at}hiroshima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cyclic nonapeptides, oxytocin and vasopressin, are neurohypophysial hormones that regulate many significant physiological processes related especially to reproduction and osmoregulation. In this study, we characterized an oxytocin-related peptide cDNA from a urochordate, Styela plicata, thought to be a sister group to vertebrates. Sequence analysis of the deduced precursor polypeptide revealed that the precursor is composed of three segments: a signal peptide, an oxytocin-like sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin/vasopressin family precursors. However, unlike other members of this family, the tunicate oxytocin-like peptide (CYISDCPNSRFWST-NH2) is a tetradecapeptide. We termed this peptide Styela oxytocin-related peptide (SOP). Furthermore, analyses of mass spectrometry, in situ hybridization, and immunohistochemistry demonstrated production of mature SOP in the cerebral ganglion. To elucidate the physiological action of SOP, we kept the tunicate for 2 d under the three different concentrations of seawater, 60, 100, and 130%, and measured the expression levels of SOP mRNA in the cerebral ganglion. The greatest expression of SOP mRNA was observed in the 60% seawater. In 60% seawater, but not in 100 or 130%, the tunicate mostly closed the atrial and branchial siphons. Therefore, we investigated the contractile effects of SOP on the siphons in vitro. SOP caused contractions in both siphons in a dose-dependent manner. Taken together, these results suggest that SOP acts to prevent the influx of a low concentration of seawater into the body and thus play an important role in osmoregulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROHYPOHYSIAL HORMONES are involved in aspects of reproduction, such as contraction of the uterus and milk ejection, in addition to aspects of osmoregulation, such as water and salt metabolism. They are classified into two groups, the cyclic nonapeptides oxytocin (OT) and vasopressin (VP). In addition to the above classical biological actions, recently, the actions of OT and VP directly in the brain have been intensively investigated and found to participate in social and maternal behaviors, memory, and learning (1, 2, 3).

The nonapeptides of the OT/VP family are widely distributed in not only vertebrates but also invertebrates (4, 5, 6, 7, 8, 9). All vertebrate species, except for the cyclostomes (hagfish and lampreys), contain at least one OT family peptide and one VP family peptide (10, 11). On the other hand, invertebrate species have one OT or VP family peptide, except for the cephalopodan octopus (12). The biological functions of the OT/VP peptides in invertebrates are very similar to those in vertebrates; they are involved in sexual and egg-laying behaviors and water metabolism (13, 14, 15, 16). Therefore, the members of the OT/VP family are fundamentally important bioactive peptides throughout the animal kingdom. Despite the conserved actions and importance of these peptides, the evolutionary lineage of the OT/VP family between vertebrates and invertebrates is obscure. The urochordates are a focal point in comparing genes in the vertebrate lineage because they are thought to be the closest relatives to the vertebrates (17, 18). To the best of our knowledge, OT/VP family peptides have never been identified in the urochordates.

To elucidate whether or not tunicates possess peptides belonging to the OT/VP family peptides, we attempted to identify cDNA encoding the tunicate OT/VP peptides. After molecular characterization of the cDNA, we revealed a new member of the OT/VP family in tunicates, and this peptide is a tetradecapeptide. In this report, we show the localization and possible biological functions of this previously unidentified peptide in the tunicate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Tunicates, Styela plicata, with body lengths of 4–6 cm were collected at the Akitsu bay (Higashi-Hiroshima) and maintained in seawater at 15 C. The experimental protocol was approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Hiroshima University, Japan.

PCR primers
All sequences of PCR oligonucleotide primers used in this study are summarized in supplemental data 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo. endojournals.org).

Molecular cloning of cDNA encoding the precursor protein
These methods were similar to a previous report (19). Total RNA from 10 pooled neural complexes (including the cerebral ganglion and neural gland) was extracted by Sepasol RNA I Super reagent (Nacalai Tesque, Kyoto, Japan), and the poly(A)+ RNA was then isolated with Oligotex-deoxythymidine (dT) 30 (TaKaRa Bio, Shiga, Japan) in accordance with the manufacturer’s instructions. All PCR amplifications were carried out in a reaction mixture containing Ex Taq polymerase (TaKaRa Bio) and 0.2 mM dNTP on a thermal cycler (Program Temp Control System PC816; ASTEC, Fukuoka, Japan). Degenerate primers were designed based on the amino acid residues of preliminary unpublished work (Muneoka, Y., personal communication; CYISDCPNSRFWST-NH2). First-strand cDNA was synthesized with the oligo(dT)-anchor primer supplied in the 3'-rapid amplification of cDNA ends (RACE) kit (3'-Full RACE Core Set; TaKaRa Bio) and amplified with the anchor primer (TaKaRa Bio) and the first degenerate primers of Styela OT-related peptide (SOP)-d-1 (supplemental data 1), corresponding to the sequence CYISDCP. First-round PCR products were reamplified with the anchor primer and the second degenerate primers of SOP-d-2 (supplemental data 1), corresponding to the sequence ISDCPNSR. Second-round PCR products were also reamplified with the anchor primer and the third degenerate primers of SOP-d-3 (supplemental data 1), corresponding to the sequence NSRFWSTG (G is a donor for amidation). All rounds of PCRs consisted of five cycles for 30 sec at 94 C, 30 sec at 45 C, and 30 sec at 72 C and 30 cycles for 30 sec at 94 C, 30 sec at 50 C, and 30 sec at 72 C (10 min for the last cycle). The third-round PCR products were subcloned into a TA-cloning vector (pGEM-T Easy) in accordance with the manufacturer’s instructions (Promega Corp., Madison, WI). The DNA inserts of the positive clones were amplified by PCR with universal M13 primers. For determination of the 5'-end sequence of cDNA, template cDNA was synthesized with an oligonucleotide primer of SOP-AS-1 (supplemental data 1); this synthesis was followed by dA-tailing of the cDNA with dATP and terminal transferase supplied in the 5'-RACE kit (Roche Diagnostics, Basel, Switzerland). The tailed cDNA was amplified with the oligo(dT)-anchor primer and gene-specific primer of SOP-AS-2 (supplemental data 1); this was followed by further amplification of the first-round PCR products with the anchor primer and gene-specific primer of SOP-AS-3 (supplemental data 1). Both first-round and second-round PCRs were performed for 30 cycles for 30 sec at 94 C, 30 sec at 55 C, and 30 sec at 72 C (10 min for the last cycle). The second-round PCR products were subcloned into the TA-cloning vector, and the inserts were amplified as described above. The nucleotide sequence was determined with an ABI PRISM Dye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) and a model 310 automated DNA sequencer (Applied Biosystems) and then analyzed with DNASIS-Pro software (Hitachi Software Engineering, Kanagawa, Japan). Universal M13 primers or gene-specific primers were used to sequence both strands.

Homology search and phylogenetic analysis
To find the most homologous genomic sequences to the tunicate precursor protein, genomic sequence data were searched on the Ensembl web site (www.ensembl.org) by tblastn using the amino acid sequence as a query. This database contains genomic sequences of five vertebrates (human, chicken, frog, and two fish), two tunicates (Ciona intestinalis and Ciona savignyi), three insects, and a nematode. The NCBI (www.ncbi. nlm.nih.gov) DNA Database was also searched in the same way. All databases used are shown in supplemental data 2. In addition to these databases, we further analyzed in a free-living tunicate (Oikopleura dioica) on the Genoscope web site (www.genoscope.cns.fr). The amino acid sequences of the tunicate precursor protein and homologous proteins were aligned using Clustal W (20) and corrected by hand. A phylogenetic tree was drawn by the neighbor-joining method (21) with the entries of known OT/VP precursor proteins listed in supplemental data 2.

Immunoprecipitation and mass spectrometry
To identify the endogenous mature peptide, immunoprecipitation using the antibody against the C-terminal peptide sequence (CPNSRFWST-NH2) of the tunicate mature peptide (SOP) was employed. The antiserum was raised in rabbit, and its titer was checked by a dot-blot assay. The neural complexes (n = 5) were dissected out, frozen immediately in liquid nitrogen, and kept at –80 C until used. The tissue was homogenized in 80% acetone/10 mM HCl. The homogenate was centrifuged at 15,000 x g for 10 min at 4 C, and the supernatant was collected and evaporated. Subsequently, the pellet was dissolved in PBS and reacted with 2 µl of the antibody for 1 h. The complexes of antigen and antibody were precipitated by protein A agarose beads, and the adsorbed materials were eluted with 0.3 M acetic acid containing 0.1% 2-mercaptoethanol as described previously (22). The immunoreactive substance was concentrated and subjected to matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) (AXIMA-CFR-plus; Shimadzu, Kyoto, Japan) as described previously (23).

In situ hybridization of mRNA
The site of mRNA expression in the neural complex was localized by in situ hybridization as described previously (24). In brief, the neural complexes (n = 5) were fixed in 4% paraformaldehyde in PBS (pH 7.3) overnight at 4 C and then soaked in a refrigerated sucrose solution (30% sucrose in PBS) until they sank. The tissues were embedded in OCT compound (Miles Inc., Elkhart, IN) and freeze-sectioned sagittally at 10 µm thickness with a cryostat at –20 C. The sections were placed onto 3-aminopropyltriethoxysilane-coated slides. In situ hybridization was carried out according to our previous method using the digoxigenin (DIG)-labeled antisense RNA probe. The probe was synthesized from PCR fragment (262 bp; supplemental data 1) with T7 RNA polymerase in a DIG RNA labeling kit (Roche Diagnostics). Control for specificity of the in situ hybridization of mRNA was performed by using a DIG-labeled sense RNA probe, which was complementary to a common sequence of the antisense probe.

Immunohistochemistry
Immunohistochemical analysis was performed as previously described (24). In brief, the neural complexes (n = 5) were fixed as described above. Sagittal sections (10 µm thickness) of the neural complex were made with a cryostat at –20 C. After nonspecific binding components were blocked, the sections were immersed with the same antiserum as used for immunoprecipitation at a dilution of 1:1000 overnight at 4 C and subsequently with rhodamine-conjugated goat antirabbit IgG. The localization of immunoreactivity in the neural complex was examined with a Nikon fluorescence microscope. The specificity of staining was assessed by substituting the antiserum with antiserum (1:1000 dilution) that had been preabsorbed with the mature peptide (CYISDCPNSRFWST-NH2) at a saturating concentration (10 µg synthetic peptide/ml) overnight before use. The specificity of the antiserum against the C-terminal nonapeptide (CPNSRFWST-NH2) of the mature peptide (SOP) was determined by a competitive ELISA. The IC50 values (concentrations yielding 50% displacement) in the competitive ELISA were estimated as follows; 0.48 pmol for SOP, 0.59 pmol for SOP-C8, and more than 1000 pmol for isotocin, (G-amide9)-SOP-N9, and an unrelated tetrapeptide, FMRF-NH2, showing the specific cross-reactivity with the C-terminal region of SOP. The sequences of SOP-related peptides are shown in Table 1Go.


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  		TABLE 1. Structures of isotocin, SOP, and synthetic SOP-related peptides

 
Real-time PCR analysis
After tunicates (n = 34) were maintained in seawater for 2 d, they were moved into three different concentrations of artificial seawater, 60, 100, and 130% and kept for 2 d. These concentrations of seawater were determined on the basis of a previous report (25). The cerebral ganglion was collected in RNAlater (Ambion, Austin, TX), total RNA was extracted by RNAqueous-4PCR (Ambion), and contamination of DNA was digested by deoxyribonuclease I supplied in the same kit. First-strand cDNA was synthesized with the oligo(dT) primer and amplified with the gene-specific primers SOP-R-S and SOP-R-AS (supplemental data 1). PCR amplifications were carried out by iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) for 10 sec at 95 C, 10 sec at 55 C, and 15 sec at 72 C. The products of PCR in each cycle were monitored using a real-time thermal cycler (MiniOpicon; Bio-Rad). The expression level of mRNA encoding the tunicate peptide in the cerebral ganglion was quantified relative to that of cytoplasmic actin mRNA as an internal control (26) (supplemental data 1). The expression data were analyzed for significance using one-way ANOVA followed by Duncan’s multiple-range test.

Contractile activity of the siphons
The atrial and branchial siphons were excised from the tunicates (n = 5). Both ends of the dissected atrial or branchial siphons (~10 mm long) were tied with threads and mounted in a 2-ml chamber filled with aerated seawater. One end of the thread was fixed on the bottom of the chamber and the other to a force displacement transducer (type 45196A; NEC-Sanei, Tokyo, Japan) connected with a strain amplifier (6M82; NEC-Sanei). The tunicate mature peptide (SOP), isotocin, and synthetic SOP-related peptides [(G-amide9)-SOP-N9 and SOP-C8] listed in Table 1Go were diluted in distilled water, and 2 µl of the solution was directly applied to the chamber at the final concentrations of 10–9–10–6 M. As the control experiment, 2 µl distilled water did not show any contractile activity. The mechanical response was recorded by a pen recorder.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of cDNA encoding the tunicate OT-like peptide
The full-length cDNA encoding the precursor protein of the tunicate peptide and the deduced amino acid sequence are shown in Fig. 1Go. The precursor cDNA consists of 907 bp, excluding the poly(A) tail, and predicts a protein of 167 amino acids. The nucleotide sequence reported in this study has been deposited in the DDBJ/EMBL/GenBank databases (accession no. AB255884). The predicted N-terminal sequence of the precursor protein contains hydrophobic amino acids, a feature characteristic of a signal peptide. The mature peptide is connected to a C-terminal protein by the Gly-Lys-Arg sequence, a signal for carboxyl-terminal amidation and a dibasic proteolytic processing site. The C-terminal protein (124 amino acid residues) possesses 14 Cys residues (Fig. 1Go). This protein may be consistent with the neurophysin domain, judging by the conservation of the positions and numbers of Cys residues as likely other OT/VP family precursor protein (supplemental data 3). Therefore, it was confirmed that the tunicate peptide (CYISDCPNSRFWST-NH2) is a novel member of the neurohypophysial hormones, although it contains 14 amino acid residues instead of 9. Additionally, the neurophysin domain is not followed by the Leu-rich core segment, which is the common feature of VP-related peptides (supplemental data 3). Furthermore, because position 8 of the peptide is a neutral amino acid (Asn), it might be argued that this tunicate peptide belongs to the OT family. We designated this peptide SOP.


Figure 1
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  		FIG. 1. Nucleotide sequence and deduced amino acid sequence of the precursor encoding the tunicate tetradecapeptide. The sequence of the tetradecapeptide is boxed. Gly (G) C-terminal amidation signal and Lys (K)-Arg (R) dibasic processing sites are underlined. The 14 Cys (C) residues are shown in a box. The asterisk represents the stop codon (TGA). The poly(A) adenylation signal AATATAAA is indicated in boldface.

 
Homology searches were performed to confirm the orthology of SOP and other OT/VP family precursors. Genomic sequences of five vertebrates (human, chicken, frog, and two fish), two tunicates, three insects, and a nematode were searched by a translated blast search (tblastn) using the amino acid sequence of the SOP precursor protein as a query (supplemental data 2). Vertebrate genomic sequences that gave the highest scores corresponded to the loci of OT/VP precursor genes. No other SOP homologs were found in the genome databases. No SOP homologous sequences were found in the invertebrate genome databases. We also searched the NCBI nucleic acid database and again found no SOP homologs other than OT/VP precursors. In addition to the databases of Ensembl and NCBI, we also analyzed in a free-living tunicate (O. dioica). However, we could not find a homologous gene in this animal.

Molecular phylogenetic analysis was performed using the vertebrate homologs found above and other known OT/VP family precursors including those of lungfish, cyclostomes, annelids, and molluscs (supplemental data 4). The branching of SOP was consistent with the phylogenetic relationship of the tunicate and other animals. Therefore, we concluded that SOP is not a paralog belonging an unknown gene family but rather is the tunicate ortholog of the OT/VP precursor family.

Identification of mature peptide in the neural complex
To detect the production of SOP as a mature peptide in the tunicate, we performed MALDI-TOF MS analysis combined with purification by immunoprecipitation. For this immunoprecipitation, antiserum against the C-terminal region of SOP (CPNSRFWST-NH2) was employed. An ion peak of mass-to-charge ratio 1675.6 (M+H)+ was detected in the extracts of the neural complex (including the cerebral ganglion and neural gland) corresponding to synthetic SOP (Fig. 2Go).


Figure 2
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  		FIG. 2. Spectra of the native peptide from the neural complex (A) and synthetic SOP (B) by the MALDI-TOF-MS analysis. The main monoisotropic ion peak was observed in mass-to-charge ratios 1675.6 and 1675.7, respectively.

 
Localization of precursor mRNA and mature peptide in the cerebral ganglion
In situ hybridization of SOP mRNA was examined in the neural complex by using an RNA probe. An intense expression of SOP mRNA was detected only in the cerebral ganglion (Fig. 3Go, A and B). The SOP mRNA-containing cells were clustered in the area surrounding the cerebral ganglion. In contrast, the SOP mRNA was not expressed in the neural gland. A control study using a sense RNA probe resulted in the complete absence of the SOP mRNA expression in the cerebral ganglion (Fig. 3Go, C and D).


Figure 3
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  		FIG. 3. Cellular localization of SOP mRNA in the neuronal complex including the cerebral ganglion (CG) and neural gland (NG). A and B, The expression of SOP mRNA was localized by in situ hybridization. SOP mRNA-containing cells were assembled in the area surrounding the cerebral ganglion; C and D, lack of hybridization of SOP mRNA with the sense probe (control) is evident (C and D). Scale bars, 100 µm.

 
After immunohistochemical analysis, dense SOP-like immunoreactive fibers were labeled in the neuropil of the cerebral ganglion (Fig. 4Go, A–C). Immunoreactive fibers derived from the neuropil converged into the anterior and posterior nerves (Fig. 4Go, A–C) but there were no immunoreactive cells in this region. A complete absence of immunoreactive product in the cerebral ganglion was observed after preincubation of the antiserum with synthetic SOP (Fig. 4DGo).


Figure 4
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  		FIG. 4. Immunohistochemical staining of mature peptide in the neuronal complex including the cerebral ganglion (CG) and neural gland (NG). A–C, SOP-like immunoreactive fibers were distributed in the neuropil of the cerebral ganglion. The square in A is shown magnified in B and C, respectively. D, No immunoreaction was observed using the antiserum preincubated with a saturated concentration of synthetic SOP. Scale bars, 100 µm.

 
Expression levels of SOP mRNA in the different concentrations of seawater
To elucidate the physiological action of SOP, the tunicates were kept in three different concentrations of seawater, 60, 100, and 130%, for 2 d, and the expression levels of SOP mRNA were assessed using real-time PCR analysis. Expression of SOP in the 60% seawater was 2.4- and 2.2-fold greater than that in the 100 and 130% seawater, respectively (Fig. 5Go). During these experiments, we found that the tunicates closed their atrial and branchial siphons in the 60% seawater unlike in the 100 and 130% seawater (supplemental data 5).


Figure 5
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  		FIG. 5. Measures of SOP mRNA in the cerebral ganglion after holding in the three different concentrations of seawater, 60, 100, and 130%, for 2 d. The expression of SOP mRNA was quantified relative to that of cytoplasmic actin mRNA. Each column and vertical line represent the mean ± SE [n = 10 (60%) or 12 (100 and 130%) samples, one sample from one animal]. **, P < 0.01 (vs. 100 and 130% seawater) by Duncan’s multiple-range test.

 
Contractile activity of SOP on the siphons
The effect of SOP on the contraction of the atrial and branchial siphons was investigated using a force displacement transducer. SOP evoked contractions with increased tonus in each siphon in a dose-dependent manner, its threshold ranging between 10–9 and 10–8 M (Fig. 6AGo). Because isotocin has approximately 43% homology with SOP, the effect of isotocin was also investigated in the same experiment. However, isotocin had no effect on the siphons at the concentration of 10–6 M (Fig. 6BGo). In addition, synthetic SOP-related peptides, i.e. N-terminal nine amino acid residues [(G-amide9)-SOP-N9] and C-terminal eight amino acid residues (SOP-C8), and a combination of both did not show any contractile activity at 10–6 M (Fig. 6BGo). The structures of SOP-related peptides are shown in Table 1Go.


Figure 6
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  		FIG. 6. Effects of SOP, isotocin, and synthetic SOP-related peptides on the siphon muscles. A, SOP evoked contractions with increased tonus in the siphon in a dose-dependent manner; B, isotocin and synthetic SOP-related peptides and their mixture had no effect at a concentration of 10–6 M. The peptides were applied at the times indicated by the arrows.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neurohypophysial hormones from the OT and VP family have been identified in both vertebrates and invertebrates. These peptides are highly conserved. All consist of nine amino acid residues; share five residues, namely Cys1, Asn5 [except for seritocin (27) and octopressin (12); Ser5], Cys6, Pro7, and amidated Gly9; and contain an intramolecular disulfide bridge among the two Cys residues (supplemental data 6) (10). In the present study, we investigated whether or not the tunicate, S. plicata, possesses a peptide that belongs to the OT/VP family of peptides. The result of cDNA cloning showed that the precursor protein is composed of three segments, a hydrophobic signal peptide, a tetradecapeptide flanked by Gly-Lys-Arg, and a neurophysin domain, similar to other peptides in the OT/VP family. In particular, the neurophysin domain in the tunicate peptide also contains the 14 conserved Cys residues, which are considered to be essential for association with neurohyophysial hormones in neurosecretory granules (10). This latter fact revealed that the tunicate tetradecapeptide is a new member of this family of neurohypophysial hormones. Because all of the neurohypophysial hormones identified before this study are nonapeptides, the identification of a neurohypophysial hormone consisting of 14 amino acid residues is a novel characterization in the animal kingdom. The neurohypophysial hormones have been classified into the OT and VP family based on the amino acid residue at position 8 (10). In the OT family, this residue is a neutral amino acid, whereas it is a basic amino acid such as Arg or Lys in the VP family. Because the amino acid residue of the tunicate tetradecapeptide is a neutral amino acid at position 8, we designated this peptide SOP. Furthermore, the neurophysin domain of SOP is not followed by the copeptin-like domain including the Leu-rich core segment that is a common feature in the precursors of VP-related peptides (supplemental data 3). This also suggests that SOP is a member of the OT-related peptides. In addition, precursor analysis shows that the unique 14- amino-acid form of SOP is a consequence of the insertion of five amino acid residues between the disulfide bridge and the Gly-Lys-Arg sequence (supplemental data 3). In the analysis of the structure-activity relationship with the siphon muscle contractions, none of the peptides, isotocin or N-terminal nine and C-terminal eight amino acid residues of SOP, showed any contractile activity, nor did mixtures of these peptides (Fig. 6Go). This result suggests that the entire structure of the tetradecapeptide may be necessary for the binding to the receptor for SOP and eliciting biological activity. Furthermore, the amino acid residues of other OT/VP family peptides at position 5 are neutral, Asn or Ser [except seritocin (27) and octopressin (12)], as described above (supplemental data 6) (10). On the other hand, the amino acid residue of SOP at this position is acidic, Asp. This is also a unique feature in addition to the extra amino acid length. Further study on the receptor for SOP will contribute to the elucidation of the ligand-receptor relationship. It was recently reported that another tunicate, C. intestinalis, contains the VP receptor-like G protein-coupled receptor mRNA in the neural complex, although whether this receptor is responsible for SOP remains to be investigated (28).

In phylogenetic studies of neurohypophysial hormones, it is accepted that the OT-related and VP-related peptides have been derived from the duplication of an ancestral gene that may have been present in Agnathans, because vasotocin is the only member of OT/VP family in the cyclostomes (10). However, to study the evolutionary lineage of neuropeptides in both vertebrates and invertebrates, the tunicates are thought to be excellent models (29). Recent phylogenetic analyses suggest that tunicate and not cephalochordates are the sister group of vertebrates (17, 18). Some neuropeptides in urochordates have been investigated to elucidate the evolutionary origin of their structures and functions in vertebrates (29, 30, 31). The characterization of the SOP cDNA is a first demonstration of the presence of a neurohypophysial hormone in the urochordates. Molecular phylogenetic analysis suggests that the SOP precursor gene has originated from an ancestral OT/VP gene before the duplication and divergence of OT and VT genes. The structure of SOP and its precursor protein revealed that there may be great diversity in the structures of hormones among the urochordates, judging by the insertion of extra amino acid residues into the mature peptide and neurophysin domain (supplemental data 3).

To elucidate the production and localization of SOP in the tunicate, we further employed mass spectrometry, in situ hybridization, and immunohistochemistry. These analyses demonstrated that SOP is produced in the cerebral ganglion but not in the neural gland. However, SOP-like immunoreactive cell bodies were not detected in the cerebral ganglion. It is possible that SOP may be actively released into the axon, and the mature peptide may not reach detectable levels in the soma. Dense SOP-like immunoreactive fibers were observed in the neuropil of the cerebral ganglion, reinforcing this possibility (Fig. 4Go). There is precedent for this in terms of localization of hypothalamic neuropeptides to soma in vertebrates; often colchicine treatment is necessary to prevent axon transport of the peptide away from the soma and allow detection via immunocytochemistry (32).

The biological functions of neurohypopysial hormones are generally related to reproduction and osmoregulation. Both VP-related peptides and OT-related peptides influence osmoregulation. In the case of lower vertebrates (fish) and invertebrates (arthropoda, mollusca, and annelida), the neurohypophysial hormones are also associated with hydromineral metabolism (13, 14, 15, 16, 33). To assess whether SOP takes part in the osmoregulation, we kept the tunicates in the three different concentrations of seawater for 2 d and measured the levels of SOP mRNA in the cerebral ganglion under these different osmotic pressures. The results showed that the expression of SOP mRNA in the 60% seawater was significantly greater than those in the 100 and 130% seawater. During these experiments, we found that the tunicates closed their atrial and branchial siphons in the 60% seawater but not in the 100 and 130% seawater (supplemental data 5). This observation suggests that SOP may be released from the cerebral ganglion and cause contractile activity in the siphons to prevent the influx of a low concentration of seawater into the body in early response. To perform this, the SOP mRNA may be highly expressed to allow continuous release of the mature peptide in late response. To investigate this hypothesis, we used a force displacement transducer to measure the effect of SOP on the contraction of the atrial and branchial siphons. SOP evoked contractions with increased tonus on both siphons in a dose-dependent manner and with a strikingly long-lasting effect. Because we could not detect SOP-like immunoreactive fibers in the siphons via immunohistochemical analysis (data not shown), SOP might act hormonally on the siphons for an extended time via body fluids and not via direct neural connections. This phenomenon concurs with the fact that peptides of the OT/VP family in vertebrates are released from the neurohypophysis directly into the blood circulation, although significant differences in osmoregulation are present between the urochordates and vertebrates due to the open and closed blood vascular systems, respectively. In vertebrates, neurohypophysial hormones released from the neurohypophysis act on the kidney to reabsorb water through aquaporins (10). Although it is unknown whether tunicates possess the same mechanism, SOP may at least directly act on the siphons as a first step of adaptation to a saline environment. Therefore, this newly identified neurohypophysial hormone in this urochordate may be involved in a primitive form of osmoregulation. As yet, we do not understand the mechanism for sensing osmotic change. Further study will elucidate the detailed mechanisms of the release of SOP and the transcriptional regulation of SOP mRNA in response to salinity.

A recent phylogenetic hypothesis (17) suggests that rather than the sessile lifestyle of tunicates being ancestral for chordates, the free-living lifestyle of cephalocordates and vertebrates is the ancestral condition. If true, tunicates are a highly derived group specifically adapted to their sessile lifestyle. Therefore, the physiological mechanisms for osmoregulation should also have been modified for this lifestyle. For example, two siphons could be a synapomorphy of tunicates, and because the tunicates often settle to estuarine environments, it should have been necessary for them to evolve new mechanisms regulating siphons to adapt to changes in salinity. The diversification of sequence (supplemental data 3), expression, and localization patterns (Figs. 2–5GoGoGoGo) and specificity for contractile activity (Fig. 6Go) of SOP might reflect this physiological evolution at the molecular and cellular levels. On the other hand, we could not find SOP homologous genes in databases of tunicates (C. intestinalis, C. savignyi, and O. dioica). We will also need to reveal whether other animals of urochordates possess the OT/VP family peptides to conclude the evolutionary lineage in the chordates.

In conclusion, we demonstrated the first characterization of cDNA encoding the tunicate tetradecapeptide, SOP, which is a new member of the OT/VP family in the urochordate. SOP produced in the cerebral ganglion may act to prevent the influx of a low concentration of seawater into the body and thereby play an important role in osmoregulation. This is the first report on this previously unidentified neuropeptide. In addition, it is the first demonstration of its biological function. It appears that SOP in this urochordate is involved in osmoregulation, just as the OT/VP family is in both vertebrates and invertebrates. The similarity in not only structure but also function implies that the primary biological function of the neurohypophysial hormones throughout the animal kingdom may have been hydromineral equilibrium. It would be interesting to elucidate how this biological role has been conserved in the tunicate despite great diversity in the sequences of SOP and its precursor. In addition, the study of SOP has the potential to provide information as to how the neuropeptides and/or peptide hormones have evolved at molecular, cellular, and physiological relationships.


   Acknowledgments
 
We are grateful to Dr. Yojiro Muneoka (Professor Emeritus at Hiroshima University, Japan) and Dr. Masaaki Ando (Hiroshima University, Japan) for their valuable discussion and encouragement. We thank Dr. Nobuo Yamaguchi and Dr. Makoto Urata (Hiroshima University, Japan) for their help in collecting and dissection of the tunicates. We also thank Dr. George E. Bentley (University of California, Berkeley, Berkeley, CA) for his critical reading of the manuscript.


   Footnotes
 
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to K.U.), the JGC-S Scholarship Foundation (to K.U.), and the Kurata Memorial Hitachi Science and Technology Foundation (to K.U.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 26, 2008

Abbreviations: DIG, Digoxigenin; dT, deoxythymidine; MALDI-FOF-MS, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; OT, oxytocin; RACE, rapid amplification of cDNA ends; SOP, Styela oxytocin-related peptide; VP, vasopressin.

Received April 28, 2008.

Accepted for publication June 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gimpl G, Fahrenholz F 2001 The oxytocin receptor system: structure, function, and regulation. Physiol Rev 81:629–683[Abstract/Free Full Text]
  2. Rose JD, Moore FL 2002 Behavioral neuroendocrinology of vasotocin and vasopressin and the sensorimotor processing hypothesis. Front Neuroendocrinol 23:317–341[CrossRef][Medline]
  3. Landgraf R, Neumann ID 2004 Vasopressin and oxytocin release within the brain: a dynamic concept of multiple and variable modes of neuropeptide communication. Front Neuroendocrinol 25:150–176[CrossRef][Medline]
  4. Land H, Schütz G, Schmale H, Richter D 1982 Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor. Nature 295:299–303[CrossRef][Medline]
  5. Land H, Grez M, Ruppert S, Schmale H, Rehbein M, Richter D, Schütz G 1983 Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA. Nature 302:342–344[CrossRef][Medline]
  6. van Kesteren RE, Smit AB, Dirks RW, de With ND, Geraerts WP, Joosse J 1992 Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis. Proc Natl Acad Sci USA 89:4593–4597[Abstract/Free Full Text]
  7. Suzuki M, Kubokawa K, Nagasawa H, Urano A 1995 Sequence analysis of vasotocin cDNAs of the lamprey, Lampetra japonica, and the hagfish, Eptatretus burgeri: evolution of cyclostome vasotocin precursors. J Mol Endocrinol 14:67–77[Abstract/Free Full Text]
  8. Hyodo S, Ishii S, Joss JM 1997 Australian lungfish neurohypophysial hormone genes encode vasotocin and [Phe2]mesotocin precursors homologous to tetrapod-type precursors. Proc Natl Acad Sci USA 94:13339–13344[Abstract/Free Full Text]
  9. Satake H, Takuwa K, Minakata H, Matsushima O 1999 Evidence for conservation of the vasopressin/oxytocin superfamily in Annelida. J Biol Chem 274:5605–5611[Abstract/Free Full Text]
  10. Acher R, Chauvet J 1995 The neurohypophysial endocrine regulatory cascade: precursors, mediators, receptors, and effectors. Front Neuroendocrinol 16:237–289[CrossRef][Medline]
  11. Hoyle CH 1999 Neuropeptide families and their receptors: evolutionary perspectives. Brain Res 848:1–25[CrossRef][Medline]
  12. Takuwa-Kuroda K, Iwakoshi-Ukena E, Kanda A, Minakata H 2003 Octopus, which owns the most advanced brain in invertebrates, has two members of vasopressin/oxytocin superfamily as in vertebrates. Regul Pept 115:139–149[CrossRef][Medline]
  13. Proux JP, Miller CA, Li JP, Carney RL, Girardie A, Delaage M, Schooley DA 1987 Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria. Biochem Biophys Res Commun 149:180–186[CrossRef][Medline]
  14. van Kesteren RE, Tensen CP, Smit AB, van Minnen J, van Soest PF, Kits KS, Meyerhof W, Richter D, van Heerikhuizen H, Vreugdenhil E, Geraerts WPM 1995 A novel G protein-coupled receptor mediating both vasopressin- and oxytocin-like functions of Lys-conopressin in Lymnaea stagnalis. Neuron 15:897–908[CrossRef][Medline]
  15. Oumi T, Ukena K, Matsushima O, Ikeda T, Fujita T, Minakata H, Nomoto K 1996 Annetocin, an annelid oxytocin-related peptide, induces egg-laying behavior in the earthworm, Eisenia foetida. J Exp Zool 276:151–156[CrossRef][Medline]
  16. Fujino Y, Nagahama T, Oumi T, Ukena K, Morishita F, Furukawa Y, Matsushima O, Ando M, Takahama H, Satake H, Minakata H, Nomoto K 1999 Possible functions of oxytocin/vasopressin-superfamily peptides in annelids with special reference to reproduction and osmoregulation. J Exp Zool 284:401–406[CrossRef][Medline]
  17. Delsuc F, Brinkmann H, Chourrout D, Philippe H 2006 Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439:965–968[CrossRef][Medline]
  18. Swalla BJ, Smith AB 2008 Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives. Phil Trans R Soc B 363:1557–1568[CrossRef][Medline]
  19. Iwakoshi E, Takuwa-Kuroda K, Fujisawa Y, Hisada M, Ukena K, Tsutsui K, Minakata H 2002 Isolation and characterization of a GnRH-like peptide from Octopus vulgaris. Biochem Biophys Res Commun 291:1187–1193[CrossRef][Medline]
  20. Thompson JD, Higgins DG, Gibson TJ 1994 CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680[Abstract/Free Full Text]
  21. Saitou N, Nei M 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425[Abstract]
  22. Ukena K, Iwakoshi E, Minakata H, Tsutsui K 2002 A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry. FEBS Lett 512:255–258[CrossRef][Medline]
  23. Ukena K, Tsutsui K 2005 A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function. Mass Spectrom Rev 24:469–486[CrossRef][Medline]
  24. Iwakoshi-Ukena E, Ukena K, Takuwa-Kuroda K, Kanda A, Tsutsui K, Minakata H 2004 Expression and distribution of octopus gonadotropin-releasing hormone in the central nervous system and peripheral organs of the octopus (Octopus vulgaris) by in situ hybridization and immunohistochemistry. J Comp Neurol 477:310–323[CrossRef][Medline]
  25. Hyodo S, Tsukada T, Takei Y 2004 Neurohypophysial hormones of dogfish, Triakis scyllium: structures and salinity-dependent secretion. Gen Comp Endocrinol 138:97–104[CrossRef][Medline]
  26. Kovilur S, Jacobson JW, Beach RL, Jeffery WR, Tomlinson CR 1993 Evolution of the chordate muscle actin gene. J Mol Evol 36:361–368[Medline]
  27. Chauvet J, Michel G, Ouedraogo Y, Chou J, Chait BT, Acher R 1995 A new neurohypophysial peptide, seritocin ([Ser5,Ile8]-oxytocin), identified in a dryness-resistant African toad, Bufo regularis. Int J Pept Protein Res 45:482–487[Medline]
  28. Deyts C, Casane D, Vernier P, Bourrat F, Joly JS 2006 Morphological and gene expression similarities suggest that the ascidian neural gland may be osmoregulatory and homologous to vertebrate peri-ventricular organs. Eur J Neurosci 24:2299–2308[CrossRef][Medline]
  29. Sherwood NM, Tello JA, Roch GJ 2006 Neuroendocrinology of protochordates: insights from Ciona genomics. Comp Biochem Physiol A 144:254–271[CrossRef][Medline]
  30. Johnsen AH, Rehfeld JF 1990 Cionin: a disulfotyrosyl hybrid of cholecystokinin and gastrin from the neural ganglion of the protochordate Ciona intestinalis. J Biol Chem 265:3054–3058[Abstract/Free Full Text]
  31. Satake H, Ogasawara M, Kawada T, Masuda K, Aoyama M, Minakata H, Chiba T, Metoki H, Satou Y, Satoh N 2004 Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family. J Biol Chem 279:53798–53805[Abstract/Free Full Text]
  32. Meister B, Villar MJ, Ceccatelli S, Hökfelt T 1990 Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: an immunohistochemical study using experimental manipulations. Neuroscience 37:603–633[CrossRef][Medline]
  33. Acher R 1996 Molecular evolution of fish neurohypophysial hormones: neutral and selective evolutionary mechanisms. Gen Comp Endocrinol 102:157–172[CrossRef][Medline]



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